1 Comment


Williams Hematology



Platelet Morphology and Biochemistry

Light Microscopic Appearance

Electron Microscopic Appearance and Biochemistry
Platelet Physiology and Biochemistry

Overview of Platelet Adhesion, Aggregation, and Platelet Thrombus Formation

Platelet Energy Metabolism

Platelet Contractile Elements and Platelet Shape Change, Secretion, and Clot Retraction

Platelet Secretion

Clot Retraction

Platelet Coagulant Activity

Platelet Membrane Glycoproteins, Platelet Adhesion, and Platelet Aggregation

Platelets and Thrombolysis

Platelet-Leukocyte Interactions, Platelet-Tissue Factor Interactions, and the Role of Platelets in Inflammation

Signaling Pathways in Platelet Activation and Aggregation
Chapter References

Platelets are small anucleate cell fragments adapted to adhere to damaged blood vessels, aggregate one with another, and facilitate the generation of thrombin. These actions contribute to hemostasis by forming a platelet plug and then reinforcing the plug by the action of thrombin converting fibrinogen to fibrin strands. To accomplish these tasks, platelets have surface receptors that can bind adhesive glycoproteins (GP); these include the GPIb/IX/V complex, which supports platelet adhesion by binding von Willebrand factor even under conditions of high shear, and the GPIIb/IIIa receptor, which is platelet-specific and mediates platelet aggregation by binding fibrinogen and/or von Willebrand factor. Other receptors for adhesive glycoproteins [GPIa/IIa (a2b1), GPVI, and GP65 for collagen; GPIc*/IIa (a5b1) for fibronectin; and GPIc/IIa (a6b1) for laminin] also contribute to platelet adhesion, but their precise contributions are less well defined. Activated platelets express surface P-selectin, which mediates interactions with leukocytes. Platelet coagulant activity results from the exposure of negatively charged phospholipids on the surface of platelets and platelet microparticles, along with release and activation of platelet factor V and perhaps exposure of specific receptors for activated coagulation factors. Platelets change shape with activation as a result of complex changes in the platelet membrane skeleton and cytoskeleton. With activation, platelets undergo release of a granule, dense body, and lysosomal contents. The activation process involves a number of receptors for agonists such as ADP, epinephrine, thrombin, collagen, thromboxane A2, and platelet-activating factor, as well as several signal transduction pathways, including phosphoinositide metabolism, arachidonic acid release and conversion into thromboxane A2, and phosphorylation of a number of different target proteins. Increases in intracellular calcium result from, and further contribute to, platelet activation. Platelet activation results in a change in the conformation of the GPIIb/IIIa receptor, leading to high-affinity ligand binding and platelet aggregation.
Platelets also act as storehouses for a variety of molecules that affect platelet function, inflammation, vascular tone, fibrinolysis, and wound healing; these agents are actively released upon platelet activation. Other vasoactive and platelet activating substances are newly synthesized when platelets are activated. Through cooperative biochemical interactions, platelets can communicate with, and are affected by, other blood cells and endothelial cells.
Quantitative and qualitative disorders of platelets result in hemorrhagic diatheses (see Chap. 117, Chap. 119, Chap. 120). In pathologic states, uncontrolled platelet thrombus formation can lead to vasoocclusion and ischemic necrosis, as, for example, in myocardial infarction and stroke (see Chap. 130 and Chap. 131). Platelets may also facilitate tumor metastasis.

Acronyms and abbreviations that appear in this chapter include: ADP, adenosine 5′-diphosphate; ATP, adenosine 5′-triphosphate; BTK, Bruton’s tyrosine kinase; cAMP, cyclic AMP; COX, cyclooxygenase; CTAP-III, connective-tissue-activating peptide III; DAG, diacylglycerol; DTS, dense tubular system; Edg, endothelial differentiation gene; EDTA, ethylenediaminetetraacetic acid; ERK2, extracellular-signal-regulated kinase 2; FcRg, Fc receptor g; GP, glycoproteins; HPS, Hermansky-Pudlak syndrome; IAP, integrin-associated protein; IP3, inositol 1,4,5-trisphosphate; ITAMs, immune-receptor tyrosine-containing activation motifs; JNK, Jun N-terminal kinase; LAT, linker-for-activator T cells; LIBS, ligand-induced binding sites; LPA, lysophosphatidic acid; MIDAS, metal-ion-dependent adhesion site; mRNA, messenger RNA; NAP1, neutrophil-activating peptide 1; NO, nitric oxide; PAF, platelet-activating factor; PAI-1, plasminogen activator inhibitor 1; PAR-1, protease-activated receptor 1; PC, phosphatidylcholine; PCR, polymerase chain reaction; PDE, phosphodiesterase; PDGF, platelet-derived growth factor; PE, phosphatidylethanolamine; PECAM-1, platelet-endothelial cell adhesion molecule 1; PF4, platelet factor 4; PG, prostaglandin; PH, pleckstrin homology; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; PSGL-1, P-selectin glycoprotein ligand 1; RIBS, receptor-induced binding sites; SH2, Src homology 2; sPLA2, secretory PLA2; TGF-b, transforming growth factor beta; TSP, thrombospondin; uPAR, urokinase receptor; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor;

On films made from blood anticoagulated with the strong calcium chelating agent ethylenediaminetetraacetic acid (EDTA) and stained with Wright stain, platelets appear as small bluish-gray, oval-to-round bodies with several purple-red granules (Plate XIV) (see Chap. 2). The mean diameter of platelets varies in different individuals, ranging from about 1.5 to 3.0 µm, approximately one-third to one-fourth that of erythrocytes. There is also considerable variability in the size of platelets in a single individual, with occasional platelets in normal blood samples having diameters greater than half the diameter of erythrocytes. Overall, platelet size appears to follow a log normal distribution. When unanticoagulated blood is used to prepare blood films, platelets undergo variable activation and spreading, and thus platelet aggregates are commonly seen; platelets from such specimens may demonstrate three or four very long and thin processes extending out from the body of the platelet (filopodia), and some platelets may be devoid of granules.
Electron microscopy reveals a fuzzy coat (glycocalix) extending 14 to 20 nm from the platelet surface, which is thought to be composed of membrane glycoproteins, glycolipids, mucopolysaccharides, and adsorbed plasma proteins (Fig. 111-1).1 Platelets move in an electric field as if they have a net negative surface charge; sialic acid residues attached to proteins and lipids are major contributors to this negative charge.2 The electrostatic repulsion created by the negative surface charge may help prevent resting platelets from attaching to each other or to negatively charged endothelial cells.

FIGURE 111-1 (A) and (B). Discoid platelets. The lentiform shape of blood platelets is well preserved in samples fixed in glutaraldehyde and critical-point-dried for study in the scanning electron microscope. The indentations apparent on the otherwise smooth surfaces of the platelets (arrows) indicate sites where channels of the open canalicular system (OCS) communicate with the cell exterior. (A × 13,200; B × 35,000.) (C), (D), and (E). Ultrastructural features observed in thin sections of discoid platelets cut in the equatorial plane (C and D) or cross-section (E). Components include the exterior coat (EC), trilaminar unit membrane (CM), and submembrane area containing the specialized filaments (SMF) of the membrane skeleton. The plasma membrane indentations form the walls of the channels of the surface-connected canalicular system (CS and OCS). The circumferential band of microtubules (MT) is seen as a continuous band beneath the plasma membrane on the equatorial section and as small open cylinders at the ends of the platelet on the cross-section. Glycogen granules (Gly) are prominent punctate structures in the cytoplasm, and residual Golgi zones (GZ) can also be identified. Organelles include mitochondria (M), dense bodies (DB), and a granules (G), many of which have regions of electron density (nucleoids). The dense tubular system (DTS), the platelet equivalent of the sarcoplasmic reticulum sequesters calcium. (C × 30,000.) (F). Platelet shape change. Platelets exposed to ADP and then fixed and examined by scanning electron microscopy. The platelets lose their discoid shape and become spiny spheres with long extensions, variably referred to as filopodia or pseudopodia (Ps). (×17,000.) (From White,786 with permission.)

The surface of the platelet has a number of indentations that are thought to be the openings of the open canalicular system, which is an elaborate channel system composed of invaginations of the plasma membrane that extend throughout the platelet (see Fig. 111-1 and “Membrane Systems” below). The contents of platelet granules can gain access to the outside when the granules fuse with either the plasma membrane or any region of the open canalicular system. Similarly, glycoproteins contained within granule membranes can join the plasma membrane after granule fusion with either the plasma membrane or the open canalicular system.
The plasma membrane is a trilaminar unit composed of a bilayer of phospholipids in which cholesterol, glycolipids, and glycoproteins are embedded.1 Platelets prepared by the freeze-fracture technique demonstrate more intramembranous particles embedded in the outer platelet membrane leaflet than in the inner leaflet, which is the reverse of findings in erythrocytes; this observation presumably reflects the many external receptors that mediate platelet interactions. The plasma membrane is thought to contain the sodium- and calcium-ATPase pumps that control the intracellular ionic environment of the platelet. Approximately 57 percent of platelet phospholipids are contained in the plasma membrane (Table 111-1). The phospholipids are asymmetrically organized in the plasma membrane; the negatively charged phospholipids are almost exclusively present in the inner leaflet, whereas the others are more evenly distributed.3 The negatively charged phospholipids, especially phosphatidylserine, are able to accelerate several steps in the coagulation sequence and so their presence in the inner leaflet of resting platelets, separated from the plasma coagulation factors, is thought to be a control mechanism for preventing inappropriate coagulation.4 During platelet activation induced by select agonists, the aminophospholipids may become exposed on the platelet surface or on the surface of platelet microparticles (see “Platelet Coagulant Activity” below).4,5 and 6


The phospholipid asymmetry in resting platelets may be maintained by an ATP-dependent aminophospholipid translocase that actively pumps phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflet.4,7 Interactions of negatively charged phospholipids with cytoskeletal or other cytoplasmic elements may also contribute to the asymmetry.8,9
The lipid composition of platelet membranes is outlined in Table 111-1. The enrichment of selected phospholipids with arachidonic acid is quite striking, furnishing a store of arachidonic acid for release and conversion into thromboxane A2 (TXA2) (see “Signaling Pathways in Platelet Activation and Aggregation” below).
The glycoproteins in the plasma membrane are discussed below.
Membrane Cytoskeleton A planar network of thin, elongated spectrin tetramers interconnected by the ends of actin filaments is present immediately below the plasma membrane and the membranes of the open canalicular system.10 Actin-binding protein (filamin-1) is able to interact with both the transmembrane glycoprotein GPIba and the actin immediately below the membrane, thus connecting these components to the spectrin network and forming a membrane cytoskeleton that probably stabilizes the membrane’s discoid shape. In addition, the association of GPIba with the membrane skeleton restricts the expansion of the spectrin network and probably helps to organize receptors into linear arrays on the platelet surface, thus enhancing receptor cooperation.11,12 Other proteins that have been found in the membrane skeleton include talin, vinculin, dystrophin-related protein, molecules implicated in signal transduction, and several isoenzymes of protein kinase C (see below).11 The protein vimentin (Mr 58,000), which is an important component of intermediate filaments, is present in platelets and may contribute to the membrane cytoskeleton. With platelet activation, GPIIb/IIIa and a2b1 may also join the cytoskeleton. Thus, interactions with the cytoskeleton determine whether receptors are free to move in the plane of the membrane; they may also have a role in moving certain receptors from the surface to the interior of platelets and vice versa via the open canalicular system.11,12 and 13 The membrane skeleton may also be important in platelet spreading after adhesion.
Microtubules The circumferential band of microtubules present below the plasma membrane probably contributes to the platelet’s discoid shape,1 but it may also be involved in platelet formation from megakaryocytes.14 On cross-section, approximately 8 to 12 separate coils are observed at the tapered ends of the platelet, but this probably represents a single coil of about 100 µm wound multiple times. Microtubules are 25-nm hollow polymers composed of 13 protofilaments made up of polymers of Mr 110,000 subunits, each composed of two proteins of Mr 55,000 (a- and b-tubulin) that associate with several high-molecular-weight proteins (microtubule-associated proteins).15,16 and 17 Approximately 60 percent of the platelet tubulin is in microtubules, and there is a dynamic equilibrium between the polymerized and free tubulin subunits.18 Motor proteins of the dynein and kinesin families are also associated with microtubules.19
Microfilaments The platelet is rich in actin, a protein that can polymerize into microfilamentous bundles (see below).10,11,20 In resting platelets, microfilaments are not prominent, but when platelets change shape, the filopodia they form contain bundles of microfilaments made up of actin and associated proteins.1,16
Peroxisomes Peroxisomes are very small organelles present in platelets. They are thought to contribute to lipid metabolism, especially plasmalogen synthesis, and may participate in the synthesis of platelet-activating factor (PAF).21 They contain acyl-CoA:dihydroxyacetone phosphate acyltransferase, which catalyzes the first step in the synthesis of ether phospholipids. Deficiencies of this enzymatic activity in platelets have been identified in the cerebro-renal Zellweger syndrome.22,23
Mitochondria Platelets contain, on average, approximately seven mitochondria of relatively small size that are involved in oxidative energy metabolism (see “Energy Metabolism” below).24 Abnormalities of mitochondrial enzymes, including NADH coenyzme Q reductase (complex I), have been implicated in the pathophysiology of aging and several neurodegenerative disorders, including some patients with Parkinson disease. Assays of platelet mitochondrial enzyme levels have been used in these studies.25,26
Lysosomes Platelets have lysosomal granules that contain acid hydrolases typical of these organelles. Among the enzymes thought to originate from platelet lysosomes are b-glucuronidase, cathepsins, aryl sulfatase, b-hexosaminidase, b-galactosidase, endoglucosidase (heparitinase), b-glycerophosphatase, elastase, and collagenase.24 When platelets undergo secretion, lysosomal contents are more slowly and incompletely released than are the contents of a granules and dense bodies.27,28 Moreover, stronger inducers of activation are required to induce release of lysosomal contents. Proteins present in lysosomal membranes (e.g., CD63) have been identified, and their appearance on the plasma membrane serves as a marker of the platelet release reaction.29,30 The elastase and collagenase activities may contribute to vascular damage at sites of platelet thrombus formation. The heparitinase may be able to cleave heparin-like molecules from the surface of endothelial cells, and the resulting soluble molecules appear to inhibit growth of smooth muscle cells.31
Dense Bodies Platelets contain, on average, approximately three to eight electron-dense organelles, 20 to 30 nm in diameter (see Fig. 111-1).1,32 The intrinsic electron density of dense bodies when viewed as unstained whole mounts derives from their high content of calcium (see Table 111-2)1,24; the granules are also dense when viewed by transmission electron microscopy because they are highly osmophilic.32 Dense granules contain high concentrations of serotonin, which is taken up from plasma by a plasma membrane carrier and then trapped in the dense bodies.32 Trapping of serotonin may occur as a result of the lower pH (~6.1) maintained in dense granules due to the action of an H+ pumping ATPase on the dense body membrane.32 Adenosine 5′-diphosphate (ADP) and adenosine 5′-triphosphate (ATP) are also highly concentrated in dense bodies.24 There is more ADP than ATP in the dense bodies (ATP:ADP = 2:3), which is the reverse of their relative concentrations in the cytoplasm (ATP:ADP = 8:1). Since there is very little connection between the pools of adenine nucleotides in the cytoplasm and the dense bodies, they have been respectively designated as the metabolic and storage pools of adenine nucleotides.24 Storage of adenine nucleotides at such a high concentration in dense bodies appears to be achieved by stacking the ATP and ADP purine rings vertically in aggregates that are stabilized by the interactions of calcium ions with the polyphosphate groups.33,34 The planar hydroxyindole rings of serotonin may also enter these stacks, helping to account for the trapping mechanism. Trapping of serotonin must differ from that of adenine nucleotides, however, since dense granule serotonin exchanges readily with external serotonin.24


The membrane of dense granules contains glycoproteins that are also found on the plasma membrane and the membranes of a granules and lysosomes, including CD36, LAMP-2, P-selectin, GPIIb/IIIa, and GPIb/IX. Since patients with Hermansky-Pudlak syndrome (HPS) (see Chap. 119) have abnormal dense bodies, it is likely that the HPS gene product, which contains two transmembrane domains, is also associated with dense granules.35 Similarly, the product of the Chediak-Higashi syndrome gene, although lacking a transmembrane domain, has been proposed to associate with the dense granule membrane.36
Release of dense granule contents from activated platelets constitutes an important positive feedback mechanism for platelet aggregation, since ADP is a potent platelet agonist and serotonin is a weak agonist (see below). ATP is a partial antagonist of ADP-induced activation, but since ATP is rapidly catabolized to ADP in plasma (T1/2 = 1.5 min), and ADP is rapidly catabolized to AMP (T1/2 = 4 min) and then to adenosine,24 a platelet inhibitor,37 it is difficult to predict the overall effect of ATP release. Adding to the complexity in vivo is the presence of an ecto-ATP diphosphohydrolase (ATPDase) (CD39; ecto-ADPase) present on endothelial and lymphoid cells, which can metabolize ATP and ADP to AMP and thus probably limits the amount of ADP present.38 ATP released from platelets may also serve as a high-energy phosphate source for platelet ecto-protein kinases, which can phosphorylate several proteins, including GPIV.39,40,41 and 42
a Granules a granules are the most abundant granules in platelets, numbering about 50 to 80 per platelet.43 They are about 200 nm in diameter on cross-section and demonstrate internal variation in electron density, often with an eccentric area of accentuated electron density, termed a nucleoid, in which b-thromboglobulin, platelet factor 4, and proteoglycans are concentrated (Fig. 111-1).1 The more electron-lucent areas contain tubular elements in which von Willebrand factor (vWF), multimerin, and factor V are preferentially localized.44 Some of the most important proteins present in a granules are listed in Table 111-2. It appears that small amounts of virtually all plasma proteins are nonspecifically taken up into a granules, and thus the plasma levels of these proteins determines their platelet levels.45,46 For example, since the a-granule pool of immunoglobulins represents the vast majority of platelet immunoglobulin, total platelet immunoglobulin is much more affected by plasma immunoglobulin levels than by changes in surface immunoglobulin45,46 (see Chap. 117).
The platelet-specific proteins (platelet factor 4 and the b-thromboglobulin family) are present in a granules at concentrations that are about 20,000 times higher than their plasma concentrations (when each is expressed as a fraction of total protein in platelets or plasma respectively) (Table 111-2).47,48 These Mr 7000 to 11,000 proteins all bind to heparin, but with varying affinities. They also share amino acid sequence homology with each other and with other members of the “intercrine-cytokine” family of molecules, such as interleukin-8 (neutrophil-activating peptide 1, NAP1), which are active in inflammation, cell growth, and malignant transformation (Fig. 111-2).49,50 and 51

FIGURE 111-2 Comparison of the amino acid sequences of platelet factor 4 (PF4), platelet basic protein (PBP), and interleukin-8/NAP1 (IL-8). Double vertical lines indicate identical amino acids (underlined) in two of the three proteins. Dots (.) denote artificial breaks inserted to improve alignment. Note that the PBP and IL-8 share the ELR sequence (amino acids 26–28 in PBP) conserved in a number of CXC chemokines, but PF4 does not contain this sequence. Cleavage of peptide bond between R9 and N10 of PBP yields low-affinity platelet factor 4 (LA-PF4/CTAP-III); cleavage of peptide bond between K13 and G14 yields b-thromboglobulin (bTG); cleavage of peptide bond between Y24 and A25 yields b-thromboglobulin fragment (bTG-F/NAP2). Cleavage points follow the amino acid indicated by the arrows. (Adapted from Niewiarowski,47 with permission.)

Platelet factor 4 (PF4) is a chemokine in the CXC family that does not contain the Glu-Leu-Arg (ELR) conserved sequence.52 PF4 binds to heparin with high affinity and can neutralize heparin’s anticoagulant activity.47,53,54 and 55 It is thought to exist as a PF4 tetramer complexed to a proteoglycan carrier.56,57 Specific lysine residues (amino acids 61, 62, 65, and 66) have been implicated in its binding to heparin. X-ray crystallography indicates that these lysines are on the surface of the PF4 tetramer and that heparin winds around this core.58,59
After PF4 is released from platelets, it binds to heparin-like molecules on the surface of endothelial cells.59 Heparin administration can mobilize this endothelial-bound pool of PF4 into the circulation.59 PF4-heparin complexes and PF4-heparin-like molecule complexes on endothelial cells have been implicated as the target antigens in heparin-induced thrombocytopenia with thrombosis.60 PF4 also binds to hepatocytes, which take it up and catabolize it.61 PF4 is a weak neutrophil and fibroblast attractant.52,62 It inhibits angiogenesis, perhaps through inhibition of endothelial cell proliferation.63 A large number of other activities have been ascribed to PF4, including histamine release from basophils64; inhibition of tumor growth65 and megakaryocyte maturation66,67; reversal of immunosuppression62,68; enhancement of fibroblast attachment to substrata69; potentiation of platelet aggregation70; inhibition of contact activation,71 and enhancement of polymorphonuclear leukocyte responsiveness to the activating peptide f-Met-Leu-Phe and monocyte responsiveness to lipopolysaccharide.72,73
The b-thromboglobulin family of proteins are CXC chemokines that contain the conserved Glu-Leu-Arg (ELR) sequence.52 They include platelet basic protein, low-affinity platelet factor 4 (connective-tissue-activating peptide III; CTAP-III), b-thromboglobulin, and b-thromboglobulin-F (NAP2) (see Table 111-2 and Fig. 111-2).48,74,75 and 76 All of these proteins share the same carboxy terminus but differ in the length of their amino termini, presumably due to proteolytic digestion of the parent molecule, platelet basic protein (Fig. 111-2). These proteins bind to heparin but with lower affinity than PF4, and thus they neutralize heparin less well. Unlike PF4, they are cleared from the circulation by the kidney rather than the liver.77 CTAP-III is a weak fibroblast mitogen, and b-thromboglobulin is chemoattractant for fibroblasts.52 b-thromboglobulin-F (NAP2) is chemotactic for granulocytes and activates them to undergo endocytosis.52,76
The biochemistry of the adhesive glycoproteins contained in a granules and others variably present in plasma and extracellular matrix is described in Table 111-3 and in other chapters (Chap. 112 for fibrinogen and Chap. 135 for vWF). Their relative concentrations in a granules varies significantly. Presumably they are localized in platelets so that when platelets undergo the release reaction they will be present at high concentrations at the site of vascular injury.


