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Williams Hematology



Endothelial Cells and Hemostasis
Thromboregulation by Vascular Endothelial Cells

Prostacyclin as a Thromboregulator

Nitric Oxide, an Endothelial Vasodilator and Inhibitor of Platelet Function

Endothelin (Et)

Inhibition of Platelet Function by Ecto-Adpase/Cd39
The Protein C Pathway


The Endothelial Protein C Receptor
Tissue Factor Pathway Inhibitor
The Endothelial Cell Fibrinolytic System

Endothelial Cell Production of Fibrinolytic Proteins

Nonfibrinolytic Functions of Plasmin

Fibrinolytic Function and Vascular Disease

Plasminogen Deficiency

Impairment of Fibrinolytic Assembly in Vascular Disease
Role of Adhesion Molecules

Molecular Changes in an Inflammatory Milieu

Adhesion Molecules in a Thrombotic Milieu
Chapter References

Blood vessels play a critical role in the control of hemostasis, thrombosis, and inflammation. Endothelial cells, which form the lining of all blood vessels, are particularly important in this process because of their intimate association with flowing blood. Endothelial cells have the unique capability to express and elaborate thromboregulatory molecules, which can be classified as early or late with respect to an endothelial cell stimulus. In addition, pro-inflammatory leukocyte adhesion molecules are expressed upon endothelial cell perturbation (Table 114-1).


Acronyms and abbreviations that appear in this chapter include: ADP, adenosine diphosphate; APC, activated protein C; Apo(a), apolipoprotein(a); ApoE, apolipoprotein(e); ATIII, antithrombin III; cAMP, adenosine 3′,5′-cyclic phosphate; CAMs, cell adhesion molecules; DDAVP, desmopressin acetate; EDRF, endothelium-derived relaxing factor; EGF, epidermal growth factor; EPCR, endothelial cell protein C receptor; ET-1, endothelin-1; ICAM-1, intercellular adhesion molecule 1; LDL, low-density lipoprotein; LFA-3, lymphocyte-function-associated antigen-3; Lp(a), lipoprotein(a); LPS, lipopolysaccharide; MAdCAM-1, mucosal addressin cell adhesion molecule 1; NK cells, natural killer cells; NO, nitric oxide; NOS, nitric-oxide synthase; PAF, platelet activating factor; PAI, plasminogen activator inhibitor; PCI, protein C inhibitor; PSGL-1, P-selectin glycoprotein ligand 1; TAFI, T thrombin activatable fibrinolysis inhibitor; TF, tissue factor; TFPI, tissue factor pathway inhibitor; TGF-b, transforming growth factor beta; TM, thrombomodulin; TNF-a, tumor necrosis factor alpha; tPA, tissue plasminogen activator; uPA, urokinase plasminogen activator; uPAR, uPA receptor; VCAM-1, vascular adhesion molecule 1; VEGF, vascular endothelial growth factor; VLDL, very low density lipoprotein; vWF, von Willebrand factor.