Multimerin comprises a family of disulfide-linked homomultimers, ranging from Mr 450,000 to many millions of daltons in size.78 The Mr 450,000 multimer is thought to be a trimer of a single subunit of either Mr 167,00079 or Mr 155,00078 that is synthesized in megakaryocytes and endothelial cells and stored in the electron-lucent region of a granules in platelets and dense-core granules in endothelial cells.80 It colocalizes with vWF in platelets but not in endothelial cells. Although multimerin’s multimeric structure is similar to that of vWF, the deduced amino acid sequence of its subunit is not homologous to that of vWF.78 The prepromultimerin subunit contains 1228 amino acids. It undergoes glycosylation and proteolysis during synthesis. It is composed of a number of domains, including an amino-terminal region that includes an RGD sequence, coiled coil sequences, epidermal growth factor-like domains, and a carboxy-terminal globular head similar to that found in the complement protein C1q. Multimerin binds both factor V and factor Va, and all of the biologically active factor V in platelets is bound to multimerin.44 With thrombin activation of platelets, factor V separates from multimerin, and is released from platelets (see below); the higher Mr multimerin multimers bind to the platelet membrane. Multimerin does not circulate in plasma at an appreciable concentration, but it may act as an adhesive extracellular matrix protein.
Fibrinogen is concentrated in a granules, as judged by the ratio of platelet/plasma fibrinogen. Megakaryocytes do not appear to synthesize fibrinogen; instead, it is taken up from plasma by a process that involves the GPIIb/IIIa receptor.81 Since fibrinogen molecules that contain altered sequences in the g chain are not stored in a granules, even when the molecules are heterodimeric (i.e., contain one normal and one abnormal g chain), it is possible that uptake requires simultaneous binding of a fibrinogen molecule to two different GPIIb/IIIa receptors via the g-chain carboxy-terminal sequence81,82 (see GPIIb/IIIa below and Chap. 119).
The vWF stored in platelet a granules appears to contribute to hemostasis because in certain pathologic states it correlates better with bleeding symptoms than does plasma vWF (see Chap. 135). vWF is made in megakaryocytes and endothelial cells (see Chap. 114 and Chap. 135). The multimeric structure of platelet vWF is thought to reflect endothelial vWF more nearly than plasma vWF, since higher Mr multimers are present (see Chap. 135).
Fibronectin is present in a granules, and, although it interacts with a number of adhesive glycoproteins and can bind to thrombin-stimulated platelets under certain circumstances, no precise role in platelet function has been identified for this adhesive protein.
Vitronectin, a molecule that binds readily to glass, also binds to plasminogen activator inhibitor 1 (PAI-1), the urokinase receptor (uPAR), collagen, and heparin and forms ternary complexes with serine proteases and serpins in the coagulation and complement systems. It is present in platelets at levels that suggest it is concentrated,83 but it does not appear to be synthesized in megakaryocytes. Mice deficient in vitronectin develop normally but demonstrate a predisposition to develop thrombosis when challenged, suggesting that vitronectin serves an antithrombotic function.84
Thrombospondin-1 is unique among the adhesive glycoproteins in blood in that it is present almost exclusively inside the platelet.85,86 It constitutes about 20 percent of the released platelet proteins. Thrombospondin-1 is synthesized by megakaryocytes, cultured endothelial cells, and other cultured cells.87,88 Although GPIIb/IIIa, aVb3, proteoglycans, integrin-associated protein, and GPIV (CD36) have all been implicated as receptors for thrombospondin, controversy remains as to its mechanism of binding to platelets.89,90,91,92,93 and 94 The phosphorylation state of GPIV (CD36) may affect its ability to bind thrombospondin.91 Thrombospondin contains an Arg-Gly-Asp (RGD) sequence, which may contribute to its binding to platelets, but other regions are probably also involved.86 The conformation of thrombospondin varies with the calcium concentration of the surrounding environment. Thrombospondin can interact with many other adhesive glycoproteins, including fibronectin and fibrinogen,95,96 and it is a component of the extracellular matrix.97 Thrombospondin appears to stabilize platelet aggregates that are formed98; it may also modulate fibrinolysis and activate latent transforming growth factor beta (TGF-b) (see below).99,100 Thrombospondin and a peptide derived from its cell-binding domain can activate platelets via binding to integrin-associated protein, a membrane protein that is a member of the immunoglobulin superfamily and is associated with both GPIIb/IIIa and a2b1 (see “Signaling Pathways in Platelet Activation and Aggregation” below).93,94,101,102
Platelets contribute about 20 percent of the factor V present in whole blood, with nearly all of it in a granules.103,104 Megakaryocytes probably synthesize factor V,105,106 and it associates with multimerin when stored in a granules. During platelet activation and release, platelet-derived factor V appears to undergo proteolytic activation by at least two separate enzymes.4,107,108 Evidence from patients with inhibitors and deficiencies of plasma and platelet factor V indicate that platelet-derived factor V has an important role in hemostasis.109,110 Platelets undergo microvesiculation when activated, and the microvesicles, which are rich in factor V, are potent promoters of coagulation.111
Protein S (see Chap. 113), PAI-1 (see Chap. 116), and a2-plasmin inhibitor (see Chap. 116) are also contained in a granules and can be released from platelets. Similarly, tissue factor pathway inhibitor (see Chap. 113), a1-protease inhibitor, and C-1 inhibitor (see Chap. 113) have also been identified in platelets.
Platelet-derived growth factor (PDGF) is a disulfide-linked dimeric molecule of Mr 30,000 that is mitogenic for smooth-muscle cells.112 Platelet a granules contain a mixture of the homodimer PDGF B-B (30 percent) and the heterodimer PDGF A-B (70 percent); the different forms appear to have different functional activities.113 PDGF may play a role in normal cell proliferation, as well as in the development of atherosclerosis, tumor growth, wound repair, and fibroproliferative responses.114,115 and 116 After it was discovered in platelets and named platelet-derived growth factor, other cells were found to produce the same factor; thus the name PDGF is misleading. PDGF is structurally related to the putative transforming protein p28sis of simian sarcoma virus,117,118 and its receptor is in the tyrosine kinase family.119
Platelet-derived endothelial cell growth factor is a protein of Mr 45,000 that stimulates endothelial cell proliferation and angiogenesis.120 This protein is thought to be present in the cytoplasm of platelets and thus is only released at the time of platelet disintegration.
Platelets contain high concentrations of vascular endothelial growth factor (VEGF), an important stimulator of angiogenesis, and can release VEGF after stimulation in vitro and during the hemostatic response to a bleeding time wound.121,122 and 123 Megakaryocytes express mRNA of the three VEGF isoforms (121, 165, and 189 amino acids),124 and by immunoblot VEGF protein bands of Mr 34,000 and 44,000 are identifiable in platelets.125 Platelets and megakaryocytes also express the gene transcript for a VEGF receptor termed KDR.126 Another endothelial growth factor structurally related to VEGF, VEGF-C, has also been identified in platelets.127 Platelet levels of VEGF have been reported to be increased in malignancies,128 and platelet VEGF has been postulated to play a role in tumor growth129 and proliferative retinopathy in sickle cell disease.130,131
Epidermal growth factor has also been identified in platelets, but the kinetics of its release upon thrombin or collagen stimulation differs from that of other granule proteins.132
An amyloid b-protein precursor protein (protease nexin II) (Mr about 115,000) is present in high concentrations in platelets and can be released with platelet activation.133,134 Unique proteolysis products of this protein are found in cerebral amyloid deposits in patients with Alzheimer’s disease, and such patients may have altered proteolysis of their platelet protease nexin II as well.135 This protein also inhibits factors IX and XI, and heparin accelerates the inhibition.136,137 and 138
Factor XIII is present in platelets but differs from plasma factor XIII in having only the “a” subunits (see Chap. 112).139,140 Platelet factor XIII accounts for about 50 percent of total blood factor XIII.139,140
Transforming growth factor beta (TGF-b) is an Mr 25,000 homodimeric protein that promotes the growth of certain cells and inhibits the growth of others.141 It also induces synthesis of extracellular matrix proteins, PAI-1, and metalloproteinases. Migration of endothelial cells is inhibited by TGF-b, but it acts as a chemoattractant for monocytes and fibroblasts. TGF-b has a wide tissue distribution and is concentrated in platelet a granules, from which it can be released with platelet activation.142 TGF-b released from platelets is inactive (latent) because it is complexed with a portion of its precursor protein, and the latter is covalently coupled to another protein, the latent TGF-b-binding protein. Activation of latent TGF-b is a complex process that appears to require the cooperation of two different cell types and involves proteolytic digestion, perhaps by plasmin, abetted by transglutaminase.141 Complex formation between latent TGF-b and thrombospondin may also activate TGF-b.100 Active TGF-b can bind to three different cell surface proteins, a proteoglycan (beta glycan) and two serine/threonine kinases.143,144 TGF-b can increase thrombopoietin production by bone marrow stromal cells, and in turn, thrombopoietin induces megakaryocyte expression of TGF-b receptors I and II, allowing TGF-b to arrest the maturation of megakaryocyte colony-forming units.145 TGF-b can also stimulate smooth muscle cells to express and release VEGF, thus perhaps supporting reendothlialization after vascular injury.146
Platelets may also release proteins that affect the uptake of oxidized low-density lipoproteins by macrophages, furnishing another potential link between platelet activation and atherosclerosis.147
Ribosomes and Messenger RNA Platelets contain only a relatively small number of ribosomes, have only remnants of a Golgi apparatus (Fig. 111-1), and have only a small amount of messenger RNA (mRNA)148,149 Since they lack nuclei, they cannot synthesize mRNA. The application of the polymerase chain reaction (PCR) to platelet mRNA has permitted the molecular biologic analysis of platelet membrane glycoproteins and select plasma proteins that are synthesized in platelets, such as von Willebrand factor.150,151 Regulated synthesis of new proteins by platelets has been reported after thrombin activation, and it appears that signaling produced by ligand engagement of GPIIb/IIIa is required to initiate the process.152,153 Signaling through GPIa/IIa (a2b1) also can initiate platelet protein synthesis.153
Open Canalicular System The surface-connected open canalicular system is an elaborate series of conduits that begin as indentations of the plasma membrane and course throughout the interior of the platelet.1,154 Tracer studies demonstrate that the open canalicular system is contiguous with the exterior of the platelet, even though elements of the open canalicular system may appear as closed vesicles or vacuoles by electron microscopy of sectioned platelets.1,154,155
The open canalicular system may serve several functions. It provides a mechanism for entry of external elements into the interior of the platelet. It also provides a potential route for the release of granule contents to the outside, eliminating the need for granule fusion with the plasma membrane itself.155 This latter function is especially important because, under most circumstances, platelet granules appear to move to the center of the platelet upon platelet activation rather than to the periphery.1,156 Controversy remains, however, regarding the relative frequency with which secretion occurs via the open canalicular system versus direct fusion with the plasma membrane.1,157
The open canalicular system also represents an extensive internal store of membrane. Both filopodia formation and platelet spreading after adhesion result in a dramatic increase in surface plasma membrane compared to the plasma membrane of resting platelets, and it is not possible for new membrane to be synthesized during the short time-course of these phenomena. Thus, the membrane of the open canalicular system most likely contributes to the increase in plasma membrane under these conditions; the membranes of a granules, dense bodies, and, to a lesser extent, lysosomes may also contribute, but only if the stimulus is sufficient to induce the fusion of these organelles with the plasma membrane (release reaction). Finally, the membrane of the open canalicular system may serve as a storage site for plasma membrane glycoproteins. For example, under certain conditions, platelet activation by thrombin leads to a consistent, selective loss of GPIb/IX from the platelet surface; electron microscopy indicates that the GPIb/IX becomes sequestered in the open canalicular system.13,158,159 Plasmin may produce a similar phenomenon.13,160 Platelet activation leads to an increase in surface GPIIb/IIIa, and, although much of this is thought to derive from a-granule membranes, at least some may come from GPIIb/IIIa receptors in the membranes of dense bodies and the open canalicular system.13,161
Dense Tubular System The dense tubular system is a closed-channel network of residual endoplasmic reticulum characterized histocytochemically by the presence of peroxidase activity.1,162,163 The channels of the dense tubular system are less extensive than those of the open canalicular system and tend to cluster in regions in close approximation to the open canalicular system.1 The dense tubular system has been likened to the sarcoplasmic reticulum of muscle because it can sequester ionized calcium and release it when platelets are activated.164,165 Calreticulin, a calcium-binding protein found in the dense tubular system, probably helps to sequester calcium.17,166 Release of calcium from the dense tubular system involves the binding of inositol 1,4,5 trisphosphate (IP3), a messenger molecule formed during signal transduction, to receptors on the dense tubular system membrane. Cyclic AMP (cAMP) inhibits calcium release from the dense tubular system, either by enhancing the calcium pumping mechanism167 or by inhibiting release induced by IP3.168
The dense tubular system membrane is also probably a major site of prostaglandin and thromboxane synthesis165,169; in fact, the peroxidase activity used to identify the dense tubular system is an enzymatic component of prostaglandin synthesis.169,170
The hemostatic system is under elaborate control mechanisms lest the response either be inadequate to meet the hemorrhagic challenge or result in inappropriate thrombosis in response to trivial provocation. Evolutionary pressures have probably favored a highly active hemostatic system, since individuals with more active hemostatic systems were more likely to avoid death from hemorrhage prior to attaining sexual maturity or during childbirth. Our active hemostatic system appears to be less well adapted to our modern age, which is characterized by long life-spans and progressive vascular disease, since the deposition of a platelet-fibrin thrombus on a damaged atherosclerotic plaque can lead to myocardial infarction or stroke.
The platelet’s major function is to seal openings in the vascular tree. It is appropriate, therefore, that the initiation signal for platelet deposition and activation is exposure of underlying portions of the blood vessel wall that are normally concealed from circulating platelets by an intact endothelial lining (Fig. 111-3 and Table 111-4). Additional parameters that probably control the platelet response are: (1) the depth of injury, with deeper damage exposing more platelet-reactive materials and tissue factor171,172,173 and 174 (see Chap. 113); (2) the vascular bed, with the blood vessels serving mucocutaneous tissues especially dependent on platelets for hemostasis, in contrast to the vascular beds in muscles and joints, which rely more on the coagulation mechanism; (3) the age of the individual, since the composition of the blood vessel wall probably changes with age; (4) the hematocrit, since increased numbers of erythrocytes enhance platelet interactions with the blood vessel wall by forcing platelets to the periphery of the bloodstream (as the erythrocytes disproportionately occupy the axial region) and by imparting radially directed energy to platelets as the erythrocytes engage in flip-flop motions175; and (5) the speed of blood flow and the size of the blood vessel, which will determine the number of platelets passing by in a given time, the amount of time a platelet has to interact with the blood vessel wall or other platelets, the rate of dilution of platelet activating agents, and the forces tending to pull a platelet from the vessel wall or another platelet (shear rate).172,175 The vasospastic response that accompanies vascular injury, to which platelets contribute by release of thromboxane A2 and serotonin, probably plays a key role in decreasing hemorrhage and facilitating platelet and fibrin deposition via its effect on blood flow. Finally, platelet thrombi appear to be able to rapidly recruit tissue factor from the blood; the tissue factor is associated with small lipid-containing vesicle and may derive from leukocyte membranes.176

FIGURE 111-3 Platelet adhesion, activation, aggregation, and platelet-leukocyte interactions. A. Platelet adhesion is initiated by loss of endothelial cells (or in the case of an atherosclerotic lesion, rupture or erosion of the plaque), which exposes adhesive glycoproteins such as collagen and von Willebrand factor in the subendothelium. Other adhesive glycoproteins are also probably exposed, and these are listed in Table 111-3. In addition, von Willebrand factor and perhaps other adhesive glycoproteins in the plasma deposit on the damaged area. Platelets adhere to the subendothelium via receptors that bind to the adhesive glycoproteins. GPIb binding to von Willebrand factor plays a prominent role, but GPIa/IIa (a2b1) binding to collagen and other platelet receptors listed in Table 111-5 probably also play a role. After platelets adhere they undergo an activation process that leads to a conformational change in GPIIb/IIIa receptors, resulting in their ability to bind multivalent adhesive proteins including fibrinogen and von Willebrand factor, with high affinity. B. Platelet aggregation occurs when the multivalent adhesive glycoproteins bind simultaneously to GPIIb/IIIa receptors on two different platelets, resulting in receptor clustering and cross-linking. C. After platelets adhere and aggregate, they bind tissue-factor-containing vesicles circulating in the plasma, expose negatively charged phospholipids on their surface (not shown), release platelet factor V (not shown) and release procoagulant microparticles (not shown). In addition, activated platelets express P-selectin on their surface, which leads to leukocyte adhesion via P-selectin glycoprotein ligand-1 expressed on the surface of leukocytes. Other interactions between platelets and leukocytes are detailed in Fig. 111-13. Thrombus formation is a dynamic cyclical process, with platelets repeatedly adhering, aggregating, and then breaking off and going downstream. Platelet-leukocyte aggregates, thrombin, thromboxane A2 (TxA2), leukotrienes (LTs), and serotonin probably also go downstream and affect the microvasculature. Ultimately the vessel either becomes fully occluded or it loses its thrombogenic reactivity, that is, it becomes passivated. (From Coller, ref. 786a with permission.)



FIGURE 111-13 Platelet-leukocyte interactions. A number of interactions can occur between platelets and leukocytes, including polymorphonuclear leukocytes (PMN) and monocytes. The interaction between platelet P-selectin and leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) is probably the most important interaction [and can lead to tissue factor (TF) synthesis by monocytes], but fibrinogen binding simultaneously to activated aMb2 on leukocytes and either GPIIb/IIIa or aVb3 on platelets may play a role under certain circumstances. Platelets can release platelet activation factor (PAF), which can interact with a PAF receptor (PAFR) on leukocytes, leading to aMb2 activation and binding of fibrinogen and factor X. Platelets also can release CXC chemokines (ENA-78 and GRO-a), and b-thromboglobulin (bTG) released by platelets can be converted by leukocyte cathepsin G (CG) into the potent chemotatic CXC chemokine, NAP-2. The chemokines, in turn, activate leukocytes by binding to the chemokine receptor CXCR2. Platelets also contain the potent immune-stimulating molecule, CD40 ligand (CD40L), and both express it on the platelet surface and release it into the circulation upon platelet activation. Not shown is the interaction between thrombospondin and CD36 molecules on both platelets and some leukocytes, or the interaction between GPIb and aMb2.

The shear rate differentially affects platelet adhesion to surfaces; vWF-dependent adhesion is most important at higher shear rates, probably because high shear rates cause conformational changes in vWF and/or platelet GPIb.177,178,179 and 180 Very high shear rates can cause platelets to aggregate via a mechanism that involves vWF binding to GPIb/IX followed by activation of GPIIb/IIIa.181,182 and 183 Platelets contribute more significantly to arterial thrombi than to venous thrombi, perhaps as a result of differences in the shear rates in the different beds.172
The subendothelial layer immediately subjacent to the endothelium contains a large number of adhesive proteins (Table 111-4),172,178 and the platelet has receptors for many of these (Table 111-3 and Table 111-5). GPIb/IX is a receptor complex that is particularly important in mediating adhesion to vWF immobilized in the subendothelium, and this receptor appears to dominate the adhesion process at high shear184 (see Chap. 135). Three different sources of vWF may contribute to the subendothelial vWF; synthesis by endothelial cells, deposition from plasma, or release from platelet a granules.178,184 Subendothelial vWF appears to associate with type VI collagen,185 but it can bind to multiple collagen types. The interaction between GPIba and vWF does not itself cause firm adhesion; rather, it results in tethering and slow translocation, probably because the bonds between vWF and GPIba not only form rapidly but also dissociate rapidly.179 Adhesion initiated by GPIba-vWF interactions are stabilized by interactions between vWF and GPIIb/IIIa.186
The biologic contributions of the interactions between the GPIc/IIa (a6b1) receptor and laminin, the GPIc*/IIa (a5b1) receptor and fibronectin, and the aVb3 receptor and vitronectin or other ligands in initiating platelet adhesion remain unknown. GPIIb/IIIa may function as an adhesion receptor for immobilized fibrinogen even in the absence of platelet activation,187,188 but platelet activation is required for GPIIb/IIIa-mediated adhesion to vWF and fibronectin.188 Platelets may interact directly with exposed collagen via GPIa/IIa (a2b1) or perhaps other receptors implicated in platelet-collagen interactions (GPIV, GPVI, p65).189 In addition,189,190,191,192,193,194,195,196 and 197 fibrinogen, fibronectin, and vWF, whether released from platelets or circulating in plasma, may also bind to collagen. In turn, these proteins may then interact with platelet GPIIb/IIIa, GPIc*/IIa (a5b1), and/or GPIb/IX, completing a sandwich mechanism initiated by collagen exposure.193
Depending on the vascular bed, available adhesive glycoproteins, and shear conditions, it is likely that various combinations of platelet receptors, including GPIba, GPIa/IIa (a2b1), GPVI, p65, and GPIIb/IIIa act in concert to transform the tethering and slow translocation of platelets initiated by GPIba interacting with vWF into stable platelet adhesion.179,196,198,199
For platelet plug formation to occur, platelets must undergo activation as well as adhesion. Figure 111-4 lists physiologic and pathologic platelet agonists that can initiate platelet activation, along with some of the signal transduction pathways that lead to activation of platelet GPIIb/IIIa receptors and their clustering. Agonists can be subclassificed as either strong (e.g., high-dose thrombin and collagen) and weak (e.g., epinephrine, ADP, serotonin) based on their ability to initiate the platelet release reaction without the added stimulation that comes with platelet aggregation. Most of these activators are released or synthesized at the site of vascular injury, resulting in a local response. In addition, cooperative biochemical interactions between erythrocytes and platelets may enhance platelet activation.200

FIGURE 111-4 Platelet activation. Many different agents and phenomena can initiate platelet activation, and these are listed as agonists. Virtually all of these are released, synthesized, or occur at sites of vascular injury, resulting in both geographical and temporal restriction of the response. These agonists can initiate aggregation either alone or in combination with one or more other agonists. A number of different signal transduction mechanisms have been defined that convert the agonist signal into a change in the conformation of the GPIIb/IIIa receptor and cytoskeletal changes that result in ligand binding, receptor clustering, and platelet aggregation. (Adapted, with permission, from the Annual Review of Medicine, Volume 43, © 1992, by Annual Reviews http://www.AnnualReviews.org.)

It has been speculated that vascular injury results in release of ADP from erythrocytes, thus leading to platelet activation. Adhesion of platelets to subendothelial structures may itself lead to platelet activation, including generation of TXA2, release of ADP and serotonin, and activation of the GPIIb/IIIa receptors on the luminal side of the platelet to their high-affinity ligand-binding states.201 These positive feedback mechanisms ensure an adequate hemostatic response. Depending on the nature of the surface to which they adhere, platelets also undergo variable spreading reactions and become anchored by a process that at least partially involves GPIIb/IIIa ligation and clustering, cytoskeletal reorganization, and tyrosine phosphorylation; these reactions also contribute to initiating the release reaction.202,203 and 204
The activated luminal GPIIb/IIIa receptors on adherent platelets may then bind vWF and/or fibrinogen and await the interaction with another platelet, which itself may have undergone activation of its GPIIb/IIIa receptors as a result of exposure to released ADP and TXA2. Alternatively, a platelet may become activated and bind vWF or fibrinogen while still circulating, in which case the platelet-ligand complex may bind directly to an activated GPIIb/IIIa receptor on the luminal surface. This process of the binding of adhesive ligands to platelet receptors repeats itself, resulting in the recruitment of additional layers of platelets, and ultimately the formation of a hemostatic plug. Intravital videomicroscopy of the mesenteric circulation of mice after endothelial cell damage demonstrates that, at least in this vascular bed, platelet thrombus formation is initially a very dynamic process, with many platelets depositing but then breaking off and moving downstream. The thrombus grows relatively slowly compared to what its growth would be if all of the platelets that deposited remained attached to the surface.205
The aggregated platelets can facilitate thrombin generation by one or more different mechanisms, including formation of microvesicles, exposure of activated factor V, exposure of negatively charged phospholipids, and perhaps activation of the contact system (see below). The thrombin thus generated further activates platelets, leading to more extensive degranulation; thrombin also further activates coagulation and initiates the deposition of fibrin strands that reinforce the platelet plug as well as serve as sites for more vWF deposition.206 Thrombin may also help to consolidate the plug by initiating platelet-mediated clot retraction (see below). Finally, thrombin affects the surface membrane receptors, downregulating GPIb/IX and upregulating GPIIb/IIIa, perhaps facilitating the transition from platelet adhesion to platelet aggregation.13,158,159,207
Release of vasoactive and mitogenic agents from platelets no doubt contributes to the inflammatory response, as does the appearance of P-selectin on the surface of platelets and endothelial cells, which is likely to localize neutrophils to the damaged region (see “Platelet-Leukocyte Interactions” below).208,209 Platelets themselves will roll on endothelial cells that have been activated to expose P-selectin on their surface,210 and at least one of the counterreceptors for endothelial cell P-selectin is platelet GPIba.180 Platelets also express CD40 ligand on their surface after activation,211 which can interact with CD40 on lymphocytes, monocytes, and endothelial cells, leading to cell activation and an enhanced inflammatory response. Finally, the platelet-fibrin thrombi resolve, most likely by a combination of embolization, fibrinolysis, and macrophage removal of debris.
Several inhibitory factors serve to balance platelet activation and prevent excessive platelet deposition (Table 111-6). The dilutional effects of flowing blood are probably most important; thus, alterations in the surface of the blood vessel that produce local areas of stasis in which platelets and coagulation factors may concentrate increase the likelihood of thrombosis.172,175 Endothelial cells can synthesize two potent inhibitors of platelet activation, prostacyclin and nitric oxide (see below and Chap. 114). Basal synthesis of prostacyclin probably is too low to influence formation of platelet aggregates, but activated endothelial cells produce more prostacyclin. Activated platelets can also facilitate prostacyclin synthesis via production and release of endoperoxide intermediates and compounds that can activate endothelial prostacyclin production via receptor-mediated mechanisms. In addition, activated platelets can release microparticles that can transfer arachidonic acid to endothelial cells.212 Thus, prostacyclin may contribute significantly to platelet inhibition at sites of injury.213 Nitric oxide, which is synthesized by endothelial cells, is a potent inhibitor of ex vivo platelet adhesion and aggregation. It is not clear, however, whether the normal basal level of nitric oxide affects platelets. Nitric oxide synthesis is probably enhanced at sites of injury, and so it may well contribute to platelet inhibition, especially since it apparently synergizes with prostacyclin.213 Endothelial cells also have CD39, an ecto-ATP diphosphohydrolase (ecto-ADPase) that can digest ATP and ADP to AMP, and thus limit the effects of released ADP.38,42 Under certain conditions, leukocytes appear to interact biochemically with platelets to limit platelet activation,214 but cathepsin G released from activated leukocytes can activate platelets.215,216


Since thrombin is such a potent activator of platelets, the control mechanisms that limit thrombin production also can be considered control mechanisms for platelet aggregation (see Chap. 113). Platelets can also become desensitized to stimulation by some agonists if they have previously been exposed to low concentrations of that agonist (homologous desensitization). It is possible that in the penumbra of released platelet agonists some platelets become inhibited by this mechanism.217,218 and 219
Since the GPIIb/IIIa receptor occupies a central role in determining the extent of platelet aggregation, it is notable that this receptor is present at extraordinarily high density on the platelet surface (receptors are probably less than 20 nm apart).220,221 and 222 This permits the receptor to rapidly initiate platelet aggregation. On the other hand, the receptor is not in its high-affinity ligand-binding state on resting platelets but rather needs to be activated by agonists, including ADP, serotonin, thrombin, collagen, and TXA2, that are localized to sites of vascular injury.204,221,222 As a result, platelets can circulate in plasma containing high concentrations of the GPIIb/IIIa ligands fibrinogen and vWF without ongoing platelet thrombus formation.
Thus, platelet adhesion is controlled by the exposure of the subendothelium, with the platelet GPIb/IX receptor for immobilized vWF and the GPIIb/IIIa receptor for immobilized fibrinogen always competent to interact with these adhesive ligands.178,187,188 In contrast, the ability of GPIIb/IIIa to mediate platelet aggregation by binding fluid-phase vWF223 or fibrinogen221,222,224 is under the control of an elaborate activation mechanism that limits the response to sites of vascular injury.
The agonists that activate the GPIIb/IIIa receptor are likely to work in combination in vivo. In fact, the mixture of agonists present in a thrombus is likely to change as the process unfolds, with perhaps collagen more important at the beginning, thrombin more important later on, and with the other agonists in varying mixtures throughout. Platelet activation induced by multiple agonists simultaneously is not simply additive; synergistic interactions are well documented (see below).225,226 Epinephrine, although a relatively weak platelet agonist itself, probably plays an important role by enhancing the platelet’s response to other agonists, including the ability to overcome aspirin-induced inhibition of platelet thrombus formation.227 Changes in epinephrine levels that can accompany cigarette smoking or vascular collapse, as during myocardial infarction, may therefore have significant effects on platelet thrombus formation.227,228 and 229 Finally, platelets can also be activated by shear stresses ex vivo; while the in vivo significance of this phenomenon remains unknown, it offers another potential link between the blood vessel narrowing produced by atherosclerotic vascular disease and platelet activation.182,183,230
Platelets have sizable stores of glycogen that can often be seen on electron microscopy (Fig. 111-1). Glycogen can be broken down into glucose 1-phosphate, and platelets can also take up glucose from their surrounding medium. Both sources of glucose can be converted to glucose 6-phosphate, which can then enter glycolysis or the hexose monophosphate shunt. Platelet glycolysis rates significantly exceed those of erythrocytes and skeletal muscle.231 Oxidative metabolism probably contributes to energy production in resting platelets, but it has been estimated that less than 1 percent of the pyruvic acid produced by glycolysis actually enters the citric acid cycle, the remainder terminating in lactate or pyruvate, which leave the platelet.232 Platelet mitochondria are capable of b oxidation of fatty acids, but it is not clear how much this contributes to energy production.233,234 and 235 Platelets can actively metabolize acetate, and this ability has been exploited to improve platelet storage conditions.236 Amino acids may also act as energy sources and feed into the citric acid cycle, but the contribution of this process to platelet energy metabolism is uncertain.
As in all cells, ATP consumption by platelets is partially devoted to maintaining ionic and osmotic homeostasis.237,238 In addition, the continuous polymerization and depolymerization of actin involves conversion of ATP to ADP, and this may account for as much as 40 percent of the ATP consumption in resting platelets.239 The inositol phosphates, which are important in signal transduction, undergo continual dephosphorylation and rephosphorylation; these reactions have been estimated to consume as much as 7 percent of the total ATP produced.240 Protein phosphorylation also occurs as an ongoing event, but its fractional use of ATP is not clear.
Depleting platelets of the metabolic pool of ATP and ADP decreases their ability to respond to stimuli, but the effect is not uniform: thus, shape change is only minimally affected, whereas there is an increasingly significant effect on platelet aggregation, a-granule and dense granule secretion, arachidonic acid liberation, and lysosome secretion.28,241
Platelet stimulation is accompanied by a marked increase in both glycolytic activity and oxidative ATP production, perhaps due to the abrupt decrease in ATP that occurs with platelet activation or the increase in cytoplasmic pH.234 The increased ATP appears to be utilized, at least in part, in phosphoinositide phosphorylation and protein phosphorylation.
The major components of the platelet contractile system are listed in Table 111-7. These elements are thought to contribute to platelet shape change, secretion, and clot retraction after platelet activation.


The platelet cytoskeleton, namely, those elements that contribute to the maintenance and change of its shape, is operationally defined as proteins that are insoluble in the presence of the nonionic detergent Triton X-100 under defined ionic conditions (see also “Membrane Cytoskeleton”).11,242,243 The cytoskeleton of resting platelets consists of the membrane skeleton described above, which lies just beneath the membrane, and a lacy cytoplasmic actin filament network, which also contains, a-actin, tropomyosin, vinculin, and caldesmon.242,244,245
With platelet activation, phosphorylated myosin joins the cytoskeleton, as does talin, and the cytoskeleton becomes an electron-dense mass of bundled filaments.242,246,247 Platelet membrane glycoproteins GPIIb/IIIa and GPIa/IIa (a2b1) also join the cytoskeleton of activated platelets, probably via some interaction between actin, vinculin, talin, and the cytoplasmic domains of the membrane glycoproteins.11,248 The tyrosine kinase pp125Fak may also play a role in the process,202,249 as may the tyrosine kinase pp60src, which is very abundant in platelets249; cortactin, an 85-kD protein that is phosphorylated on tyrosine; and small GTP-binding proteins such as Rho, Rac, and Cdc 42.10,11,250
Platelets contain calpains, which are calcium-dependent, sulfhydryl, neutral proteases composed of two subunits that preferentially cleave cytoskeletal proteins, in particular actin-binding protein (filamin-1) and talin,250,251 but have also been reported to cleave the cytoplasmic domain of GPIIIa, and a number of molecules involved in signaling, including kinases and phosphatases [see “Calcium-Dependent Proteases (Calpains)” below]. It has been proposed that calpains are involved in cytoskeletal reorganization upon platelet activation and perhaps binding of ligand to GPIIb/IIIa.252,253 Calpains have also been implicated in platelet spreading, microparticle formation, and the generation of platelet coagulant activity.250,254,255
Platelet shape change occurs in response to many different agonists. It involves loss of the platelet’s normal discoid shape (about 1.5 to 2.5 µm diameter and about 0.5 to 0.9 µm width) and transformation to a spiny sphere with long, thin filopodia extending several µm out from the platelet and ending in points that are as small as 0.1 µm in diameter (Fig. 111-1).1,256 Although the reason platelets undergo shape change is unclear, one possibility is that it reduces electrostatic repulsion even without reducing surface charge density; thus, the tip of a platelet can approach and make contact with a surface or a cell, with the great bulk of the repulsive surface charge now at a distance.2
Actin fibril formation, which is an important component in shape change, is a complex, energy-requiring process that depends on nucleation, polymerization, helix winding, and filament bundling.242 The proteins listed in Table 111-7 either facilitate or inhibit these processes. In resting platelets, actin monomers and small, thin actin filaments predominate (60 percent), but with activation, actin polymerization occurs and monomeric actin decreases to 20 to 40 percent of total actin.20,242 Actin filaments in resting platelets are relatively stable because their barbed ends (the ends from which they can grow by adding additional actin monomers) are capped with the protein CapZ (Fig. 111-5).10

FIGURE 111-5 Control of platelet actin assembly. Rest: Forty percent of the actin in the resting cell is filamentous. The rest of the actin is soluble (60 percent) and is in a 1:1 complex with b4-thymosin. Filaments are stable because they are capped on their barbed ends by capZ. Active: Shape change begins when calcium rises into the micromolar level and gelsolin becomes active. Gelsolin binds to actin filaments, interdigitates, and causes filaments to fragment. After fragmentation gelsolin remains bound to the barbed filament end. Assembly of actin begins when capping proteins are dissociated from the barbed ends of the filament fragments formed in the rounding step by polyphosphoinositides (ppIs) and when the actin-related protein (ARP) 2/3 complex in platelets is activated to nucleate de novo filaments. Actin monomers, stored in complex with b4-thymosin, are the source of the actin for this polymerization event. Transfer of actin from b4-thymosin to the barbed ends of actin filaments is facilitated by profilin. Once assembly is complete, capZ recaps the barbed filament ends. (From Hartwig,10 with permission.)

The initial step in shape change is the activation of gelsolin, which then both severs existing actin filaments and caps the newly created barbed ends. This increases the number of actin filaments by an estimated tenfold and substitutes gelsolin for CapZ as the capping protein.10 Severing of actin filaments that interact with the planar lattice of actin-binding protein (filamin-1), GPIb, and spectrin in the membrane cytoskeleton releases the constraints on the spectrin network. This allows the membrane skeleton to swell (but not produce filopodia) (Fig. 111-6) by incorporating into the plasma membrane the membranes from the open canalicular system, and later the membranes from the granules that release their contents.

FIGURE 111-6 Control of platelet shape change. Resting platelets are small discs. Platelets convert from discs in two steps. In the first, a calcium transient activates platelet gelsolin. Gelsolin binds, severs, and caps actin filaments. The fragmentation of endogenous filaments causes the cell to become spherical. In the second step, spherical cells protrude lamellae and filopods. Actin filament assembly drives the protrusion of cellular processes. (From Hartwig,10 with permission.)

The protrusive force for filopodia development comes from subsequent actin polymerization on the newly severed actin filaments, including those attached to the plasma membrane. Uncapping of the actin filaments appears to be accomplished by the inactivation of gelsolin by phosphoinositides (ppIs) that are produced during platelet activation, including phosphatidylinositol 3,4 bisphosphate (PI3,4P2), PI4,5P2, and PI3,4,5P3.10 The uncapped actin filaments act as nuclei onto which actin monomers (which are maintained in an available pool by association with thymosin-b4) can assemble. Profilin accelerates actin polymerization by facilitating the transfer of actin from the actin-thymosin-b4 complex to the barbed ends of the actin filaments. Other proteins that have been implicated in organizing the tips of the filopodia where the actin bundles attach to the plasma membrane are the small GTPase Cdc 42, the exchange protein WASP (which is abnormal in Wiskott-Aldrich syndrome) (see Chap. 119), vinculin, VASP, zyxin, and profilin.17 As the filopodia form, the platelet’s granules and organelles move to the center, surrounded by the microtubule coil, resulting in an increase in electron density. Activation of myosin via phosphorylation of myosin light-chain kinase, contributes to the inward contractile force by its interaction with the actin fibers.
After platelets adhere to surfaces, they undergo variable degrees of spreading and activation. The patterns of spreading and activation depend primarily on the protein surface on which they spread, with collagen consistently inducing the most activation.195,257 Activation can result in release of granule contents and exposure of activated GPIIb/IIIa receptors on the luminal surface of the platelets, where it is strategically located to bind adhesive glycoprotein ligands that can recruit additional platelets.201 If the surface density of platelets is sufficient, the platelets can also enter into lateral associations, which appear to depend on GPIIb/IIIa, leading to syncytial development. In general, platelet spreading results in the development of broad lamellipodia rather than spikelike filopodia.10,250 The different morphologies of platelet spreading reflect differences in the organization of the orthogonal network of actin filaments. In turn, these differences reflect the different signals initiated by the adhesion process, and both ppIs and the small GTPase molecules Rac and Rho appear to be particularly important in this process.17 Pleckstrin, a platelet protein that is phosphorylated during platelet activation, appears to participate in this process by binding to ppIs and affecting Rac via an exchange factor.258 Signaling after adhesion results from the assembly of protein complexes on the cytoplasmic surfaces of the receptor(s) involved in the adhesion process. These complexes then initiate local cytoskeletal rearrangements as well as the generation of signaling molecules that probably act throughout the platelet.153,204
Membrane glycoproteins are affected by cytoskeletal rearrangements associated with platelet shape change and spreading. Activation of platelets in suspension under certain conditions results in movement of GPIb/IX receptors from the surface of platelets to the open cannalicular system.158,159 With adherent platelets, the GPIb/IX internalization is much slower.17 The initial effect of activation on GPIIb/IIIa is an increase in receptors on the plasma membranes as the GPIIb/IIIa receptors in a granules, and perhaps dense bodies and the open cannalicular system, join the plasma membrane. After activation, more GPIIb/IIIa molecules become associated with the cytoskeleton, and this presumably reflects ligand-induced GPIIb/IIIa clustering, resulting in the development of protein complexes, including cytoskeletal proteins, on the cytoplasmic surface of the receptor.204 When platelets are adherent, and ligand-coated beads bind to GPIIb/IIIa receptors, the beads are transported to the center of the platelets, indicating that the cytoskeleton can move GPIIb/IIIa receptors that have ligand attached to them.259,260 Finally, it has been suggested that the GPIIb/IIIa activation process itself, in which GPIIb/IIIa adopts a high-affinity binding state, is due to the loss of the basal constraints imposed on GPIIb/IIIa by the cytoskeleton.261
The contractile mechanism involving actin and myosin is thought to mediate granule secretion and clot retraction, but the details remain obscure.32,242 After the initial platelet shape change, actin becomes organized centrally into thick filamentous masses, where it probably associates with myosin filaments.242 The contractile response is thought to be initiated by an increase in cytosolic calcium, which results in the formation of a calcium-calmodulin complex that then activates myosin light-chain kinase; phosphatases and cAMP-dependent kinase A can modulate the response (see below). The centralization of organelles in a contractile ring correlates well with secretion.1 There is controversy, however, as to whether platelets secrete the contents of their granules by fusion with the open canalicular system in the center of the platelet or by direct fusion with the plasma membrane.1,157
A two-step model for granule secretion has been proposed in which the first step is docking of granules to the inner leaflet of the plasma membrane and the second step is the fusion of the lipid bilayers.32,262 The docking process is thought to involve small GTPases, notably rab 3, which is phosphorylated when platelets are activated.263 The rab 3 is thought to form complexes with SNARE proteins present on both the granule and plasma membrane, including syntaxins 2 and 4, leading to the development of a 7S docking complex.262 The docking reaction does not require ATP, but the subsequent priming reaction, which prepares the complexes for membrane fusion, is energy dependent.264 The 7S docking complex interacts with other proteins, including N-ethylmaleimide-sensitive factor (NSF) and both a- and g-synaptosomal-associated proteins (SNAPs), forming a large 20S fusion complex. Phosphoinositides and cytoskeletal proteins, including myosin light-chain kinase, also participate in the priming reactions.265 In the fusion step, it has been proposed that SNAP interactions with NSF activates the NSF ATPase activity, resulting in release of SNAREs from the 20S complex. Calcium plays an important role, probably acting in concert with a homologue of synaptotagamin, in creating fusion pores between the membranes, and the process is aided by synaptophysin and synaptogyrin (or their homologues).266
When blood initially clots in the test tube, the fibrin mesh extends throughout, trapping virtually all of the serum in a gel-like state. If platelets are present, within minutes to hours, the clot retracts, extruding a very large fraction of the serum.267 This process is thought to mimic in vivo phenomena that result in consolidation of thrombi and perhaps enhancement of wound healing. Clot retraction has also been implicated in decreasing the efficiency of thrombolysis, which may partially account for the resistance of platelet-rich thrombi to fibrinolytic agents.268 Although the platelet requirement for clot retraction is indisputable, and temporal studies strongly incriminate an actinomyosin contractile mechanism,269,270 no model describing the details of the process has gained acceptance.271 Proposed mechanisms include movement of platelet filopodia along fibrin strands, tugging of fibrin strands by filopodia, and internalization of fibrin by the action of the membrane skeleton.269,270,271,272 and 273 Platelet GPIIb/IIIa is required for clot retraction, as demonstrated by studies of patients with Glanzmann thrombasthenia (see Chap. 119) and studies of normal platelets in the presence of agents that block the GPIIb/IIIa receptor.272,274,275 Results with these agents demonstrate, however, differences in their ability to inhibit clot retraction that do not correlate with their ability to block fibrinogen binding to platelets. Moreover, fibrinogen lacking the g-chain sequence that mediates binding to platelet GPIIb/IIIa is still capable of supporting clot retraction.276,277 Thus, while GPIIb/IIIa is required for clot retraction, the process is not a simple reflection of fibrinogen binding to GPIIb/IIIa.
In resting platelets, the negatively charged phospholipids, including phosphatidyl serine, are almost exclusively present in the inner leaflet. The mechanisms responsible for this assymetry are not clear but may involve unidirectional enzymatic movement by an aminophospholipid translocase or an association between the negatively charged phospholipids and elements in the cytoplasm, including cytoskeletal elements and their accompanying proteins.4,7,9,278,279 The mechanisms responsible for the redistribution of negatively charged phospholipids to the platelet surface are also not fully understood but probably involve a calcium-activated plasma membrane enzyme of Mr 37,000 that counters the assymetric distribution (“scrambalase”) (Fig. 111-7).279,280 and 281 Since platelet activation with certain agonists results in the formation microparticles, which are particularly rich in surface-exposed negatively charged phospholipids, it is possible that the molecular reorganization of the membrane that produces microparticles also results in surface exposure of negatively charged phospholipids on both the microparticles and the residual platelet membrane. Microparticles also are rich in factor Va and thus actively support thrombin generation.5,212,282