Thromboregulators acting at the initial stages of thrombus formation interfere with platelet deposition or regulate the contractile state of the blood vessel. They include nitric oxide, eicosanoids, and the ectoADPase/CD39. Nitric oxide is a highly reactive, and therefore evanescent, gas produced by endothelium that acts as a potent vasodilator and inhibitor of platelet aggregation. The endothelial cell eicosanoids, similarly, are fatty-acid-derived hydrocarbons that block platelet aggregation and induce vascular relaxation. The endothelins are a family of endothelium-derived peptides that strongly stimulate vascular constriction over prolonged periods of time. Endothelial cell ectoADPase is a membrane-associated protein that metabolizes adenosine diphosphate (ADP) in the primary platelet releasate, thus preventing recruitment of additional platelets to the initial hemostatic plug.
The late thromboregulators act to regulate thrombin generation, neutralize thrombin, or lyse thrombi. Endothelin is a long-acting vasoconstrictor. Antithrombin III, a natural anticoagulant, is a circulating inhibitor of thrombin and factor Xa, which employs endothelial cell heparan proteoglycans as cofactors. Tissue factor pathway inhibitor is a protein that inhibits factor VIIa tissue factor activity. The thrombomodulin/endothelial cell protein C receptor (EPCR)/protein C system of the vascular wall is integrally involved in the regulation of hemostasis through direct anticoagulant effects on thrombin. Cellular signals resulting from thrombin-mediated activation of protein C and interaction of activated protein C with the EPCR appear to be linked to the inflammatory system. The fibrinolytic system is inextricably intertwined with the vascular endothelium, as endothelial cells not only synthesize and secrete elements of the fibrinolytic system under specific circumstances but also regulate the formation of plasmin. Impairment of fibrinolytic synthetic and assembly systems may play a central role in the etiology of occlusive vascular disease.
In the setting of inflammation, alterations in thromboregulatory balance are evidenced by increased expression of tissue factor and modulation of the thrombomodulin/EPCR/protein C system. Under the same circumstances, endothelial cell adhesion molecules, in addition, constitute a special class of glycoproteins that mediate physical interactions between endothelial cells and leukocytes. Such glycoproteins include members of two molecular families, the cell adhesion molecules (CAMs—MadCAM-1, ICAM-1, VCAM-1, and PECAM-1) and the selectins (P- and E-selectin). In concert, these molecules create a dynamic and changeable interface that modulates a panoply of interactions between the endothelium and various classes of circulating leukocytes.
The endothelium acts as a dynamic interface between flowing blood and the vessel wall. It is subject to unique physical forces, circulating factors, and cell–cell interactions that create region-specific phenotypes. In addition to its role in maintaining vascular permeability, the endothelium serves to regulate the fluid state of blood by displaying thromboresistance and profibrinolytic properties.
In vivo, endothelial cells are highly heterogeneous. They undergo “transdifferentiation,” a process whereby they acquire specialized characteristics in response to signals from the local microenvironment. Thus, small- and large-vessel endothelial cells in vivo, and even endothelial cells from different tissues within the same organ, may express distinct surface molecules, show different membrane specializations such as fenestrae, and exhibit varying synthetic capabilities.
Since the early 1970s, endothelial cells cultivated in vitro, mainly from large-vessel umbilical veins, have served as an important and informative model in vascular biology. However, cultured endothelial cells have significant limitations. First, they exist in an active, replicative mode compared to their in vivo counterparts. In addition, cultured endothelial cells tend to lose their specific regional characteristics and acquire dedifferentiated properties with repeated passage. With the introduction of transgenic animal models, comparisons can be made between in vivo and in vitro studies.
Thromboregulation1,2 refers to a group of processes by which blood cells in the circulation and cells of the vessel wall interact to facilitate or inhibit thrombus formation. It is accomplished through cell proximity or contact and can be cell-associated or involve released compounds generated during agonist exposure. Thromboregulatory systems function to prevent or reverse platelet accumulation, activation of coagulation factors, and formation of fibrin, thereby maintaining blood fluidity1,2,3,4,5,6,7,8,9 and 10 (Fig. 114-1).

FIGURE 114-1 Schematic depiction of some endothelial cell thromboregulatory properties. Secreted products are represented as arrows. Cell-surface-associated molecules appear as boxes. Endothelial cell synthetic products are shaded. “Early” thromboregulators modulate activation of platelets and blood vessel contractility. “Late” thromboregulators modify activation of the coagulation cascade or fibrinolytic system. “Inflammatory” thromboregulators are those whose expression or activity are directed by inflammatory mediators. ADPase, endothelial cell ectoADPase; PGI2, prostacyclin; NO, nitric oxide; ET, endothelin; HS, heparan sulfate; AT III, antithrombin III; uPA, urokinase plasminogen activator; uPAR, uPA receptor; Ann II, annexin II; tPA, tissue plasminogen activator; Plg, plasminogen; TM, thrombomodulin; PC, protein C; TF, tissue factor; VIIa, factor VIIa; TFPI, tissue factor pathway inhibitor; CAMs, cellular adhesion molecules.