FIGURE 111-7 Schematic representation of platelet calcium homeostasis and the reactions leading to procoagulant expression. Thrombin activates protease-activated receptor 1 (PAR-1), which interacts with G proteins and stimulates phospholipase Cb (PLCb) to split phosphatidylinositol-4,5-bisphosphate (PIP2) into 1,2-diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). Collagen binds to the integrin receptor GPIa/IIa (a2b1) and activates platelets through tyrosine phosphorylation of the Fc receptor g chain (FcRg-chain), which is associated with the membrane glycoprotein GPVI. This leads to tyrosine phosphorylation and activation of PLCg2, which, like PLCb, splits PIP2 and produces IP3. The IP3 opens Ca2+ channels in the intracellular membrane system called the dense tubular system, which serves as an intracellular Ca2+ store. ATP-driven Ca2+ pumps, both in dense tubular system membranes and in the plasma membrane, carry Ca2+ out of cytosol by active transport. This can be inhibited by specific Ca2+-ATPase inhibitors. Further platelet activation via diacylglycerol (DAG) and protein kinase C and activation of phospholipase A2 with formation of thromboxane A2 are not shown. Activation of the phospholipid scramblase by Ca2+ ions and a concomitant Ca2+-mediated inhibition of ATP-dependent aminophospholipid translocase that serves to maintain the normal phospholipid asymmetry lead to translocation of negatively charged phosphatidylserine to the outer membrane bilayer. This is a prerequisite for formation of the complexes of coagulation factors on the platelet surface. A Ca2+-induced activation of the proteolytic enzyme calpain is important for the formation of microvesicles. Protein tyrosine phosphatase activities are associated with calpain activation and microvesiculation in an as-yet-unknown manner. The microvesicles express a procoagulant surface. Substances that activate or inhibit the various processes are labeled with + or – respectively. FVa and FXa indicate factors Va and Xa, respectively. (Reproduced from Solum278 with permission.)

Microparticle formation can be induced in vitro by activation of platelets with ionophore A23187, complement C5b-9, or the combination of thrombin and collagen; by adding tissue factor to recalcified platelet-rich plasma; or by high shear stress.212,283,284 Incubation of platelets with sera from patients with heparin-induced thrombocytopenia can also produce microparticles,285 perhaps accounting, in part, for the thrombosis that is sometimes associated with this disorder. Elevations of cytosolic Ca++, calpain activation, cytoskeletal reorganization, protein phosphorylation, and phospholipid translocation have all been implicated in microparticle formation. Inhibiting GPIIb/IIIa, and perhaps aVb3, decreases tissue-factor-induced platelet coagulant activity and microparticle formation in the presence or absence of fibrin,283 whereas inhibiting GPIb inhibits tissue factor-induced microparticle formation only in the presence of fibrin.286
The biologic relevance of microparticles is supported by the finding of increased circulating levels of platelet microparticles in patients with activated coagulation and fibrinolysis, diabetes mellitus, sickle cell anemia, human immunodeficiency virus infection, unstable angina, and patients with respiratory distress syndrome.212,287 Microparticles can bind to fibrin thrombi via one or more of the receptors present on their surface [GPIIb/IIIa, GPIb/IX, GPIa/IIIa (a2b1), and P-selectin].288
Microparticles bind factor VIII, factor Va, and factor Xa, allowing them to form both the tenase and prothrombinase complexes on their surface.212 They can also bind protein S, which could serve an anticoagulant function. In addition, microparticles can activate platelets by supplying arachidonic acid. In a similar manner, they can activate endothelial cells and monocytes, resulting in enhanced monocyte attachment to endothelial cells, a potential contributor to atherosclerosis.212
Platelet activation leading to increased platelet coagulant activity shares several features with cell apoptosis, including surface exposure of negatively charged phospholipids and membrane blebbing leading to microparticle formation. Platelets contain the apoptosis-related proteins procaspase-3 and procaspase-9, as well as the caspase activators APAF-1 and cytochrome c.289,290 There are conflicting data, however, regarding the relative roles of caspases and calpains in the development of platelet coagulant activity.289,290
There is incontrovertible evidence that platelets accelerate thrombin formation, but the precise mechanisms involved remain controversial.4,292,293,294,295 and 296 The effect of platelets on activation of factor X by factors IXa and VIIIa and the activation of prothrombin by factors Xa and Va have been extensively studied.4,292,296 Both reactions are accelerated by platelets, most dramatically when the platelets have been activated by thrombin or other agonists (see Chap. 112). Platelets also are able to accelerate factor VIII activation by thrombin.297 It is likely that factor VIIIa on platelets acts as a binding site for factor IXa and that factor Va on platelets acts as a binding site for factor Xa.298 A separate receptor for factor IXa may also exist. What remains unclear is whether factors VIIIa and Va bind to specific receptors on platelets or whether they bind nonspecifically to negatively charged phospholipids, most particularly phosphatidylserine, that join the outer leaflet of the platelet plasma membrane bilayer when platelets are activated.279,293,294,298 The assembly of the factor IXa/factor VIIIa/platelet complex increases the catalytic efficiency of factor X activation (kcat/Km) by a factor of 2.4 × 106.4 In addition, the binding of activated coagulation factors to the surface of platelets appears to protect them from inactivation by inhibitors in plasma and platelets.4 The relatively large platelet pool of factor V,103,105 which appears to be complexed to multimerin,78 and the ability of platelet proteases to activate it108,299 also probably contribute to platelet coagulant activity. The importance of platelet factor V in normal hemostasis can be inferred from the presence of a bleeding diatheses in patients with Quebec platelet syndrome (related to proteolysis of platelet a-granule proteins, including factor V) (see Chap. 119), as well as in a patient with abnormal platelet factor V.296
The physiologic importance of microparticle formation is supported by observations on patients with significant bleeding diatheses and defects in platelet microparticle formation (see Chap. 119).294,296,300 Platelets from the most intensively studied patient had impaired ability to accelerate both factor X and prothrombin activation, did not bind factor Va normally, and did not expose negatively charged phospholipids normally. In flow chamber studies, the patient’s platelets did not support normal fibrin deposition. The defect in microparticle formation appears to be the primary abnormality, since the patient’s erythrocytes also failed to undergo normal vesiculation in response to the calcium ionophore A23187.294,301
In addition to the platelet’s role in accelerating the activation of factor X and prothrombin, there are other connections between platelets and the coagulation system. These include: (1) the presence of fibrinogen in a granules and perhaps on the surface of platelets, where it is strategically located for interactions with locally generated thrombin4,81; (2) the presence of intracellular vWF and the binding of extracellular vWF to platelets, with the potential colocalization of factor VIII attached to the vWF (see Chap. 135); (3) the presence of factor XI or a factor XI-like protein associated with platelet membranes4,302,303; (4) the presence of cytoplasmic factor XIII (see Chap. 112); (5) the presence in platelets of inhibitors of coagulation (a1-protease inhibitor, C-1 inhibitor, tissue factor pathway inhibitor, the thrombin inhibitor protease nexin I, and the factors IXa and XIa inhibitor protease nexin II or b-amyloid precursor protein)4,136; and (6) the ability of activated platelets to facilitate factor XI activation and prevent its inactivation by a1-protease inhibitor.4
Platelet membrane glycoproteins mediate the interactions between the platelet and its external environment. Receptors can receive signals from outside the platelet and send signals inside. In addition, receptors can receive signals from inside the platelet that affect their external domains. Platelet glycoprotein receptors are derived from several different receptor families (integrins, leucine-rich glycoproteins, immunoglobulin cell adhesion molecules, selectins, tetraspanins, and seven transmembrane domain receptors) (see Table 111-5). One member of the integrin family, GPIIb/IIIa, is unique to platelets; the leucine-rich glycoproteins GPIb/IX and GPV appear to have highly restricted expression, including primarily platelets and cytokine activated endothelial cells.304,305 and 306
Integrin receptors are heterodimeric complexes composed of an a subunit containing three or four divalent cation-binding domains and a b subunit rich in disulfide bonds. Both subunits are transmembrane glycoproteins and are encoded by different genes. There are at least 14 a subunits and 7 b subunits.220,307,308 and 309 Three major families of integrin receptors are recognized based on the b subunit: b1, b2, and b3. Integrins are widely distributed on different cell types, and each integrin demonstrates unique ligand-binding properties. Integrin receptors mediate interactions between cells and between proteins and cells; they are also involved in protein trafficking in cells. Integrin receptors can also transduce messages from outside the cell to inside the cell, and from inside the cell to outside the cell.
GPIIb/IIIa (Fibrinogen Receptor; aIIbb3; CD41/CD61) The GPIIb/IIIa complex, a member of the b3-integrin receptor family, is the dominant platelet receptor, with 80,000 to 100,000 receptors present on the surface of a resting platelet.220,221,309,310 Another 20,000 to 40,000 receptors are present inside platelets, primarily in a-granule membranes, but also in dense bodies and the membranes lining the open canalicular system; these receptors are able to join the plasma membrane when platelets are activated and undergo the release reaction.311,312 and 313 On average, GPIIb/IIIa receptors are less than 20 nm apart on the platelet surface and thus are among the most densely expressed adhesion/aggregation receptors present on any cell type. By electron microscopy, the receptors appear to have a globular head of 8 × 12 nm, composed of the amino termini of both subunits, and two 18-nm tails representing the carboxy-terminal regions of each subunit, including their transmembrane and intracytoplasmatic domains.314 Biochemical studies of GPIIb/IIIa proteolytic digestion fragments, however, are not entirely consistent with this structural model.315
GPIIb/IIIa shares the same basic structural features of all integrin receptors (Table 117-5). GPIIb, the a subunit, is a transmembrane protein with four calmodulin-like domains that are able to bind divalent cations (see Fig. 119-5). The mature protein contains 1008 amino acids and consists of a heavy (a) chain which is extracytoplasmatic, and a light (b) chain that contains the transmembrane and intracytoplasmatic domains.220,309,316 GPIIIa, the b subunit, contains 762 amino acids and has a characteristic cysteine-rich region near its transmembrane domain.220,309,317 The genes coding for GPIIb and GPIIIa are very close to each other on chromosome 17 at q21.32 but are not so close as to share common regulatory domains.318,319 Both proteins are made in megakaryocytes and join to form a calcium-dependent, noncovalent complex in the rough endoplasmic reticulum (see Fig. 119-2).309,320 They subsequently undergo further processing in the Golgi apparatus, where the carbohydrate structures undergo maturation and the pro-GPIIb molecule is cleaved into its heavy and light chains.309,321,322 Approximately 15 percent of the mass of both GPIIb and GPIIIa is composed of carbohydrate.323 The mature GPIIb/IIIa complex is then transported to the plasma membrane or the membranes of a granules or dense bodies. If GPIIb and GPIIIa do not form a proper complex, either because of a structural abnormality or the failure to synthesize one of the subunits, the glycoproteins that are synthesized are rapidly degraded and so are not expressed on the membrane surface (see Chap. 119).219,220
The GPIIb/IIIa receptors in a granules appear to cycle to and from the plasma membrane.324 This recycling helps to explain the ability of GPIIb/IIIa to take up fibrinogen from plasma and transport it to a granules, where it is concentrated.81,83
On resting platelets, GPIIb/IIIa will support adhesion of platelets to immobolized fibrinogen, but it has low affinity for fibrinogen in solution; when platelets are activated with ADP, epinephrine, thrombin, or other agonists, however, GPIIb/IIIa binds fibrinogen relatively strongly.221,222,224 The signal-transduction mechanisms that mediate activation are discussed below. Activation induces changes in the GPIIb/IIIa receptor itself that are responsible for the change in fibrinogen-binding affinity,325,326 but changes in the microenvironment surrounding GPIIb/IIIa may also be involved. The cytoplasmic domains of GPIIb and GPIIIa have been implicated in controlling the activation process.204,327,328
The precise areas of GPIIb/IIIa that are involved in binding fibrinogen are not known with certainty, but data from ligand cross-linking studies and peptide inhibition studies, and deductions from patients with mutations in GPIIb/IIIa that affect fibrinogen binding (see Chap. 119), suggest that the regions in GPIIIa between amino acids 109 to 171 and 211 to 222 are important (see Fig. 119-4).329,330,331 and 332 These regions of GPIIIa contain sequences that are similar to those of the metal-ion-dependent adhesion site (MIDAS) present in the I domain of some integrin a subunits, including a DxSxS motif (Asp 119, Ser 121, Ser 123), lending support to the importance of this region in cation and ligand binding.333,334 Based on studies of other integrin a subunits, a b propeller model structure with 7 blades has been proposed for this family of subunits; the fundamental features of this structure may be applicable to GPIIb.335 Each blade of the propeller radiates out from the center and is composed of a repeat sequence that includes four beta strands that are linked by loops that either extend above (between strands 2 and 3 of the same blade, and between strand 4 and strand 1 of the next blade) or below (between strands 1 and 2, and between strands 3 and 4) the plane of the propeller (Fig. 119-5). A 3 dimensional version of this image is available from the Williams Hematology website (http//www.williamshematology.com). The loops that extend below the propeller contain the E-F hand-like calcium-binding sequences, which appear to be important in receptor biosynthesis, whereas the loops that extend above the propeller appear to be involved in ligand binding. Data from patients with Glanzmann thrombasthenia appear to be in accord with this model (see Chap. 119). Divalent cations are required for the binding of all ligands, but there are differences in the optimal cation (calcium, magnesium, manganese) and its concentration, depending on the ligand.222,224,336
Data from other integrin receptors identified a cell recognition sequence composed of Arg-Gly-Asp (RGD) in the ligand fibronectin.337,338 Fibrinogen contains one RGD sequence near the carboxy terminus of each of the two Aa chains (amino acids 572 to 574) and another at amino acids 95 to 97.339 In addition, the carboxy-terminal 12 amino acids of each of the two g chains (amino acids 400 to 411) contains a sequence that includes Lys-Gln-Ala-Gly-Asp-Val, and it appears that the Lys and Gly-Asp form a molecular mimic of the RGD sequence.340 The g-chain sequence appears to be the most important in the binding of fibrinogen to platelets, but the RGD sequences may also participate.188,341,342 and 343 Small, synthetic peptides containing the RGD or g-chain sequence inhibit the binding of fibrinogen to platelets, and these observations have been exploited to produce therapeutic agents to inhibit platelet thrombus formation (see Chap. 131).344 Similarly, monoclonal antibodies that inhibit binding of ligands to GPIIb/IIIa have been developed and a murine/human chimeric Fab fragment of one of them has been shown to be effective as an antiplatelet agent (see Chap. 131).344
The binding of fibrinogen to GPIIb/IIIa appears to be a multistep process221: (1) the initial interaction is divalent cation-dependent and most likely involves the g-chain carboxy-terminal regions188,342,343; (2) subsequent interactions, which may include internalization of the fibrinogen,345 render the binding irreversible, even when divalent cations are removed346; (3) binding of fibrinogen induces changes in the receptor that can be recognized by antibodies (ligand-induced binding sites; LIBS)241; and (4) binding of fibrinogen to GPIIb/IIIa induces changes in fibrinogen that can be recognized by antibodies (receptor-induced binding sites, RIBS) and may involve exposure of the Aa chain Arg-Gly-Asp-Phe (RGDF) sequence at amino acids 95 to 98.347,348
Binding of fibrinogen to platelet GPIIb/IIIa leads to platelet aggregation, presumably via cross-linking of GPIIb/IIIa molecules on two different platelets by fibrinogen.349,350 The dimeric and relatively rigid structure of fibrinogen, and the location of the binding sites at the extremes of the g chains, are all consistent with such a model since the two binding sites on a single fibrinogen molecule are probably more than 45 nm apart. Soon after fibrinogen binds, it can be dissociated from the platelet by chelating the divalent cations, but the binding becomes irreversible within an hour.346 Fibrinogen binding alone is not sufficient for platelet aggregation, but the events necessary after fibrinogen binding, which probably include ligand- and/or cytoskeletal-mediated receptor clustering, are not well understood.1,346,349,351 After ligands bind to GPIIb/IIIa, “outside-in” signaling through GPIIb/IIIa can occur, resulting in a number of phosphorylation events, changes in the platelet cytoskeleton, and even initiation of protein translation.153,204
In addition to fibrinogen, several other adhesive glycoproteins that contain RGD sequences can bind to GPIIb/IIIa on activated platelets, including vWF, fibronectin, vitronectin, and thrombospondin.89,352 There are subtle differences in the binding of each of these ligands with regard to divalent cation preference and competent activating agents.325 The binding of all of these other ligands can also be inhibited by RGD-containing peptides, indicating a common requirement for the interaction between the RGD sequence in the protein and the RGD-binding site in GPIIb/IIIa.353,354
Platelet aggregation measured in an aggregometer ex vivo depends upon fibrinogen binding to GPIIb/IIIa. It is less clear whether fibrinogen is the most important ligand supporting platelet aggregation in vivo, since studies performed in model systems under flowing conditions indicate that vWF is the major ligand at higher shear rates.223 Even in the aggregometer, vWF can partially substitute for fibrinogen if the fibrinogen concentration is very low.355
In contrast to the requirement for platelet activation in order for platelets to bind soluble fibrinogen (or other adhesive glycoproteins), unactivated platelets will adhere to fibrinogen immobilized on a surface.187,188,356 This activation-independent adhesion may be due to alterations in the structure of fibrinogen when it is immobilized on a surface.348,357 Alternatively, it may result from there always being a few GPIIb/IIIa receptors that are transiently in the proper conformation to bind fibrinogen, and the favorable kinetics achieved as a result of the high local density of fibrinogen that accompanies immobilization on a surface.
Fibrinogen and/or fibrin have been identified on the surface of damaged blood vessels; thus it is possible that GPIIb/IIIa mediates platelet adhesion under those circumstances.358 In contrast, GPIIb/IIIa on unactivated platelets does not appear to be able to mediate adhesion to vWF or fibronectin188; if platelets are activated, however, GPIIb/IIIa can support adhesion to these glycoproteins.353 In models of platelet accumulation under flowing conditions, GPIIb/IIIa acts in synergy with GPIb/IX, von Willebrand factor, and fibrinogen at the apex of thrombi, where shear forces are greatest.186,198,199 GPIIb/IIIa has also been implicated in platelet spreading after adhesion,201,203 and it is necessary for clot retraction (see above) and the accumulation of platelet a-granule fibrinogen.81,83
Less well defined roles for GPIIb/IIIa have been suggested in the binding of plasminogen359 and factor XIIIa360 to platelets; calcium transport across the platelet membrane (see below)361,362 and 363; IgE binding to platelets leading to parasite cytotoxicity364; and interaction with the Borellia species spirochetes that cause Lyme disease.365
GPIa/IIa (Collagen Receptor; VLA-2; a2b1; CD49b/CD29) The GPI/IIa (a2b1) receptor, a member of the b1 integrin family, is widely distributed on different cell types and can mediate adhesion to collagen.189,192,193 and 194,366,367,368 and 369 The GPIa (a2) subunit contains a region of 191 amino acids inserted in the amino-terminal region (I domain) that is homologous to similar regions in other proteins that are known to interact with collagen, including vWF and cartilage matrix protein.370 This region has a metal-ion-dependent adhesion site (MIDAS domain) and probably mediates the interactions with collagen.
Platelet adhesion to collagen mediated by GPIa/IIa (a2b1) is enhanced in the presence of magnesium or manganese and is inhibited by calcium, and thus the conditions in human blood, where calcium is abundant and magnesium is only present at low levels, do not provide optimal cation concentrations for GPI/IIa function.189,193 GPIa/IIa (a2b1) can, however, mediate platelet adhesion to collagen in heparinized blood.193,194 Regions of collagen type I have been implicated as potential binding sites for GPIa/IIa (a2b1)371; the peptide sequences 502–516 of collagen type I alpha 1 chain, containing a Gly-Glu-Arg sequence, may be of particular importance,372 but other interactions may also be important.373 In type III collagen, amino acids 522–528 of fragment alpha 1 (III) CB4 contains a binding region for GPIa/IIa (a2b1).374 Evidence from patients with diminished platelet GPIa/IIa (a2b1) supports a role for this receptor in hemostasis (see Chap. 119).369,375
There are at least three alleles for a2 that differ at nucleotides 807 (T or C) and 1648 (G or A) (Fig. 111-5). The 807 substitution does not affect the amino acid sequence, but the 1648 substitution causes a change from Glu to Lys, resulting in the Brb and Bra alloantigens. Allele 1 (T-G) is present in 39 percent of individuals, allele 2 (C-G) in 53 percent, and allele 3 (C-A) in 7 percent (Fig. 111-8).376,377 Individuals with allele 1 have higher GPIb/IIa (a2b1) platelet density than individuals with allele 2, and individuals with allele 3 have the lowest density. The density of GPIb/IIa (a2b1) receptors correlates with platelet deposition on collagen under flow. Individuals with allele 1 have been reported to be at increased risk of developing myocardial infarction378,379 and stroke.380

FIGURE 111-8 Polymorphisms of integrin a2. The left panel shows a cartoon of the three alleles and their frequency. The nucleotide position in the cDNA is shown above. The 807 T/C substitution is silent, not altering an amino acid; the 1648 G/A substitution causes an amino acid alteration (glutamic acid to lysine) and is responsible for the Brb and Bra alloantigens respectively. The middle panel indicates the association of the different genotypes containing alleles 1 and 2 (807 T and 807 C respectively) with platelet receptor density, shown as arbitrary units on the horizontal axis. The right panel is a schematic drawing indicating platelet deposition to immobilized collagen under shear stress according to the a2 genotypes 1,1 and 2,2. (From Bray,377 with permission.)

GPIa/IIa (a2b1) is probably linked to the membrane skeleton and is competent to mediate adhesion on resting platelets.12 Its ligand specificity appears to be determined by the cell on which it is expressed, since on platelets it appears to function only as a collagen receptor, whereas on endothelial cells it functions as a laminin receptor as well as a collagen receptor.381,382 Engagement of GPIa/IIa (a2b1) is capable of initiating platelet protein synthesis.153
GPIc*/IIa (Fibronectin Receptor; a5b1; VLA-5; CD49e/CD29) GPIc*/IIa (a5b1) is a b1-integrin receptor that is expressed on a wide variety of different cells and mediates adhesion to fibronectin.337,338 It is important in interactions with extracellular matrix, and data from cells other than platelets indicate a role for this receptor in developmental biology and tumor metastasis. The RGD sequence in fibronectin is crucial for cell adhesion, but other regions in fibronectin probably also contribute. RGD-containing peptides can inhibit cell adhesion mediated by GPIc*/IIa (a5b1). As with other integrin receptors, the adhesion depends on the presence of divalent cations. GPIc*/IIa (a5b1) is competent to mediate adhesion of resting platelets to fibronectin.383,384 The biologic role of this receptor on platelets is not clear. Although it may be involved in hemostasis and/or thrombosis, it is also possible that its function is restricted to megakaryocyte binding to marrow matrix, since it seems to serve this function on other hematopoietic precursors.385 GPIc*/IIa (a5b1) is not the only fibronectin receptor on platelets, since with appropriate activation, GPIIb/IIIa can also bind fibronectin.89
GPIc/IIa (Laminin Receptor; a6b1; VLA-6; CD49f/CD29) Platelet adhesion to laminin can be mediated by the GPIc/IIa (a6b1) integrin receptor.178,386,387 The adhesion is best demonstrated with magnesium and manganese; calcium does not support adhesion. This receptor is competent on resting platelets, but its role in platelet physiology is not clear. An Mr 67,000 laminin receptor has also been identified on platelets; this receptor is present on other cells as well.388
aVb3 (Vitronectin Receptor; CD51/CD61) The aVb3 receptor shares the same b3 subunit as GPIIb/IIIa (see Fig. 119-3).317 The aV subunit and GPIIb have 36 percent sequence identity.389 It differs dramatically, however, from GPIIb/IIIa in its platelet surface density, since there are only about 50 to 100 aVb3 receptors per platelet.336 The aVb3 receptor can mediate adhesion to vitronectin, but only in the presence of magnesium or manganese, not calcium.336 It also can mediate interactions with fibrinogen, vWF, and thrombospondin.90,356,390,391 Activated aVb3 may also uniquely mediate adhesion to osteopontin, a protein found in high concentrations in atherosclerotic plaques.392 Its role in platelet physiology is not defined, but it may contribute to the development of platelet coagulant activity.283
The aVb3 receptor is also present on endothelial cells,341,391 osteoclasts,393 and other cells; it has been implicated in bone resorption,394 endothelial-matrix interactions,341 lymphoid cell apoptosis,395 neovascularization,396 tumor angiogenesis,397 and both smooth-muscle cell migration and intimal hyperplasia after vascular injury.398 Binding of adhesive proteins to aVb3 can be inhibited by RGD-containing peptides.
The presence or absence of aVb3 on the platelets of patients with Glanzmann thrombasthenia can help localize the abnormality to either GPIIb (if aVb3 is present in normal or increased amounts) or GPIIIa (if aVb3 is reduced or absent) (see Chap. 119).
GPIb/GPIX (CD42) GPIb is composed of GPIba (CD42b) (610 amino acids) disulfide-bonded to GPIbb (CD42c) (122 amino acids) (Fig. 111-9).177,304,399,400 The GPIba gene is on the short arm of chromosome 17, and the GPIbb gene is on the long arm of chromosome 22. A genetic polymorphism in GPIba affects the number of repeating 13-amino-acid units (1, 2, 3, or 4) and produces changes in the Mr of GPIba.401 The two-repeat variant is most common, but there is considerable ethnic variation in the frequency of the different numbers of repeats. This Mr polymorphism has been linked to the Sib and Ko alloantigens, which have been localized to a Thr®Met variation at amino acid 145 of GPIba, with Met associated with either three or four repeats and Thr associated with either one or two repeats (Fig. 111-10 and see Chap. 138).377 Two reports suggest an association between the alleles with the larger number of repeats and coronary artery disease,402,403 but inconsistent results have been reported on the impact of these polymorphisms on the risk of cerebrovascular disease.403,404 Two other GPIba polymorphisms have been described: (1) C or T at position 5 from the ATG start codon (RS system), and (2) a nucleotide dimorphism at the third bases of the codon for Arg 358.400,405,406 A C at position 5 is present in only 8 to 17 percent of individuals and more closely resembles the sequence surrounding the ATG start codon (Kozak sequence) considered optimal for translation. In fact, this polymorphism is associated with higher levels of platelet surface GPIb and may be a risk for early myocardial infarction.407 GPIb has been implicated as a target antigen in autoimmune thrombocytopenia and in quinine and quinidine-induced thrombocytopenia (see Chap. 117).

FIGURE 111-9 Schematic view of the platelet GPIb-IX-V complex. Key structural features of the complex are shown. The leucine-rich repeats of the four polypeptides are drawn based on the structure determined for the porcine ribonuclease inhibitor, a protein made up entirely of leucine-rich repeats. The depicted polypeptide arrangement is based on the published stoichiometry determined by monoclonal antibody binding and on the associations determined for the polypeptides. A caveat about this depiction: the quantity of GPV on the platelet surface has only been determined using 2 GPV monoclonal antibodies, which could lead to overestimates or underestimates of true polypeptide number. In addition, no quantitation has ever been performed to indicate that every GPV molecule on the platelet surface is associated with the complex. Complexes of greater complexity having the same stoichiometry are also possible. Diamonds on stalks represent N-linked carbohydrates and circles on stalks represent O-linked carbohydrate. (From Lopez et al,400 with permission.)

FIGURE 111-10 Polymorphisms of platelet GPIba. The four alleles of GPIba that have been defined by the VNTR, Ko, and Kozak polymorphisms. The 4 variable number of tandem repeat (VNTR) polymorphisms are defined as A-D, according to the number of 39 base pair repeats: A has four repeats, B has three repeats, C has two repeats, and D has one repeat. The Ko polymorphism results from a T to C nucleotide substitution that alters the amino acid from methionine (met) to threonine (thr). The “*” indicates the location of the nucleotide substitution, which generates a sequence that better conforms to the consensus Kozak sequence. (From Bray,377 with permission.)

GPIba has a large number of O-linked carbohydrate chains terminating in sialic acid residues,408 and the latter contribute significantly to the negative charge of the platelet membrane.2 Electron micrographic analysis indicates that GPIb exists as a long flexible rod (60 nm) with two globular domains of about 9 and 16 nm.409 Thus, GPIb probably extends much further out from the platelet’s surface than does GPIIb/IIIa, which may account for its primacy in platelet adhesion, as well as the increased risk of cardiovascular disease in individuals with longer GPIb molecules due to an increased number of 13-amino-acid repeats. The long extension may also make it susceptible to conformational changes induced by shear forces.304 The extracellular region of GPIba is readily cleaved by a variety of proteases, including platelet calpains,410 yielding a soluble fragment named glycocalicin that circulates in normal plasma at 1 to 3 µg/ml.411 Levels of plasma glycocalicin correlate with platelet production and thus can been used to differentiate thrombocytopenia due to decreased platelet production from thrombocytopenia due to increased platelet destruction.412,413
GPIbb has a free sulfhydryl group in its cytoplasmic domain.414 In addition, its cytoplasmic domain can undergo phosphorylation of serine 166 by cAMP-dependent kinase, which may affect actin polymerization and GPIb function.304,415 The cytoplasmic domain of GPIba connects GPIb to actin-binding protein, thus connecting GPIb to the platelet cytoskeleton.12,180,416 Alterations in the cytoskeleton can affect GPIb functional activity.417,418 and 419 The cytoplasmic domain is probably also a site of palmitic acid attachment, which may enhance attachment to the lipid bilayer.420 The GPIba cytoplasmic domain also has a site that binds protein 14-3-3z, which has been implicated in GPIb-mediated intracellular signaling that results in GPIIb/IIIa activation.180 GPIb also appears to be in close proximity to FcgRIIA and the Fc receptor g chain, two receptors that can initiate signaling.421,422
GPIb appears to exist on the surface of platelets in a one-to-one complex with GPIX (160 amino acids).423,424 and 425 The function of GPIX is unknown, but it is probably required for efficient surface expression of GPIb.426 GPIba has seven leucine-rich repeats in the amino-terminal region of its extracellular domain, whereas GPIbb and GPIX have one each.177,399,425 These repeats are consensus sequences of 24 amino acids with seven regularly spaced leucines; well-defined disulfide loop sequences flank the repeats.304 Similar leucine-rich repeats are present in a variety of other proteins; crystallographic studies of one of these proteins (porcine ribonuclease inhibitor) identified a unique b sheet-a helix recurring structure for each of the leucine-rich repeats.427 Immediately C-terminal to the leucine-rich repeat flanking sequence in GPIba is a 19-amino-acid sequence rich in negatively charged amino acids that contains three sulfated tyrosines.180,400
GPIb mediates platelet interaction with vWF, with the binding site in the vWF AI domain (residues 480–718).400 Both Asp 514 to Glu 542 and the region encompassing Glu 596 and Lys 599 have been proposed as recognition sites.180,400 This region is distinct from the RGD-containing region of vWF that mediates binding to GPIIb/IIIa (see Chap. 135).
The vWF-binding domain on GPIba has been localized to the first 300 amino acids, and three different regions appear to contribute: the anionic, sulfated-tyrosine sequence; a portion of the carboxy-terminal leucine-rich repeat flanking sequence; and the leucine-rich repeats themselves (Fig. 111-6 and Fig. 111-8).400,428,429,430 and 431 Support for the importance of the carboxy-terminal repeat region in vWF binding also comes from the observation that patients with pseudo (platelet-type) von Willebrand disease, whose platelet GPIb/IX complex has enhanced affinity for vWF, have mutations within this region (Fig. 111-11 and see Chap. 119). Plasma vWF will not bind to GPIb under static conditions unless the antibiotic ristocetin or the snake venom botrocetin is added. The mechanism by which ristocetin induces vWF binding to GPIb is unclear but appears to involve changes in platelet surface charge, and requires dimerization of ristocetin molecules.184,304,432 Botrocetin binds to vWF, exposing the site that binds to GPIb.433 Peptide studies implicate the anionic, sulfated tyrosine region of GPIb as the binding site for botrocetin-treated vWF.304

FIGURE 111-11 The region in GPIba from amino acids 209–302. Disulfide bonds between Cys209 and Cys248 and between Cys211 and Cys264 create two loops. Mutations in the loop between Cys209 and Cys248 result in enhanced interaction between von Willebrand factor and GPIb, producing platelet-type von Willebrand disease (see Chap. 119). This loop has also been implicated in regulating von Willebrand factor binding to GPIb. The region between amino acids 235–279 has been implicated in ristocetin-induced binding of von Willebrand factor, whereas the region between amino acids 271–285 has been implicated in botrocetin-induced von Willebrand factor binding. The region between amino acids 269 and 287 bears resemblance to the thrombin-binding peptide hirudin, and so it has been postulated to be part of the thrombin-binding region. (From Lopez,304 with permission.)

Unlike GPIIb/IIIa, which requires intact, activated platelets to bind to vWF, GPIb-mediated vWF binding does not require platelet activation or even platelet metabolic integrity, since formaldehyde-fixed platelets are readily agglutinated in the presence of vWF and either ristocetin or botrocetin.184 This observation forms the basis of the assay of plasma vWF activity (see Chap. 135).
Platelets will adhere to vWF when the latter is immobilized on a surface, even in the absence of ristocetin or botrocetin.178,184,434 Under these circumstances, the vWF may undergo a conformational change that allows for direct interactions. It may not, however, be necessary to propose a change in vWF conformation, since the interaction between vWF and GPIb appears to have both high association and dissociation rates, permitting tethering and translocation on a surface coated with vWF but minimal interaction in fluid phase.179 Similarly, vWF associated with fibrin can interact with platelet GPIb without ristocetin or botrocetin.206,435
Shear stress is an important factor in GPIb-mediated adhesion of platelets to immobilized vWF and subendothelial surfaces.177,178,434,436,437 Platelets deficient in GPIb or platelets in which GPIb has been blocked with monoclonal antibodies178,436 adhere poorly to subendothelial surfaces at all shear rates, but the defect in blood from patients with von Willebrand disease is manifest primarily at higher shear rates.178 In what may be a related phenomenon, subjecting platelets to high shear stresses can induce platelet aggregation, which is mediated by vWF binding to GPIb, followed by platelet activation and GPIIb/IIIa-dependent platelet aggregation.181,183,438 Whether the shear rates generated in vivo in stenotic blood vessels are of sufficient magnitude and duration to produce a similar degree of platelet activation is unknown. It is also uncertain as to whether the effect of shear is acting on GPIb, on vWF, or on both177,179,183,304 but shear-induced changes in the structure of vWF, leading to a more extended conformation, have been defined.439
GPIba also functions as a binding site for thrombin.304,440,441 The region between amino acids 216 and 240 has been proposed as the binding site, but the region between amino acids 269 and 287 has also been suggested because of its similarity to hirudin, a thrombin-binding protein.304,429 Sulfation of the three tyrosine residues in the latter region is particularly important for thrombin binding.180 The functional significance of the binding of thrombin to platelet GPIb is not established, but GPIb has been proposed as the high-affinity binding site for thrombin.440,442 If true, it appears that not all GPIb molecules serve this function, since there are only about 50 high-affinity thrombin-binding sites and about 25,000 GPIb molecules per platelet.440,441 Platelets lacking GPIb (Bernard-Soulier syndrome) do, in fact, have blunted responses to thrombin (see Chap. 119). One possible model is that binding of thrombin to GPIb facilitates its effect on one or more of the other thrombin receptors, but this is speculation.
GPIb has also been demonstrated to interact with P-selectin in a cation-independent manner.180 Although GPIb shares a number of features with the P-selectin ligand, PSGL-1 (both are sialomucins and have analogous anionic/sulfated tyrosine sequences) the interaction between GPIb and P-selectin appears to be more like the interaction between P-selectin and heparin.180 In inflamed mesenteric venules in animals, platelets are observed to roll on the activated endothelium,443 and so it is possible that platelet GPIb interacts with endothelial P-selectin in this interaction.180
GPV Glycoprotein V is an Mr 82,000 protein composed of 544 amino acids that contains 15 leucine-rich repeats.444,445,446 and 447 GPV appears to form a noncovalent complex with GPIb/IX, but since the number of GPV molecules on the surface of platelets is approximately 50 percent of the number of GPIb and GPIX molecules,448 it has been suggested that the basic unit consists of two GPIb molecules, two GPIX molecules, and one GPV molecule.304,400 GPV is deficient in platelets from patients with Bernard-Soulier syndrome (see Chap. 119), who have defects in GPIb or GPIX, but GPV is not required for surface expression of the GPIb/IX complex.449 A soluble fragment of Mr 69,000 is cleaved from GPV by thrombin, but cleavage does not correlate with thrombin-induced platelet activation.450 Platelets from mice lacking GPV appear to respond more actively to thrombin and ADP than wild-type mice, raising the possibility that GPV inhibits platelet activation.451 The platelets from these mice also adhere to immobilized vWF and can bind vWF in the presence of botrocetin, indicating that GPV is not required for the interaction between vWF and the GPIb/IX/V complex.451
PECAM-1 (CD31) Platelet-endothelial cell adhesion molecule 1 (PECAM-1) is an Mr 130,000 transmembrane glycoprotein of the immunoglobulin gene family with six immunoglobulin-like domains of the C2 group.452 In addition to platelets and endothelial cells, it is expressed on monocytes, myeloid cells, and some lymphocyte subsets (see Chap. 13). There are approximately 8000 PECAM-1 molecules on the surface of platelets.453
Crosslinking of PECAM-1 molecules on the platelet surface enhances platelet adhesion and aggregate formation under low shear conditions and enhances ADP- and PAF-induced platelet aggregation.454 These effects correlate with tyrosine phosphorylation of PECAM-1 and suggest that PECAM-1 is a costimulatory agonist, working in concert with platelet GPIIb/IIIa.454 Phosphorylated PECAM-1 can recruit a protein tyrosine phosphatase (SHP-2), which may participate in the enhanced platelet function found when PECAM-1 is activated.455
In endothelial cells, PECAM-1 is localized to the contact areas between endothelial cells, where it is likely to be involved in controlling transmigration of leukocytes.456 It appears to be capable of both homotypic and heterotypic adhesive interactions, with the latter perhaps mediated by glycosaminoglycan interactions with a region in the second immunoglobulin domain.457 An antibody to PECAM-1 decreases neutrophil accumulation and myocardial infarct size in a rat model of ischemia-reperfusion injury.458
FcgRIIA (CD32) The FcgRIIA is a low-affinity immunoglobulin receptor of Mr 40,000 that is widely distributed on hematopoietic cells (see Chap. 13). Three different mRNA transcripts (A, B, and C) make similar FcgRIIA receptors,459 and these are preferentially expressed on different cells. In addition, a polymorphism within FcgRIIA, H131R, affects the binding of different IgG subclasses.460
The FcgRIIA on platelets may bind immune complexes generated in certain diseases.461,462 It may also provide a second binding site for antibodies that bind to platelets via their antibody-binding site (see “CD9” below). This second interaction can potentially lead to bridging between platelets, with the antibody binding to an antigen on one platelet and an FcgRIIA receptor on another platelet.463 It is also possible that antibodies can bind to both an antigen and FcgRIIA receptor on a single platelet. These interactions can lead to platelet activation, because crosslinking of FcgRIIA can initiate tyrosine phosphorylation, phosphoinositol metabolism, activation of phospholipase Cg2, calcium signaling, and cytoskeletal rearrangements.464,465 FcgRIIA expression on platelets shows considerable variation among individuals (about 600 to 1500 molecules per platelet), and this variation correlates with FcgRIIA-mediated function.462 This variation in receptor density may explain individual differences in immune-mediated disorders such as heparin-induced thrombocytopenia with thrombosis.466 The H131R polymorphism may also have clinical significance, since the homozygous H/H genotype is overrepresented in patients with heparin-induced thrombocytopenia,467 but the R/R 131 genotype may confer a higher risk of developing thrombosis in patients with heparin-induced thrombocytopenia.468 The R/R genotype may also be associated with the likelihood of requiring splenectomy in patients with immune thrombocytopenia.469 FcgRIIA has also been suggested to be in close proximity to the GPIb/IX/V complex,304 and it appears that the signal transduction that accompanies vWF binding to GPIb may be mediated through FcgRIIA.470 Cooperation between FcgRIIA and C1q receptor has also been reported.471
Fc Receptor g-chain Platelets contain the Fc receptor g (FcRg) chain,472 although they lack the Fc receptors for Fce R1, FcgRI, and FcgRIII that normally form signaling complexes with FcRg chain in other cell types. In platelets, the FcRg chain exists as an Mr 20,000 homodimer believed to be involved in signal transduction. The FcRg chain, along with FcgRIIA, are the only known platelet proteins with immune-receptor tyrosine-containing activation motifs (ITAMs). Much of the understanding of the role of proteins with ITAM-containing domains comes from studies in cells other than platelets, where phosphorylation of the ITAM domain serves to recruit proteins with Src homology 2 (SH2) domains,473,474 many of which are involved in signal transduction. FcRg-chain is associated with GPVI475 and GPIba422 and may activate intracellular signaling pathways in response to platelet adhesion to collagen and vWF respectively. Phosphorylation of the ITAM sequence of FcRg-chain recruits syk,476 activates PI-3 kinase,477 and appears to be involved in activation of PLCg478 and polyphosphoiniositide hydrolysis (see “Signaling Pathways in Platelet Activation and Aggregation” below).
ICAM-2 (CD102) Intracellular adhesion molecule 2 (ICAM-2), a member of the immunoglobulin family of receptors that is also present on endothelial cells, is a ligand for the b2-integrin aLb2 (LFA-1) on lymphocytes and myeloid cells.479 Approximately 2600 ICAM-2 molecules are present on platelets, distributed on the membrane surface and open canalicular system.479 Platelet ICAM-2 may contribute to platelet-leukocyte interactions (see “Platelet-Leukocyte Interactions” below).
GPVI GPVI is an Mr 62,000 transmembrane glycoprotein of 316 amino acids.191 Its extracellular region contains two immunoglobulin C2-like domains, and its transmembrane domain contains an Arg residue that may make a salt bridge with the FcRg-chain. For a discussion of the role of GPVI as a receptor for collagen, see “Collagen Signaling” below.
Human Leukocyte Antigens (HLA) HLA class I molecules are expressed on the surface of platelets. They are discussed in Chap. 138.
P-selectin (GMP140, PADGEM, CD62P) P-selectin is an Mr 140,000 glycoprotein that is present in the membrane of a granules in resting platelets and joins the plasma membrane when platelets are activated.208,209,480 Approximately 13,000 molecules are detected by antibodies on the surface of activated platelets. The expression of P-selectin on circulating platelets has, therefore, been used as an indicator of in vivo activation of platelets.30,325 It is also present in the Weibel-Palade body membranes of endothelial cells; as in platelets, it joins the plasma membrane when endothelial cells are activated.208,480
P-selectin has a modular structure in which the amino-terminal region has a calcium-dependent lectin domain that binds carbohydrates. Adjacent to the lectin domain is an epidermal growth factor domain, followed by nine repeats that are homologous to complement regulatory proteins (“sushi” domains), a transmembrane domain, and a cytoplasmic domain (Fig. 111-12).209,480 The cytoplasmic domain contains serine, threonine, tyrosine, and histidine residues that can be phosphorylated. In addition, a cysteine residue becomes acylated with stearic or palmitic acid. Alternatively spliced forms of P-selectin may be produced in which sushi domains are omitted. The selectin family also includes E-selectin (ELAM-1; CD62E), which is expressed on the surface of activated endothelial cells, and L-selectin (LAM-1; CD62L), which is on myeloid and lymphoid cells.481