The physiologic defense systems that render endothelial surfaces antithrombotic can be overwhelmed by excessive shear stress, injury, increased turbulence, and inflammation.11 The endothelial cells are thereby transformed into a prothrombotic and antifibrinolytic phenotype.12 This transformation is accompanied by up-regulation of leukocyte and endothelial cell adhesion molecules,13 increased expression of tissue factor, and accumulation of monocytes/macrophages in the vessel wall.11 Such events commonly occur at the site of fissured or fractured atherosclerotic plaques in the coronary and cerebrovascular circulation9,11 and involve exposure of tissue factor to the blood.14
The early thromboregulatory systems are the eicosanoids (PGI2, PGD2), nitric oxide (NO), and the ecto-ADPase/CD39 systems. Because they operate very early in the hemostatic/thrombotic cascade, they represent attractive targets for therapeutic intervention, including up-regulation, administration, and gene therapy.
The discovery of thromboxanes by Hamberg, Svensson, and Samuelsson in 1975 initiated a new era in platelet biochemistry and physiology.15 It meant that the activated platelet releasate contains two vasoconstrictors (thromboxane and serotonin) and an agonist for platelet aggregation (thromboxane), operative through ADP release. Subsequently, an agent derived from endothelium that caused vasodilatation and inhibited aggregation was discovered. Initially named PGX, it was later designated as PGI2, or prostacyclin.16,17 and 18
There is a wide range of agonists for eicosanoid production in endothelial cells. They can be hormonal, biochemical, or physical, such as shear stress. Exposure to stimuli increases intracellular calcium levels, which in turn activates phospholipases such as A2 and C. The phospholipases catalyze formation of free arachidonate from membrane phospholipids. In some tissues, activity of phospholipase A2 may be rate limiting for eicosanoid production. Oxygenation and cyclization of free arachidonate are catalyzed by a microsomal enzyme known as PGH synthase-1 (also known as cyclooxygenase or COX-1). A cyclic endoperoxide, PGG2, then forms and is reduced to PGH2 via peroxidase activity in COX-1. Although endoperoxides are biochemically active, they are transformed to eicosanoid end products within 5 min. The major and most important COX-1 product of endothelial cells is prostacyclin (PGI2), which is catalyzed by the isomerase PGI-synthase. Other endothelial cell isomerases catalyze formation of PGE2a, and PGD2. PGD2 acts in a manner similar to PGI2, and on the same receptor. With a half-life of 3 min, PGI2 undergoes chemical hydrolysis to 6-keto-PGF1a. The synthesis of PGG2/H2 is common to many tissues, but subsequent processing is specific for a given tissue. For example, in platelets, thromboxane-synthase catalyzes metabolism of PGH2 to thromboxane A2 (Fig. 114-2).2,10,15,16,17,18 and 19 Endothelial cells do not contain cytosolic enzymes that catalyze oxygenation of arachidonate to lipid hydroperoxides, i.e., the lipoxygenases. However, via transcellular metabolism, endothelial cells take part in production of lipoxygenase products.1,2,19

FIGURE 114-2 Transformation of released arachidonate to prostaglandins and prostacylin in endothelial cells as catalyzed by cyclooxygenase enzymes.29,284 In response to prothrombotic or inflammatory stimuli, the essential fatty acid arachidonate is released from cell membrane phospholipids by phospholipase A2 (PLA2). Regulatory enzymes of this pathway are the cyclooxygenase, COX-1, which functions mainly in the endoplasmic reticulum, and COX-2 which functions principally in the nucleus. COX-1 and COX-2 catalyze insertion of two molecules of oxygen into arachidonate to form endoperoxide PGG2, which is then peroxidized to the endoperoxide PGH2. The latter is the precursor common to all eicosanoids. The endothelial eicosanoids are depicted herein.