FIGURE 111-12 Structures of the selectins, L-selectin (CD62L), E-selectin (CD62E), and P-selectin (CD62P). (Modified from McEver,787 with permission.)

P-selectin can mediate the attachment of neutrophils and monocytes to platelets and endothelial cells. Thus, neutrophils and monocytes may be recruited to sites of vascular injury where platelets deposit and become activated (see “Platelet-Leukocyte Interactions” below). Recognition of ligand by P-selectin requires specific carbohydrate and protein structures. Fucose and sialic acid are important carbohydrate components, with sialyl-3-fucosyl-N-acetyllactosamine (SLex; CD15S) a preferred ligand structure.482,483,484 and 485 Myeloid and tumor cell sulfatides may also act as ligands for P-selectin.486,487 P-selectin glycoprotein ligand 1 (PSGL-1), a mucin-like transmembrane glycoprotein homodimer (Mr 220,000) expressed on neutrophils, monocytes, and lymphocytes, is an important ligand for P-selectin.488,489 and 490 Both sulfation of tyrosine residues contained in an anionic region and branched fucosylation of O-linked carbohydrates are required for optimal binding to P-selectin. Binding of P-selectin to PSGL-1 on monocytes can trigger tissue factor synthesis.491
In intact blood vessels, the rapid on and off rates of the interactions between PSGL-1 on neutrophils and P-selectin on endothelial cells allows leukocytes to roll on the endothelium, the first step in leukocyte transmigration (see Chap. 67).492 The rapid upregulation of surface P-selectin after endothelial cell activation allows for a quick response. Platelets have been reported to roll on activated endothelium, and this appears to result from an interaction between endothelial P-selectin and perhaps platelet GPIba.180,443 An antibody to P-selectin can image thrombi, and an antibody that blocks P-selectin binding can inhibit fibrin deposition in experimental systems.493
A cDNA encoding a soluble form of P-selectin, lacking the transmembrane domain, has been identified, and an increase in plasma levels of soluble P-selectin has been reported in thrombotic and inflammatory disorders.494
CD9 CD9 is a protein of 228 amino acids that contains four putative transmembrane domains, making it a member of the tetraspanin superfamily.495,496 CD9 is also present on endothelial cells, smooth muscle cells, cultured fibroblasts, some lymphoblasts, eosinophils, basophils, and other cells. CD9 is present at high density on the platelet surface (about 40,000/platelet).497 It colocalizes with GPIIb/IIIa on the inner surface of a granules in resting platelets and on pseudopods of activated platelets.498 Binding of monoclonal antibodies specific for CD9 to platelets results in platelet aggregation by triggering phosphatidylinositol metabolism via a mechanism that also requires binding to the platelet FcgRII receptor.499,500 and 501 The platelet activation induced by the binding of such antibodies requires external calcium and results in an association between CD9 and GPIIb/IIIa.502
CD63 (Granulophysin, LAMP-3) CD63, an Mr 53,000 protein member of the tetraspanin superfamily, appears to be present in both lysosomal and dense granule membranes in platelets.29,503 CD63 is also present in Weibel-Palade bodies in endothelial cells, the lysosomal membranes of a variety of other cells, as well as the membranes of melanosomes. It joins the surface membrane when platelets are activated, making it a useful marker for platelet activation.29,30 CD63 appears to be markedly reduced or absent from the dense bodies of patients with Hermansky-Pudlak syndrome,503 who have oculocutaneous albinism and a defect in platelet dense bodies (see Chap. 119). The amino acid sequence of CD63 has been deduced from cDNA cloning.504
Platelet-Endothelial Cell Tetra-Span Antigen (PETA-3; CD151) PETA-3 is an Mr 27,000 glycoprotein member of the tetraspanin superfamily.505,506 It is present on platelets, endothelial cells, and many other cells.507 Antibodies to PETA-3, like those to CD9, can initiate platelet aggregation by binding to both PETA-3 and FcgRIIA.505 The role of PETA-3 in platelet physiology is uncertain, but it may participate with FcgRIIA as a signal transduction complex.505
GPIV (CD36) GPIV (CD36) is an Mr 88,000 glycoprotein that is highly, but variably, expressed on platelets (about 20,000 copies per platelet).92,308,508,509 and 510 Biochemical data suggest that it may form dimers and multimers.511 Increased platelet surface expression of GPIV (CD36) has been described in patients with myeloproliferative disorders.512 It is also present on monocytes, endothelial cells, hematopoietic cell lines, and melanoma cells. It has been proposed as a platelet receptor for thrombospondin513 and collagen,514,515 but the functional significance of these interactions remains unclear because individuals with inherited deficiencies of GPIV (CD36) (Naka-negative) do not have a bleeding disorder (see Chap. 119).516 GPIV (CD36) may play a role in the thrombospondin-mediated interaction reported between platelets and sickle erythrocytes517 and in the binding of Plasmodium falciparum-infected erythrocytes to endothelial cells and monocytes.518 It has also been implicated in monocyte binding of oxidized LDL and myocardial uptake of long-chain fatty acids.519
The nucleotide sequence of GPIV cDNA encodes a protein of 471 residues with an Mr of 53,000 and ten potential N-linked glycosylation sites.518 It is unusual in having two putative transmembrane domains and two short cytoplasmic tails. The cytoplasmic regions may associate with intracellular tyrosine kinases of the src family and undergo phosphorylation.520 Moreover, the phosphorylation status of the extracellular region of the protein may control its ligand-binding properties,91 offering a potential explanation for some of the variable results obtained under different conditions.91,92,521
LAMP-1 and LAMP-2 (CD107a, CD107b) LAMP-1 and LAMP-2 are lysosome-associated membrane proteins that are about 30 percent homologous. They are integral membrane glycoproteins of 110 and 120 kDa respectively that are contained within lysosomal membranes.522 When platelets undergo the release reaction, they join the plasma membrane. Each protein has two extracellular disulfide-bonded loops containing 36 to 38 amino acids. The loops are separated by a region rich in proline and serine that shares homology with the hinge region of IgA. There are multiple N-linked glycosylation sites on each glycoprotein, and they contain more than 60 percent carbohydrate. Among the carbohydrate residues are polylactosaminoglycans that may possess sialylated Lewisx structures, which are thought to interact with selectins (see above).
C1q Receptors Platelets have several receptors for C1q, an Mr 460,000 glycoprotein composed of six globular domains attached to a short collagen-like triple helix.523,524 One is for the collagen-like domain (cC1qR, Mr 60,000 to 67,000 nonreduced and 72,000 to 75,000 reduced), and another is for the globular domain (gC1qR, Mr 28,000 to 33,000).525,526 A third receptor of Mr 126,000 enhances phagocytosis.527 C1q circulates with C1r and C1s as a calcium-dependent complex, but interaction with immune complexes leads ultimately to dissociation of the complex and release of free C1q, with its collagen-like domain exposed. cC1qR has sequence homology to calreticulin and can modulate platelet-collagen interactions at low collagen concentrations. It may also localize immune complexes, and when cross-linked by aggregated C1q, it can initiate platelet activation, aggregation, secretion, and expression of platelet coagulant activity.528 Thus, the binding of C1q monomers to platelets inhibits collagen-induced platelet aggregation but has little effect on platelet adhesion to collagen.529 C1q multimers support platelet adhesion and can induce aggregation via activation of GPIIb/IIIa.528 C1q can also augment platelet aggregation induced by aggregated IgG.471 The gC1qR is present on platelets and other cells, including endothelial cells, where it functions as a receptor for high-molecular-weight kininogen.526 It may, therefore, participate in contact activation.
67-kD Laminin Receptor An Mr 67,000 protein identified as a laminin receptor on several different cells has also been detected on platelets. It can mediate platelet adhesion to laminin under certain conditions.388 The relative roles of this receptor and the integrin receptor GPIc/IIa (a6b1), which also mediates the interaction between platelets and laminin, are unknown.
GMP-33 An Mr 33,000, predominantly a-granule membrane protein has been identified that joins the plasma membrane when platelets undergo the release reaction. Approximately 4000 antibody molecules directed against GMP-33 bind to unactivated platelets, and 19,000 bind to activated platelets.530 The function of GMP-33 is unknown.
Leukosialin, Sialophorin (CD43) Leukosialin, a glycoprotein of Mr 90,000, may act as a ligand for ICAM-1.531 It is expressed on myeloid and some lymphoid cells. Abnormalities in leukosialin have been described in Wiskott-Aldrich syndrome (see Chap. 119).
The interactions between platelets and the fibrinolytic system are complex, and Table 111-8 contains a partial listing.532,533 and 534 Both profibrinolytic99,359,535,536,537,538,539 and 540 and antifibrinolytic268,360,541,542,543,544,545 and 546 effects of platelets have been described, and so it is difficult to predict the net effect. Since platelet-rich thrombi are known to resist thrombolysis in animal models, the antifibrinolytic effects of platelets appear to predominate in vivo.547


The effects of fibrinolytic agents on platelets are also complex, with considerable evidence that fibrinolytic agents can activate platelets soon after administration,548,549,550,551,552,553 and 554 via either a direct effect of plasmin555,556 or an indirect effect through the paradoxical generation of thrombin.534,557,558,559 and 560 A direct effect of tissue plasminogen activator on fibrinopeptide release from fibrinogen has also been described.561
Stimulation of platelets by thrombolytic agents may prolong the time required for reperfusion and may contribute to reocclusion after successful reperfusion.172,532 In animal models and in humans, potent antiplatelet agents can, in fact, speed reperfusion, abolish reocclusion, and diminish the size of myocardial infarcts.562,563,564 and 565
With prolonged use of thrombolytic agents, however, there may be inhibition of platelet function via a variety of mechanisms,160,551,554,566,567,568,569,570,571,572,573,574,575 and 576 which might contribute to some of the hemorrhagic phenomena and the prolonged bleeding times observed with this therapy. The inhibition may be caused by the thrombolytic agents making the platelets refractory to further stimulation.
Leukocytes can bind to activated platelets and in model systems transmigrate through a platelet monolayer (reviewed in Coller577). These interactions may be important at sites of vascular injury where leukocytes have been shown to deposit on adherent and aggregated platelets. Many mechanisms of platelet-leukocyte interactions have been defined, but the initial interaction appears to be mediated primarily by the interaction between P-selectin (CD62P) expressed on the surface of activated platelets and P-selectin glycoprotein ligand-1 (PSGL-1) on the surface of neutrophils and monocytes (Fig. 111-13).480,482,578,579,580,581 and 582 Activated aMb2 on leukocytes can interact with fibrinogen via a region on the g chain (amino acids 190–202,583 perhaps with the cooperation of amino acids 377–395584), and thus fibrinogen bound to platelet GPIIb/IIIa and/or aVb3 may also support leukocyte binding to platelets. Platelets can synthesize and release PAF, which can activate leukocyte aMb2, as can the CXC chemokines released by activated platelets (ENA-78, GRO-a) and produced by the action of leukocyte cathespin G on b-thromboglobulin secreted by platelets (neutrophil-activating peptide-2, NAP-2) (reviewed in Weber and Springer585). Thrombospondin may also serve as a bridging molecule between CD36 receptors, which are expressed on both platelets and mononuclear cells.586 Platelets also have ICAM-2 on their surface, which is a ligand for the leukocyte integrin receptor aLb2479; although this ligand-receptor interaction appears to have only a minor role in platelet-leukocyte adhesion, it may be more important in leukocyte tethering.585
Animal models and studies of human tissue demonstrate that within hours after vascular injury, leukocytes became enmeshed in platelet thrombi and/or transiently form a monolayer on top of adherent or aggregated platelets.587,588 The initial surface association of leukocytes with platelets is usually transient, lasting less than a day. Since leukocytes, and in particular, monocytes, contain and can produce tissue factor, especially when P-selectin binds to PSGL-1, the platelet-leukocyte interaction may be important in initiating coagulation.
Platelet aggregates that form in animal models of vascular injury and in vitro systems stain positively for tissue factor antigen even when the perfusion is as brief as 5 min.176 Since this is too short a time for significant protein synthesis to occur, the tissue factor appears to be derived from circulating microvesicular structures.
Several clinical observations support a potential role for platelet-leukocyte interactions in vascular disease, including the presence of circulating platelet-leukocyte aggregates in patients with unstable angina589 and after coronary artery angioplasty590; in the latter situation, the presence of such aggregates appears to confer a worse prognosis for ischemic vascular complications.590
Transcellular metabolism of eicosanoids can result in production of unique products (see Chap. 114) and leukocytes can modify platelet activation.591 In a complemetary fashion the intimate relationship between leukocytes and platelets allows the latter to contribute to the inflammatory response, including the release of chemokines that can activate leukocytes; platelet-derived growth factor, which can affect fibroblast and smooth-muscle cells; transforming growth factor beta (TGF-b), which both stimulates and inhibits cellular growth; and PF-4, which can prime neutrophils and has antiangiogenic activity. Platelets also contain FcgIIA receptors that can localize IgG and immune complexes, resulting in complement activation. Finally, platelets express CD40 ligand on their surface after activation, and this molecule can interact with CD40, a member of the tumor necrosis factor receptor family, on leukocytes and endothelial cells, leading to their activation and their elaboration of a number of proinflammatory molecules.211,592,593
Platelets generally circulate in a quiescent state but are poised to be activated in response to a variety of agonists that become available at sites of vascular injury or ruptured atherosclerotic plaques. A number of different phenomena occur with platelet activation, and these are listed in Table 111-9. Agonists differ in their intrinsic ability to produce these phenomena, and added complexity derives from differences in dose responses to each agonist and the synergistic effects of agonists used in combination. Agonists are diverse (Fig. 111-4) and include small and large soluble molecules, enzymes, and immobilized adhesive glycoproteins. They can be classified as either “strong” or “weak,” depending on whether full activation, including the release reaction, can be initiated without the augmenting effect of platelet aggregation itself (Fig. 111-4). Low doses of strong agonists behave like weak agonists. Most agonists are released, synthesized, or formed at the site of vascular injury and this undoubtedly serves to localize the response.


Agonists bind to receptors of two general categories: seven-transmembrane, G-protein-coupled receptors and receptors that can initiate phosphorylation of target proteins (Fig. 111-14). In both cases, a sequence of signaling events ultimately leads to platelet activation. Physiologic responses of platelets to agonists are listed in Table 111-9, with all of them leading to activation of the GPIIb/IIIa receptor to a high-affinity ligand binding state leading to subsequent platelet aggregation. Moreover, binding of ligands to platelets and platelet aggregation itself further propagates signals that are required for stabilization of the platelet aggregates and clot retraction. In this section, the major agonists, receptors, and signaling pathways involved in early stages of platelet activation that lead to shape change, granule secretion, and platelet aggregation, as well as post-aggregation signaling events are described.

FIGURE 111-14 General scheme of agonist activation of platelets. Agonists stimulate specific platelet receptors such as seven-transmembrane G-protein-coupled receptors (GPCR) (left) or receptors coupled to tyrosine kinases (right). GPCRs activate a heterotrimeric G-protein by converting the a subunit to a GTP-bound state. The a subunit, after separating from the bg subunit, binds and activates phospholipase Cb (PLCb). Similarly, some tyrosine kinases (e.g., Syk) that are activated by other receptors activate PLCg. PLCb or PLCg catalyze the hydrolysis of phosphatidylinositol bisphosphate (PIP2), which generates diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 acts on specific receptors to increase intracellular Ca2+, whereas DAG facilitates the activation of protein kinase C (PKC). These events promote dense granule fusion with the plasma membrane and release of ADP and other bioactive molecules and the activation of the major platelet integrin, GPIIb/IIIa. The exact signaling pathways leading to granule release and integrin activation are not well defined.

Many, but not all, platelet agonists initiate platelet activation by binding to seven transmembrane heterotrimeric, G-protein-coupled receptors. When such receptors are activated, the Ga subunit exchanges GDP for GTP and dissociates from the bg complex. The free Ga subunit, and in some cases, the bg complex can activate some relatively common downstream pathways and initiate positive feedback loops (Fig. 111-7 and Fig. 111-14). Activation of these pathways is usually intertwined. One common pathway involves the activation of one or more isozymes of phospholipase C (PLC), leading to phosphoinositide hydrolysis. Three classes of PLC (b,g, and d), have been described, and multiple isozymes exist within each class.594 The best-studied PLCs in platelets include PLCb and PLCg2. PLCb is often activated downstream of the seven transmembrane, G-protein-coupled, receptor family, whereas PLCg2 can be activated by phosphorylation on tyrosine, which is a downstream signal from other types of agonist receptors (Fig. 111-14). PLC of either type hydrolyzes phospholipids between the glycerol backbone and the phosphate moiety; both PLCb and PLCg2 are inositol-lipid specific. The hydrolysis of one particular phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), by either class of PLC is critical in platelet function, since it results in the formation of two important products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to specific receptors on the dense tubular system, causing release of Ca2+ into the intracellular space. Increases in intracellular Ca2+ are important for activation of a number of signalling enzymes and proteins involved in cytoskeletal reorganization (see below). Increases in calcium are also important in granule fusion and the release reaction. DAG binds to protein kinase C (PKC) and participates in its conversion to an active enzyme. For many agonists, activation of one or more of the multiple isozymes of PKC is an obligatory step in the conversion of GPIIb/IIIa to a high-affinity fibrinogen receptor and subsequent platelet aggregation.595,596 and 597 The precise mechanism by which PKC causes GPIIb/IIIa activation, however, remains unclear. One consequence of protein kinase C activation is to cause the release of ADP from dense granules to the extracellular space. Released ADP then acts on its own seven transmembrane G-protein coupled receptor and/or a ligand-gated calcium channel to potentiate the action of numerous agonists.
Activation of a number of receptors also leads to the activation of phospholipase A2 (PLA2), which releases arachidonic acid from membrane lipid stores. Arachidonic acid is then rapidly converted in the dense tubular system to the prostaglandin products, PGH2 and TXA2, which are themselves potent activators of platelet aggregation (see below).
ADP ADP is present in platelet dense granules and is secreted when platelets are activated by adequate concentrations of most, if not all, agonists. Another source of ADP is the red blood cell; damaged red blood cells or those subjected to high shear stress may release ADP and increase the local ADP concentration. ADP is an important physiological agonist not only because it can induce platelet aggregation independent of other agonists but because secreted ADP contributes significantly to the full aggregation response induced by many other agonists. This has been convincingly demonstrated in experimental systems in which secreted ADP is rapidly degraded or inhibited. Moreover, submaximal concentrations of ADP synergize with other agonists, and this has been most studied with epinephrine (see below). ADP also induces or contributes to a variety of responses in platelets: shape change, granule release, TXA2 production, activation of GPIIb/IIIa, and platelet aggregation.598 Recent pharmacologic and cloning and sequencing studies suggest that ADP exerts its full effect on platelets through at least three receptors (reviewed in Kunapuli598) (Fig. 111-15). Two of these receptors, P2Y1 and P2TAC, are G-protein-coupled and are responsible for most of the physiologic effects of ADP, and the third, P2X1, is a ligand gated ion channel.

FIGURE 111-15 ADP activation of platelets. ADP potentially activates three separate receptors on platelets, a ligand-gated Ca2+ channel called P2X1, and 2 seven-transmembrane G-protein-coupled receptors, P2Y1, and a postulated receptor termed P2TAC. P2Y1 activates Gaq, which then activates PLCb, whereas P2TAC is believed to activate Gai, which inhibits adenylyl cyclase. Current evidence suggests that activation of both P2Y1 and P2TAC are required for maximal aggregation by ADP. Preliminary data suggest that P2X1 may also be important.

The platelet P2Y1 receptor has been cloned and sequenced599 and, like most heterotrimeric G-protein-coupled receptors, is predicted to span the membrane seven times. Data from experiments with inhibitors to P2Y1 and mice lacking P2Y1 suggest that stimulation of this receptor is necessary, but not sufficient, to induce platelet aggregation.600 P2Y1 probably couples to heterotrimeric G-proteins containing Gaq. The importance of Gaq can be inferred from the observation that platelets from mice that do not express Gaq do not aggregate in response to ADP601 and that a patient has been described with partial Gaq deficiency in association with abnormal platelet function and a bleeding diathesis (see Chap. 119). Activation of PLCb and subsequent phosphoinositide hydrolysis initiated by P2Y1 has been linked to both shape change and platelet activation.
The other G-protein-coupled ADP receptor on platelets, P2TAC, can be identified by its distinct pharmacologic properties602,603 but it has not yet been cloned. This receptor is also likely to be a seven transmembrane spanning protein, but it probably is coupled to Gai rather than Gaq (reviewed in Kunapuli598). Gai activation causes inhibition of the adenylyl cyclases, a class of enzymes that produce cAMP. Because cAMP activates type A kinases, which inhibit platelet activation by a variety of effects, it is an attractive notion that inhibition of cAMP production promotes platelet aggregation. However, a decrease in cAMP levels alone is insufficient to activate platelets.604,605 Synergistic effects between the signaling pathways of the P2Y1 and P2AC receptors (and perhaps the P2X1 receptor discussed below) appear to be sufficient to induce platelet aggregation.
Platelets contain mRNA for a third ADP receptor, P2X1. Receptors of the P2X family are ligand-gated ion channels rather than G-protein-coupled receptors.606 The amino acid sequence of P2X1 suggests that it spans the plasma membrane twice and is largely extracellular.607 ADP binding to this receptor is proposed to cause a rapid Ca2+ influx. However, Ca2+ influx induced by stimulation of this receptor appears to be insufficient in and of itself to induce platelet shape change or aggregation.604 It may, however, cooperate in a positive manner with the other platelet ADP receptors.
Several antiplatelet agents inhibit ADP-induced platelet activation. Thus, metabolites of ticlopidine and clopidogrel appear to inhibit the P2TAC receptor (see Chap. 131), whereas soluble CD39 catabolizes ADP.608 Several patients with apparently inherited defects in ADP-induced platelet aggregation have been described (see Chap. 119).
Epinephrine When added to platelet-rich plasma, epinephrine uniquely initiates a first phase of aggregation without first inducing shape change; after a plateau period, a second wave of aggregation occurs. The ability of epinephrine to synergize with other agonists such as ADP is well documented, but there is controversy as to whether epinephrine, in the absence of released ADP or thromboxane A2, is sufficient to initiate platelet aggregation.609,610 and 611 Epinephrine can cause an elevation in intracellular calcium, even in aspirin-treated platelets,612 possibly by opening an external channel or causing release of calcium from membrane sources613,614; it does not appear to mobilize intracellular calcium or generate measurable amounts of IP3. Epinephrine is a potent inhibitor of adenylyl cyclase and thus prevents formation of cAMP, but the reduction in cAMP caused by epinephrine is unlikely to be sufficient to mediate platelet aggregation.615,616 and 617 Analysis of the purified epinephrine receptor and its nucleotide sequence identify it as a seven-transmembrane, G-protein-coupled, a2 adrenergic receptor of Mr 64,000.618,619
Prostaglandin H2/Thromboxane A2 (TXA2) The metabolism of arachidonic acid to TXA2 is a fundamental pathway contributing to agonist-induced platelet activation and aggregation. TXA2 is a potent platelet agonist that stimulates its own seven-transmembrane-spanning G-protein-coupled receptor. Many agonists stimulate the release of arachidonic acid from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the plasma membrane.620 Most arachidonic acid is released by the action of PLA2, but some is also released by: (1) the concerted actions of PLC and DAG kinase, followed by PLA2, or (2) the action of PLC followed by the action of DAG lipase. PLA2 is a cytosolic enzyme, with multiple isoforms in platelets.621 PLA2 acts on the C2 position of triacylglycerols such as PC and PE to form free arachidonic acid and the resulting lysophospholipid. PLA2 also converts phosphatidic acid into lysophosphatidic acid, a potent platelet agonist. Some PLA2 isozymes are activated by the rise in platelet intracellular Ca2+ that occurs during agonist-stimulated activation, whereas other isozymes are activated in a Ca2+-independent manner.
Arachidonic acid is subsequently metabolized by prostaglandin H2 synthase 1 (cyclooxygenase-1 or COX-1) in the dense tubular system, to prostaglandin (PG)G2, and then to PGH2.622 Thromboxane synthase next converts PGH2 to TXA2, which is spontaneously and rapidly converted to the inactive metabolite, TXB2.623 TXA2 and its precursor, PGH2, can both stimulate platelet thromboxane receptors to induce platelet aggregation.623,624 An inducible cyclooxygenzase enzyme (COX-2) is present in many cells involved in mediating the inflammatory response, but only trace amounts are present in platelets.625 Cyclooxygenase (COX) inhibitors such as aspirin inhibit platelet function by inhibiting COX-1 and decreasing TXA2 production.626,627
Pharmacologic studies have suggested the existence of two distinct TXA2 receptor subtypes based on differing affinities for agonist ligands. It appears that the low-affinity binding sites mediate platelet aggregation and granule secretion, whereas the high-affinity sites are associated with platelet shape change.628 Only one thromboxane A2 receptor, however, has been cloned to date.628a Two alternatively spliced forms of the receptor have been reported,628a TXRa cloned from placenta, and TXRb cloned from the endothelium, which differ only at the carboxy terminus.628a The alternative splicing significantly affects the function of these two receptors, as TXRb but not a undergoes agonist-induced internalization.629 Although both TXRa and TXRb mRNA can be detected in platelet lysates, it appears that TXRa is the dominant form.630 The TXA2 receptor has been localized to the platelet plasma membrane,631 and on SDS-polyacrylamide gel electrophoresis it migrates as a broad band of Mr 55,000 to 57,000,632,633 due to variability in glycosylation.630 The thromboxane receptor is coupled to Gaq and Ga13634,635 and possibly Ga11,636 Ga12,636a and Gai2.637 Studies of TXA2 receptor-deficient mice demonstrate that this one gene locus is responsible for most, if not all, biological effects attributed to TXA2 receptor subtypes.638 Bleeding times in these mice are prolonged, confirming the importance of this pathway in normal hemostasis. Moreover, platelet aggregation in response to collagen but not ADP is delayed, demonstrating the importance of TXA2 production in collagen-induced platelet aggregation.
A significant portion of PGH2/TXA2-induced platelet aggregation is actually mediated by secreted ADP, since ADP scavenger systems reportedly block aggregation induced by a stable PGH2/TXA2 analogue either partially (30 percent)639 or totally.640
Thrombin Thrombin is derived from the inactive zymogen, prothrombin, which circulates in plasma. When acted upon by the prothrombinase complex (factor Xa, factor Va, Ca2+), assembled on activated platelets and other cells, prothrombin is cleaved into thrombin641 (see Chap. 112), one of the most potent platelet agonists. The proteolytic activity of thrombin is required for its role as a platelet agonist.642 Thrombin activates the protease-activated receptor 1 (PAR-1), a seven-transmembrane G-protein-coupled receptor on platelets and other cells,643,644 by cleaving an extracellular 41 amino acid peptide from the N-terminus of the receptor (Fig. 111-16). Removal of this peptide results in a new amino-terminus, which acts as a “tethered ligand” by binding to another region of PAR-1 to activate the receptor and initiate signal transduction. The 41 amino acid cleavage product of PAR-1 can also induce platelet aggregation by a poorly defined mechanism.645

FIGURE 111-16 Above. Model for thrombin-protease activated receptor 1 (PAR-1). The receptor’s LDPR sequence interacts with thrombin’s subsites, and the receptor’s YEPFWEDEE sequence binds to thrombin’s anion-binding exosite. Thrombin cleaves the receptor between R41 and S42. Below. After cleavage, the new amino terminus acts as a tethered ligand and inserts into the membrane, initiating activation. (From Vu et al.787a Reprinted by permission from Nature (353:674–677). Copyright 1991 Macmillan Magazines Limited.)

Surprisingly, platelets from PAR-1 knockout mice respond almost normally to thrombin. These unexpected results led to the search for, and discovery of other members of the PAR family, including PAR-3646 and PAR-4.647 A full response of platelets to thrombin appears to require at least two thrombin receptors, PAR-1 and PAR-4 on human platelets,647,648 and PAR-3 and PAR-4 on mouse platelets.647,649
When platelets are exposed to subaggregating concentrations of thrombin, they become relatively insensitive to the addition of an aggregating concentration of thrombin, a process termed homologous desensitization. Part of this mechanism involves rapid PAR-1 internalization, but other biochemical changes probably are also involved.650 Trafficking of the thrombin receptor to lysosomes is dictated by sequence in the cytoplasmic tail of PAR.651
Thrombin can bind to GPIba, and platelets from patients lacking the GPIb/IX complex (Bernard-Soulier syndrome) have decreased thrombin-induced platelet aggregation (see Chap. 119). A region on GPIba with three sulfated tyrosines and a large number of anionic amino acids (with homology to the high-affinity thrombin inhibitor hirudin) contains the thrombin binding site. Despite these data, it is not clear that the binding of thrombin to GPIba initiates a signal or is of physiologic significance.
Platelet Activating Factor Platelet-activating factor (a mixture of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine652) is a phospholipid ether produced by platelets, leukocytes, and other cells. PAF is a potent platelet agonist and proinflammatory mediator.653 Cellular responses to PAF are mediated by a specific seven-transmembrane, G-protein-coupled receptor.654,655 PAF induces G-protein-dependent inhibition of adenylyl cyclase and activation of PLC,656 which causes phosphoinositide turnover, leading to the activation of PKC and an increase in intracellular Ca2+.655 PAF also indirectly activates PLA2, which causes release of arachidonic acid from the platelet membrane.657 All of these effects contribute to the overall platelet response to PAF.
Serotonin Platelets serve as the major storage site in the circulation for serotonin because they have an extensive capacity to take it up actively into dense granules. The release of serotonin from the dense granules during platelet activation may amplify platelet aggregation and granule release. The receptor that mediates serotonin’s effects on platelet function has been classified pharmacologically as a 5HT2 receptor,658 and the 5HT2-receptor-blocking compound ketanserin antagonizes serotonin’s stimulatory effects on platelets.659 The receptor has been cloned from platelets and shown to be essentially identical to the seven-transmembrane, G-protein-coupled, 5-HT2A receptor present in the brain frontal cortex.660,661 Two naturally occurring amino acid substitutions have been identified in the receptor.662 Platelets from patients heterozygous for the H452Y polymorphism have a blunted calcium response when stimulated with serotonin compared to platelets from patients homozygous for H452.662 Many studies have been performed correlating platelet serotonin transporter activity and 5-HT2A receptors with a number of neuropsychiatric disorders.663,664,665,666 and 667 There is some concern, however, about the correlation between 5-HT2A receptors on platelets and those in the brain.668
Addition of serotonin in micromolar concentrations to platelets in vitro causes elevation of intracellular calcium, PLC activation, protein phosphorylation, and mild aggregation.669,670 In whole blood, serotonin does not itself cause platelet aggregation, but it does enhance aggregation induced by ADP and thrombin.671 Serotonin released from platelets can cause vasoconstriction of blood vessels that have suffered endothelial damage,672 further promoting thrombus formation. Inhibition of serotonin’s action has a favorable effect in animal models of thrombosis and vascular damage, but it is not clear whether the benefit derives from effects on platelet aggregation or vasoconstriction.673
Vasopressin Vasopressin interacts with platelets to induce shape change, aggregation, and dense granule release.674 These events follow an induced rise in intracellular calcium and PLC activation.675 The platelet binding site is classified pharmacologically as a V1-type receptor,676 and radiolabeled vasopressin binds to it with a KD of 1 to 10 nM.677 Unlike the case with V2 receptors that activate adenylate cyclase, the V1 receptors appear to activate phospholipase C,678 perhaps via coupling through Gaq11.679 There are fewer than 100 binding sites for vasopressin on the platelet.680 There is controversy as to whether physiologic concentrations of vasopressin are sufficiently high to activate platelets directly681,682; even if vasopressin does not directly activate platelets, it may be able to enhance platelet activation induced by other agonists.
Thrombospondin and Integrin-Associated Protein Thrombospondin (TSP), a large disulfide-bonded trimer (subunit Mr 160,000), is both a platelet a-granule protein and an extracellular matrix protein present in the subendothelium. TSP is rapidly released from platelets upon thrombin stimulation. In addition to its role as an adhesive protein, TSP also functions as an agonist to stimulate GPIIb/IIIa-mediated platelet aggregation.683,684 Multiple potential TSP receptors are present on platelets, including GPIV (CD36), GPIIb/IIIa, aVb3, and integrin-associated protein (IAP). Of these receptors, IAP is most strongly implicated as the major signaling receptor in response to TSP. IAP was first discovered as a protein that copurifies with integrins, including GPIIb/IIIa,683 aVb3,685 and a2b1.686 The sequence of IAP indicates that it has a single immunoglobulin-like extracellular domain, five-membrane spanning regions, and a short cytoplasmic tail.102,684,685 IAP probably generates signals independent of integrins and affects integrin function via downstream effectors. IAP couples physically and functionally to the large G-protein, Gai,687 which is of note, since all known large G-proteins couple to seven- instead of five-transmembrane spanning receptors. Further downstream signaling probably involves tyrosine kinases, including Syk, Lyn, and Fak, as well as activation of PLCg2.683
How other TSP binding sites on platelets contribute to the overall response induced by TSP is not clear. GPIV copurifies with several tyrosine kinases, including Fyn, Lyn and Yes.688 However, whether TSP binding to GPIV activates these kinases and whether they then contribute to the observed platelet response is unknown.
Collagen Upon vascular injury, collagens in the subendothelium become exposed to flowing blood and promote both platelet attachment and activation, thereby contributing to normal hemostasis. Collagen is also thought to be one of the most thrombogenic substances in atherosclerotic plaques, and upon plaque rupture is believed to contribute to platelet aggregation and thrombus formation, leading to ischemic damage.689 The types of collagen present in the subendothelium include: I, III, IV, V, VI, VIII, and XIII,690 the most abundant being types I and III (more than 95 percent). Under conditions that mimic physiologic blood flow, platelets adhere tightly to collagen types I, III, and IV, weakly to types VI, VII, and VIII, and not at all to type V. However, under static conditions, platelets can adhere to types II and V collagens as well as to the other collagens listed above.691
Collagen-induced platelet activation probably involves multiple receptors (Fig. 111-7 and Fig. 111-17). The best characterized collagen receptor on platelets is the GPIa/IIa (a2b1) integrin (see “GPIa/IIa” above). The I, or inserted, domain in the Ia (a2) subunit is homologous to a number of collagen-binding domains in other proteins and probably mediates adhesion of the receptor to collagen. Another platelet collagen receptor, which appears to be important in collagen-induced signaling, is GPVI. This Mr 62,000 glycoprotein from the immunoglobulin superfamily191,692,693 and 694 functions in concert with the Fc receptor g chain (FcRg), with the latter initiating intracellular signaling.695 Other collagen receptors on platelets include GPIV (CD36)510 and an Mr 65,000 protein (GP65).696 The potential interrelation of all these collagen receptors is unknown, but there is considerable evidence that they probably work in concert, perhaps by assembling intracellular proteins into complexes, since interactions of collagen with both GPIa/IIa (a2b1) and GPVI are required for a full platelet response.375,697,698

FIGURE 111-17 Collagen activation of platelets. Collagen binds to at least three receptors on platelets, glycoprotein IV (GPIV), GPIa/IIa (a2b1), and GPVI. GPIV appears to play little or no role in collagen activation of platelets. However, collagen binding to both a2b1 and GPVI is required to activate the tyrosine kinase, Syk. GPVI is physically and functionally coupled to the FcRg chain, which contains an ITAM motif. The tyrosines (Y) within the ITAM motif become phosphorylated (P), most likely by Fyn. The tyrosine kinase Syk binds to the phosphorylated ITAM motif through SH2 domains. Syk itself becomes phosphorylated (probably by Fyn or Lyn tyrosine kinases) and activated, leading to the activation of Bruton’s tyrosine kinase (BTK). BTK might be the kinase that directly phosphorylates and activates PLCg2, although this is not certain. PLCg2 hydrolyzes PIP2, forming IP3 and DAG as described earlier.