The cloning of an early response gene from 3T3 fibroblasts demonstrated that the cDNA was highly homologous to COX-1.20 It then became apparent that there are two forms of COX, COX-1 (constitutive) and COX-2 (induced as an intermediate-early gene in monocytes, macrophages, neutrophils, and endothelial cells).21 COX-2 is inducible in endothelial cells by prothrombotic, inflammatory, or mitogenic stimuli and in neutrophils by inflammatory stimuli.22,23,24,25,26,27 and 28 In a given species, there is approximately 60 percent homology between deduced amino acid sequences. COX-1 contains 576 residues as compared to 587 for COX-2. There is a C-terminal sequence of 18 amino acids in COX-2 that is absent in COX-1. Antibodies directed at this C-terminal sequence can identify COX-2 by Western blot. The catalytic activities of both COX enzymes are similar, and all amino acids critical for COX-1 activity are conserved in COX-2. The active site in COX-1 is, however, slightly larger than that of COX-2, which is important for design of COX inhibitors. COX-2 contains mannose and an additional N-glycosylation site at the 18–amino acid C-terminal sequence. An N-glycosylation site at Asn410 is required for COX-1 to fold into an active conformation. The gene for COX-1 is located on chromosome 9 and is 22 kb long, and the gene for COX-2 is located on chromosome 1 and is 8 kb long. Transcription of COX-2 proceeds via several signaling mechanisms, including cAMP/protein kinase A, protein kinase C, tyrosine kinases, and pathways activated by growth factors, endotoxin, and cytokines.21,29,30
Activity of PGI2 as a muscle relaxant can be demonstrated following infusion of the parent molecule or its analogs. The inhibitory action of PGI2 and analogs is due to an interaction with its receptor on vascular smooth muscle cells and platelets. The biochemical effects of PGI2 are mediated mainly through G proteins and result in an increase in intraplatelet concentrations of cyclic AMP9 that lead to abolition of shape change, absence of platelet secretion, and impaired binding of von Willebrand factor and fibrinogen to the platelet surface. PGI2 also inhibits platelet adhesion to subendothelium—especially at high shear rates.9 Decreased PGI2 production has been described in thrombotic thrombocytopenic purpura (TTP).31 Thus far, it has not been possible to utilize prostacyclin or its analogs as therapeutic agents because of side effects, such as diarrhea.17
Nitric oxide is a colorless gas, slightly soluble in water and highly reactive with O2 to form nitrogen dioxide, NO2. It has a half-life of about 6 s and can also form the stable cation NO+. NO is the first gas to be characterized as an intracellular messenger. Nitric oxide accounts for the action of what was originally termed the endothelium-derived relaxing factor (EDRF).32,33 Upon release from endothelial cells, NO induces vasodilation, regulates normal vascular tone, and inhibits platelet aggregation. Overproduction of NO may be involved in the hypotension that accompanies endotoxic shock. In the pulmonary bed, abnormally low levels of NO may contribute to the etiology of pulmonary hypertension.34
In vascular endothelial cells, NO is formed from L-arginine by nitric-oxide synthase (NOS) in the presence of NADPH and oxygen.33 L-arginine is then converted to citrulline and nitric oxide. In endothelial cells, the isoform of NO synthase (eNOS or the NOS3 gene product) functions constitutively, but is further activated by receptor-dependent agonists that elevate intracellular calcium. Important stimuli include ADP, thrombin, bradykinin, and shear stress.9 Shear forces induce transcriptional activation of the eNOS gene because its promoter contains a shear response consensus sequence (GAGACC).9 The NO that forms activates guanylate cyclase, thereby generating cyclic GMP. NO is then oxidized to nitrite and then to nitrate, which can be measured in blood samples. Nitric oxide in the circulation is rapidly inactivated by erythrocytes.34 Inhalation of nitric oxide has a vasodilatory effect on the pulmonary vasculature. In patients with congestive heart failure and pulmonary congestion, NO inhalation decreases pulmonary hypertension and increases pulmonary ventilation.35
Interestingly, production of NO by endothelial cells appears to be impaired in the presence of the thiol-containing amino acid, homocysteine. Cynomolgus monkeys with diet-induced hyperhomocysteinemia (11 µM) showed reduced blood flow in the lower extremity and an impaired response to endothelial-cell-dependent vasodilators.36 Similarly, production of NO by endothelial cells in vitro is significantly inhibited in the presence of homocysteine,37 possibly by a mechanism involving impairment of the enzyme glutathione peroxidase.38
There are two groups of isoforms of NOS: One is constitutively synthesized and regulated by Ca2+ and calmodulin. The second is cytokine-inducible and posttranscriptionally regulated.32 Most NOSs are cytosolic, whether inducible or constitutive. A membrane-bound, constitutive NOS isoform containing a myristoylation consensus sequence has been isolated from bovine aortic endothelial cells.32 Endothelial NOS is Mr 144,000 and shares 57 percent amino acid sequence identity with neuronal NOS. The cofactor (6R)-tetrahydro-L-biopterin (H4B) participates in inducible and constitutive NOS isoform reactions. It is thought that H4B stabilizes the enzyme in a manner allowing for maximum activity of the NOS subunit to which the pterin binds.32 Biological reactions controlled by NO include vasodilation, regulation of normal vascular tone, and inhibition of platelet aggregation.39
Platelet aggregation and serotonin secretion in response to thrombin can be blocked via formation of endothelial NO.40 This action of NO as an inhibitor is unaffected by aspirin, indicating that it is not due to participation of endothelial eicosanoids.40
In addition to the constitutive isoform of NOS (eNOS, the NOS3 gene product), endothelial cells stimulated by agonists such as cytokines will express the inducible form of NO synthase, iNOS, which is the NOS2 gene product. In this manner, NO can further inhibit platelet reactivity and reduce basal vessel tone via relaxation of vascular smooth muscle. In biochemical terms, this is due to binding of NO to the heme prosthetic group of guanylyl cyclase. The inhibitory effect of NO on platelet secretion can be monitored by surface expression of P-selectin. The ability of NO to inhibit mobilization of intracellular platelet calcium results in reduction of the conformational changes in platelet membrane GPIIb/IIIa—a requirement for fibrinogen binding and subsequent aggregation. Other effects of NO, such as inhibition of leukocyte adhesion to the endothelial surface, inhibition of smooth muscle cell migration, and reduction of smooth muscle cell proliferation, all suggest that secretion of NO into the microenvironment is a major component of the response to vascular injury.9,11
In addition to producing two important vasodilators (PGI2, NO), endothelial cells also synthesize endothelins—potent vasoconstrictors. Endothelins are a group of 21–amino acid peptides, produced in a broad spectrum of cells.9 Endothelin-1 (ET-1) is not stored in the cell but forms from an inactive precursor, preproendothelin-1. Shear stress, hypoxia, or ischemia induce transcription of the gene encoding preproendothelin-1. Preproendothelin-1 is cleaved by an ET-1-converting enzyme, thereby forming the active peptide. ET-1 emerging from the activated endothelial cell binds to a G-protein-coupled receptor in smooth muscle. This binding increases the cytosolic calcium concentration, thus promoting smooth muscle contraction. When concentrations of other thromboregulators, such as NO, are decreased, the action of ET-1 may be amplified and produce greater vasoconstriction.
Excessive ET-1 has also been implicated in the hepatorenal syndrome—a form of renal failure observed in patients with severe liver disease. This disorder is characterized by intense and prolonged renal vasoconstriction. The hypoxia, oxidant injury, and endotoxemia that commonly characterize end-stage liver disease are probably the stimuli for endothelin production. The renal vasoconstriction has been attributed to activation of the sympathetic and renin-angiotensin systems in the kidney.9,41,42
In addition to the platelet inhibition by PGI2 and NO, endothelial cells can inhibit platelet function by the action of endothelial cell CD39, an ectoenzyme with ADPase and ATPase activities.7,43 The enzyme belongs to the E-type ATP diphosphohydrolase (ATPase) family, members of which degrade nucleotide tri- and/or diphosphates (apyrase, EC3.6.1.5).1,6,7 and 8,43 A soluble CD39 molecule has been generated by removing the NH2-terminal and COOH-terminal portions of CD39, including the two transmembrane regions.8 ADP released from activated platelets is metabolized by CD39, thereby inhibiting ADP-induced platelet activation, release, and aggregation (Fig. 114-3). Recombinant soluble CD39 can also inhibit ADP-induced platelet aggregation in vitro, and intravenous injection in mice decreases platelet aggregation in response to ADP and other agonists. It thus has potential as an antithrombotic agent.