Glycoprotein VI exists in a stable physical complex with the FcRg chain. In GPVI-deficient platelets, the FcRg chain is also absent.475 The addition of either collagen or an antibody that can cross-link GPVI induces tyrosine phosphorylation of the FcRg chain.475 The kinases contributing to this event are likely to be the nonreceptor kinases Fyn and/or Lyn.699 Tyrosine phosphorylation of the immune-receptor tyrosine-based activation motif (ITAM) on the FcRg chain increases the affinity of the ITAM for SH2 domains, resulting in the recruitment of proteins with SH2 domains to the FcRg chain. The nonreceptor tyrosine kinase Syk contains two adjacent SH2 domains and a tyrosine kinase domain. In platelets from normal mice, Syk physically associates with the FcRg chain and becomes phosphorylated and activated after collagen stimulation,475 whereas in platelets from mice lacking FcRg chain, collagen is unable to induce Syk phosphorylation and activation.695 Similarly, in platelets lacking GPVI or in platelets in which the GPIa/IIa (a2b1) integrin is blocked, collagen-induced Syk phosphorylation is also inhibited, demonstrating that GPVI, GPIa/IIa (a2b1), and Syk all participate in the platelet response to collagen. The b subunit of the GPIa/IIa (a2b1) receptor also has tyrosines spaced in a manner reminiscent of an ITAM motif, and thus it is possible that Syk might also associate with this collagen receptor. In addition to Syk,700 Src701 also becomes tyrosine phosphorylated in response to collagen. Although Src is an abundant kinase in platelets, its role in platelet signaling is unclear, since mice lacking Src do not suffer from any obvious bleeding disorder.702 Syk, on the other hand, appears to play a critical role in collagen activation of platelets, since platelets from mice lacking Syk do not aggregate or undergo secretion in response to collagen.695
Collagen stimulation of platelets also results in tyrosine phosphorylation and activation of PLCg2,703 and, as discussed above, activation of this enzyme causes phosphoinositide hydrolysis, leading to GPIIb/IIIa activation. PLCg2 activation occurs downstream of Syk, as evidenced by the findings that collagen is unable to activate PLCg2 in platelets pretreated with a Syk-selective inhibitor697 or in platelets from Syk knock-out mice.695 It is unknown whether Syk activates PLCg2 directly, but Bruton’s tyrosine kinase (BTK) might be positioned between Syk and PLCg2 because patients lacking BTK not only exhibit the B-cell deficiency X-linked agammaglobulinaemia but also show reduced platelet responsiveness to collagen and diminished phosphorylation of PLCg2.704
GPIV (CD36) can also bind collagen and antibodies to GPIV partially inhibit platelet adhesion to collagen.704a There is currently no evidence that GPIV contributes to collagen-induced signaling in platelets, since platelets from patients lacking GPIV respond normally to collagen.704b
Platelets stimulated with collagen exhibit several distinct responses. While elevated cAMP levels normally inhibit platelet aggregation, collagen-stimulated platelets are relatively resistant to inhibition by cAMP.705 This may be related to the fact that collagen stimulates the PLCg isotype, which is insensitive to cAMP-mediated inhibition, whereas other agonists such as thrombin stimulate PLCb, which is inhibited by cAMP. Another difference has been noted with the phosphatase inhibitor, phenylarsine oxide, which inhibits collagen, but not thrombin or ADP-induced platelet aggregation.706 This suggests that one or more phosphatases are critical in collagen-induced platelet aggregation.
Von Willebrand Factor—GPIb/IX/V The GPIb/IX/V complex functions as a signaling receptor upon vWF binding, resulting in platelet activation and GPIIb/IIIa-mediated aggregation. Thus, ristocetin-mediated interaction of vWF with platelets results in PIP2 metabolism, activation of PKC, and an increase in intracellular Ca2+. Shear forces can also initiate signaling through the binding of vWF to GPIb/IX/V.707 GPIb/IX/V signaling causes arachidonic acid to be released and metabolized to form TXA2. These proaggregatory events are specifically inhibited by monoclonal antibodies that recognize GPIb, but not antibodies to GPIIb/IIIa.708 This signaling appears to be mediated in part by an Mr 29,000 protein termed 14-3-3z that binds directly to the cytoplasmic domains of both GPIba and GPIbb and may have PLA2-related properties709,710 (Fig. 111-9). The binding site for 14-3-3z on GPIba has been mapped to the last 15 residues of the C-terminus of the cytoplasmic domain, with the last 5 residues being especially important.711 Protein 14-3-3z exists as a dimer, allowing it to link GPIb molecules.712 The helix I region of 14-3-3z (residues 202–231) is required for binding to GPIba, and another region binds the serine/threonine kinase c-RAF,712 suggesting a direct link to the Raf/MEK/MAPK signaling pathway.
The GPIb/IX/V complex also appears to be involved in transmitting at least one cAMP-dependent inhibitory signal. Thus, elevated cAMP, which activates protein kinase A, induces phosphorylation of GPIbb on serine 166.713 Elevated cAMP also normally inhibits agonist-induced platelet actin polymerization. However, in platelets from patients with Bernard-Soulier syndrome, which lack GPIb/IX/V, actin polymerization proceeds normally after collagen stimulation, even when cAMP is elevated, suggesting that cAMP-mediated phosphorylation of GPIbb may be required for the cAMP-mediated inhibition.714
Lysolipid Phosphate Activators: Lysophosphatidic Acid and Sphingosine-1-Phosphate Platelets possess cell surface receptors for the lysolipid phosphates, lysophosphatidic acid (LPA)715 and sphingosine-1-phosphate,716 which stimulate platelet aggregation. The receptor(s) for these lipids are believed to be members of the endothelial differentiation gene (Edg) family of G-protein-coupled receptors; they can activate PLC and initiate tyrosine kinase phosphorylation.715,717 Lysolipid phosphates are synthesized by both de novo and agonist-stimulated pathways.718 Platelets accumulate sphingosine-1-phosphate due to a lack of sphingosine-1-phosphate lyase activity and high levels of sphingosine-1-phosphate kinase activity. One pathway for LPA formation involves agonist-stimulated production of phosphatidic acid, either by PLD or the sequential actions of PLC and DAG kinase. Phosphatidic acid in the outer portion of the plasma membrane or in platelet-derived microvesicles is hydrolyzed by secretory PLA2 (sPLA2) to LPA,719,720 which then can act on its extracellular receptors to induce platelet aggregation. Mild oxidation of LDL generates LPA, and the LPA component of oxidized LDL in the lipid-rich thrombogenic core of atherosclerotic lesions exposed during plaque rupture may be a potent stimulus for platelet activation.721 LPA levels in plasma are approximately 0.2 µM, whereas they are 10 µM in serum, reflecting mainly the secretion of LPA-precursors by activated platelets.712 Phosphatidic acid in platelet-derived microparticles is believed to be a substrate for sPLA2 and the principle source for plasma LPA. Plasma LPA circulates bound to albumin, activates endothelial cells, stimulates fibroblast proliferation and smooth-muscle cell contraction, and is a monocyte chemoattractant. LPA is degraded by cell-associated phosphatases located on platelets and other cells. Thus, LPA activates platelets and activated platelets at sites of vascular injury may provide a source of LPA to recruit monocytes and promote smooth-muscle cell accumulation and extracellular matrix deposition.
Calcium Elevation of intracellular Ca2+ has a multitude of effects on platelet physiology. The concentration of Ca2+ in resting platelets (100 to 500 nM) is very low compared to the plasma concentration of Ca2+ (about 2 mM). Exposure of platelets to most agonists is accompanied by a rapid, transient rise in the intracellular free Ca2+ concentration to micromolar levels, followed by a quick return to normal resting levels. The cytoplasmic Ca2+ concentration at any given time is a result of the rates of passive Ca2+ influx, active Ca2+ extrusion across the plasma membrane, and both active release and/or uptake of Ca2+ by the dense tubular system (DTS), which is a Ca2+ storage depot in platelets analogous to the sarcoplasmic reticulum in muscle. Active Ca2+ extrusion and uptake of Ca2+ are mediated by several pumps (see Fig. 111-7). The cytosolic pool of Ca2+ has a rapid turnover due to a plasma membrane Na2+/Ca2+ antiporter, whereas the dense tubular system contains a more slowly exchanging pool regulated by a Ca2+/Mg2+ ATPase, a pump that also appears to be located in the plasma membrane.722 During agonist stimulation, most Ca2+ enters the platelet cytosolic compartment through receptor-operated calcium channels (reviewed in Geiger and Walter723) in the plasma membrane. Release of intracellular Ca2+ from the DTS storage sites also occurs rapidly in response to agonist stimulation, due in large part to IP3 that is generated as part of the phosphoinositide cycle.724 GPIIb/IIIa may also participate in Ca2+ entry.725
Elevations of Ca2+ induce numerous downstream events, including activation of Ca2+-sensitive forms of PLA2726 and PKC727; calmodulin-dependent enzymes such as myosin light-chain kinase, which phosphorylates myosin light chain728 and promotes cytoskeletal rearrangements required for platelet shape change; and gelsolin, which facilitates actin severing and rearrangement, secretion, and aggregation. In addition, Ca2+ probably plays a direct role in the membrane fusion events that result in degranulation and the release reaction. Calcium-dependent proteases or calpains also become activated and play an important role in post aggregation events (see below).
Phosphoinositide 3-Kinases Phosphoinositide 3 kinases (PI3 kinases) are a family of lipid kinases that phosphorylate the D-3 hydroxy group of the myo-inositol ring of phosphoinositides (reviewed in Zhang et al729 and Rittenhouse730). Class I PI3 kinases are heterodimeric proteins containing both adaptor and catatlytic subunits, which utilize phosphatidylinositol (Ptdlns), Ptdlns(4)P, and Ptdlns(4,5)P2 as substrates to form Ptdlns(3)P, Ptdlns(3,4)P2, and Ptdlns(3,4,5)P3 respectively. Platelets contain two isoforms of PI3 kinase, designated class Ia and class Ib, that have distinct subunits and regulatory features. The catalytic subunit of class Ia PI3 kinases is an Mr 110,000 to 120,000 protein; the adaptor subunit, p85, has two SH2 domains, a breakpoint cluster region homology domain, a proline-rich region, and a single SH3 domain. Members of this class of PI3 kinases possess intrinsic serine-threonine protein kinase activity in addition to lipid kinase activity, and they appear to be regulated, at least in part, by binding of the p85 subunit to tyrosine-phosphorylated proteins. Following platelet activation, PI3 kinase can be coimmunoprecipitated with the tyrosine kinases Src and Syk. Class Ib PI3 kinases have been isolated from platelets and neutrophils and contain a catalytic subunit, p110g, that is activated by the bg subunit of heterodimer G proteins. Both isoforms of PI3 kinase appear to associate with the cytoskeleton in agonist-activated platelets.
In platelets, 3-phosphorylated phosphoinositides are produced in response to a variety of agonists, including thrombin, TXA2, LPA, ADP, and collagen, and may mediate early signaling events that precede GPIIb/IIIa activation as well as late events involved in stabilizing fibrinogen binding and platelet aggregation.729,730 and 731 Thrombin stimulates rapid accumulation of Ptdlns(3,4,5)P3 and Ptdlns(3,4)P2732 and late production of Ptdlns(3,4)P2; the latter requires fibrinogen binding to GPIIb/IIIa and calpain activity.733 Collagen promotes the association of the p85 adaptor subunit via its SH2 domains with tyrosine-phosphorylated forms of FcRg chain and the regulatory protein, linker-for-activator of T cells (LAT), and may thereby modulate Pl 3-kinase activity.734 FcgRIIA-induced platelet aggregation required PI3 kinase activity, which appears to be upstream of PLCg2 in the pathway.735
Many of the biological actions of PI3 kinases are mediated by their phospholipid products, which bind to specific sequences in proteins. The pleckstrin homology (PH) domains (about 100 amino acids long) present in pleckstrin and other platelet proteins involved in signal transduction, recognize either PI(3,4)P2 or PI(3,4,5)P3 (reviewed in Leevers, Vanhaesebroeck, and Waterfield736). Binding of PI(3,4,5)P3 to the amino terminal PH domain in PLCg enhances its activity.737 PI(3,4,5)P3 binding to PH domains in BTK738 targets BTK to the plasma membrane, where it is further phosphorylated and activated.739 PI(3,4)P2 or PI(3,4,5)P3 binding to the PH domains in the serine/threonine kinase AKT (or protein kinase B) changes the conformation of AKT, permitting it to become activated by phosphorylation on serine and threonine by AKT-kinase (PDK1).740,741 AKT activation is biphasic, occurring before and after platelet aggregation733; AKT may therefore play multiple roles in platelet activation.733
Small G Proteins Some members of the Ras superfamily of small GTPases modulate integrin receptor activation.742 Small GTPases, like their large G-protein counterparts, cycle between a resting, GDP-bound state, and an active, GTP-bound state. In their active state, small GTPases can interact with, and activate downstream signaling molecules, thus acting as molecular switches. One small GTPase, R-Ras, has been shown to activate GPIIb/IIIa when both are expressed in CHO cells—cells that are normally unable to activate this integrin by a signal transduction cascade.743
The small G-protein, RhoA has an established role in stress fiber formation in nucleated cells.744 In platelets RhoA is required for vinculin-dependent focal adhesion formation in platelets adherent to fibrinogen745, but not for GPIIb/IIIa activation.745
Preliminary data suggest that a third small G-protein, H-Ras, may function to suppress integrin activation.746 H-Ras in other cells is often part of a pathway involving the serine/threonine kinases Raf, MEK and MAPK. Whether a Ras/Raf/MEK/MAPK pathway is inhibitory in platelets is not entirely clear, however, since a soluble MEK inhibitor attenuates rather than enhances platelet aggregation to low concentrations of collagen and arachidonic acid.747 Moreover, Ras is activated by platelet agonists748, so the exact role of Ras in platelet activation remains to be determined. Based on studies in nucleated cells, PEA-15, a small protein that contains a death effector domain (DED),749 may act in concert with an R-Ras dependent pathway, to oppose the inhibitory effects of H-Ras on integrin function. Thus it appears that the small G-proteins have the capacity to up- and down-regulate the activation state of GPIIb/IIIa.
After ligand binding, integrin clustering, and platelet aggregation, calcium-dependent proteases or calpains become activated. Calpain activation also requires a rise in intracellular calcium. The most important and well-studied class of calpains in platelets are the µ-calpains, which are activated by micromolar concentrations of calcium, although m-calpains, which require millimolar concentrations of calcium for activation, also exist. Each form of calpain consists of an Mr 80,000 catalytic subunit paired with an Mr 30,000 subunit. Activated µ-calpains cleave numerous molecules in cells that affect platelet function, including actin binding protein (releasing GPIb, talin, and the cytoplasmic domain of GPIIIa from the membrane skeleton), and some forms of PKC (reviewed in250). In addition, calpain appears to be upstream of, and able to induce, the activation of the small G-proteins Rac and RhoA.250 These small G-proteins have profound effects on cytoskeletal structure, Rac being involved in lamellipodia formation750 and Rho being linked to stress fiber formation.744 Thus calpains, by their effects on structural and signaling molecules, appear to affect the signaling and cytoskeletal rearrangements that occur following platelet aggregation.
The cytoplasmic domains of GPIIb and GPIIIa appear to control the activation state of the GPIIb/IIIa receptor. When recombinant GPIIb/IIIa that either lacks the GPIIb cytoplasmic domain entirely, or contains mutations in the highly conserved GFFKR sequence in the cytoplasmic domain is expressed in a cell line, the GPIIb/IIIa is in an active conformation that can bind ligand.751,752 This activation probably occurs because of a disruption of an interaction between the two cytoplasmic domains and/or with associated proteins that maintains the integrin in a resting conformation. Evidence for an interaction between the GPIIb and GPIIIa cytoplasmic domains comes from studies suggesting that there is a salt bridge between Arg995 in GPIIb and Asp723 in GPIIIa751 and that peptides from specific regions of the GPIIb and GPIIIa cytoplasmic domains (amino acids 999–1008 of GPIIb and 721–740 of GPIIIa) can bind to each other.753 Interestingly, the GPIIb and GPIIIa peptide complex also binds Ca2+.753 Finally, introduction into platelets of a lipid-modified GPIIb cytoplasmic domain peptide (amino acids 989–995) activates GPIIb/IIIa and initiates platelet activation and TXA2 formation.754 Presumably this peptide displaces the normal GPIIb from its complex with GPIIIa, and thus these data support the hypothesis that disruption of the normal interaction between the cytoplasmic domains of GPIIb and GPIIIa facilitates integrin activation.
The state of GPIIb/IIIa activation may also be affected by molecules associated with the integrin. Thus, overexpression of the GPIIIa cytoplasmic domain in cells suppresses integrin activation, presumably because it competes with the normal GPIIIa for binding of molecules necessary to induce the activated state. CD98 may be one of the molecules involved in this process.755 The interaction of GPIIb/IIIa with cytoskeletal elements has been proposed to maintain the integrin in a resting conformation; in this model, platelet activation releases these constraints and allows GPIIb/IIIa to assume its active conformation. Numerous molecules have been identified that appear to associate directly with the GPIIb or GPIIIa cytoplasmic domains or with other parts of the integrin. For example, CIB, an Mr 22,000 Ca2+ binding protein associates with the GPIIb cytoplasmic domain and may play a role in ligand-induced signaling through GPIIb/IIIa.755a,755b756 b3-endonexin757 and the cytoskeletal proteins talin758 and myosin759 have been reported to bind to the GPIIIa cytoplasmic domain, and b3-endonexin757 and talin760 have been reported to activate GPIIb/IIIa. In addition, integrin-associated protein, a five-transmembrane-spanning molecule,685 CD9761,762 and CD151 (PETA-3),763 members of the tetraspanin receptor family, likely associate with other portions of GPIIb/IIIa and coisolate with the integrin. Thrombospondin binding to IAP683,685 and antibody-mediated cross-linking of either CD9 or CD151 to the platelet Fc receptor cause platelet activation764 with resultant activation of GPIIb/IIIa.
Platelet aggregation is commonly described as progressing through two phases, an initial reversible aggregation phase, which is often the response observed with low concentrations of agonists, followed by a stronger, irreversible phase. The irreversible phase of aggregation correlates with TXA2 production and platelet secretion of ADP. Fibrinogen binding to GPIIb/IIIa and the platelet-platelet contacts that occur during the initial phase of aggregation initiate specific signal transduction events, resulting in positive feedback loops that promote irreversible aggregation, maintain secretion, and initiate later events like clot retraction.765
Fibrinogen or vWf binding to the extracellular region of GPIIb/IIIa transmits long-range conformational changes to the integrin cytoplasmic domains that induce signaling from outside the platelet to inside the platelet (outside-in signaling).766,767 These conformational changes, along with integrin clustering,768 are likely to be the bases for outside-in signal transduction through GPIIb/IIIa, perhaps by altering the association of the cytoplasmic domains with one another and presumably initiating recruitment of proteins with enyzmatic acitivity into cytoplasmic complexes.
When platelets are aggregated in response to one of multiple agonists, the GPIIIa cytoplasmic domain becomes phosphorylated on tyrosine.759,769 Two sites of potential tyrosine phosphorylation exist on the GPIIIa cytoplasmic domain, and both may be utilized. Several molecules have been identified that bind specifically to the tyrosine-phosphorylated cytoplasmic domain of GPIIIa. A synthetic GPIIIa cytoplasmic domain peptide containing phosphate groups on the two candidate tyrosines binds to the contractile protein myosin,759 and this interaction may facilitate the transmission of cytoskeletal tension from inside the platelet to outside and thus initiate clot retraction (see above). Recombinant, mutated GPIIIa that cannot be phosphorylated is unable to support extensive clot retraction when expressed in a cell line.759 Other proteins that bind to the diphosphorylated GPIIIa cytoplasmic domain include the adapter protein SHC,770 which also becomes tyrosine-phosphorylated during platelet aggregation.770 Therefore, it is possible that SHC may link diphosphorylated GPIIIa to the Ras/Raf/MAPK pathway.770,771
Mice containing mutated GPIIIa molecules that cannot be phosphorylated exhibit a mild bleeding disorder as evidenced by occasional rebleeding of tail cuts. Moreover platelets derived from these mice form abnormally loose thrombi when activated by shear forces.765
Other GPIIIa cytoplasmic domain binding proteins have been described, including skelemin, a member of a family of proteins that regulate myosin,772 and talin, which has binding sites on both GPIIb and GPIIIa, providing another potential linkage of GPIIb/IIIa to cytoskeletal elements.758
Some signaling events that occur downstream of GPIIb/IIIa require only integrin clustering, whereas other events require clustering, ligand binding, and/or platelet aggregation. For example, the tyrosine kinase Syk becomes activated in response to GPIIb/IIIa clustering, independent of cytoskeletal assembly, whereas activation of the tyrosine kinase FAK requires integrin clustering, ligand binding to GPIIb/IIIa, and cytoskeletal assembly.772a Activation of Syk downstream of GPIIb/IIIa leads to phosphorylation of Vav1, a guanine nucleotide exchange factor for Rac, and lamellipodia formation in a cell line. Syk and Vav1 cooperate to activate Jun N-terminal kinase or JNK, extracellular-signal-regulated kinase 2 (ERK2) and AKT.772a These pathways are also likely to be involved in postaggregation events in the platelet.
Prostaglandins Prostaglandins that inhibit platelet activation include prostaglandin PGE2 and PGI2 (also called prostacyclin) (reviewed in Majerus773 and Moncada and Whittle774). In the vasculature, the endothelium produces PGI2 and PGE2, which help to maintain vascular patency.775 Inhibition is initiated by the binding of these prostaglandins to their own specific G-protein-coupled receptors. Prostaglandin receptor occupancy converts Gas to the GTP-bound, active form, which activates adenylyl cyclase. Adenylyl cyclase catalyzes the formation of cAMP. The exact amount of cAMP present in the cell is also determined by its rate of breakdown by phosphodiesterase (PDE). Therefore, agents that inhibit PDE such as theophilline, caffeine, and the drug cilostazol also elevate cAMP levels in platelets and other cells. Cyclic AMP then activates protein kinase A, which phosphorylates specific target proteins. The exact mechanism by which PKA inhibits platelet activation probably involves more than one pathway. One mechanism may involve phosphorylation and inhibition of the IP3 receptor, which would repress agonist-induced intracellular Ca2+-mobilization.776 Phosphoinositide metabolism is also affected, since the activities of both PLC and PLA2 are suppressed.777 PGE1 phosphorylation of GPIbb also inhibits actin polymerization (see above). Moreover, cAMP also inhibits Raf kinase.778 Finally, the small G-protein, Rap 1b, is phosphorylated by PKA.779 However, the functions of Raf kinase and Rap1b in platelets are unclear.
Nitric Oxide Nitric oxide (NO) is synthesized from L-arginine by NO synthase in endothelial cells, platelets, and other cells. The formation of NO is enhanced at sites of shear stress and by platelet agonists (e.g., thrombin or ADP),780 and it readily diffuses into platelets.781,782 Similar to PGI2 or PGE2, NO pretreatment of platelets inhibits platelet activation and can reverse platelet aggregation soon after initiation. However, NO works not by elevating cAMP but instead by increasing cGMP.783 NO synthase activity in platelets increases during platelet activation, suggesting that NO production is a normal mechanism to limit platelet aggregation. NO and PGI2 act together synergistically to inhibit platelet activation.784
CD39 (ATP Diphosphohydrolase; ADPase) Vascular endothelium regulates platelet function by producing prostacyclin and nitric oxide, as well as by expressing CD39, a plasma-membrane-associated ATP diphosphohydrolyase (ATPDase; ecto-ADPase) that converts extracellular ATP and ADP to AMP.42,785 CD39 limits the platelet-activating effects of ADP released by damaged tissues, red blood cells, and activated platelets; furthermore, AMP generated by CD39 is degraded by ecto-5′ nucleotidase to adenosine, an antagonist of ADP-induced platelet activation. CD39 is an Mr 95,000 cell-surface glycoprotein expressed on endothelial cells, activated NK cells, B cells, and T cells. It contains two putative transmembrane regions separated by an extracellular domain with six glycosylation sites and apyrase-like regions that confer the ATPDase activity. A soluble recombinant form of CD39 inhibits platelet aggregation and recruitment in vitro and may have potential as an antithrombotic agent in vivo.608

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Copyright © 20017 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology


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