FIGURE 114-3 Blockade of ADP-induced platelet reactivity by purified soluble CD39. The response to increasing concentrations of ADP is shown on the left. The response to ADP as inhibited by increasing quantities of purified soluble CD39 is shown on the right. The experiment demonstrates that blockade of platelet reactivity by purified soluble CD39 is far greater than the reduction of platelet activation when ADP as an agonist is diluted 10-fold. Thus, in the presence of only 3.3 µg/ml soluble CD39 platelet aggregation induced by 10 µM ADP was abruptly terminated, with the curve rapidly returning to baseline.8

The protein C pathway plays a critical role in the prevention of thrombosis as described in detail in Chap. 113. This pathway is initiated on the endothelial cell surface when thrombin combines with the endothelial receptor protein thrombomodulin (TM). Although thrombin is capable of slowly activating protein C, this reaction is markedly inhibited in the presence of physiologic concentrations of calcium ions. Once thrombin is bound to TM, the rate of protein C activation is dramatically enhanced44 and is dependent on the presence of calcium. The detailed biochemistry of this activation reaction has been reviewed elsewhere.45,46 In large vessels, an additional protein, the endothelial cell protein C receptor, can bind protein C and further augment its activation by the thrombin–TM complex.47 Presumably the activated protein C (APC) can dissociate from EPCR and interact with protein S on either the endothelial cell or platelet surface to exert its function. The function of APC per se will not be discussed here, as it can be found in detail elsewhere in this book (see Chap. 113) and in other reviews.45,46,48
In addition to functioning as a cofactor for protein C activation, TM has many other effects on thrombin. When thrombin is bound to TM, it is no longer able to clot fibrinogen, activate platelets, activate factors V and VIII,45 or interact with the protease-activated receptors.49,50 Thus, TM acts as a direct anticoagulant. The rates of inactivation of thrombin by its inhibitors antithrombin III (ATIII) and protein C inhibitor (PCI) are also enhanced when thrombin is complexed with TM.51 This leads to an estimated half-life of thrombin in the complex of 2 to 3 s.
TM also has functions that appear to oppose its many anticoagulant functions. TM promotes the activation of a procarboxypeptidase B by thrombin. This carboxypeptidase, also referred to as thrombin activatable fibrinolysis inhibitor or TAFI,52 causes partial inhibition of fibrin degradation by plasmin, presumably by removing carboxy-terminal lysine residues from fibrin, thereby decreasing the binding of fibrin to certain forms of plasminogen and plasmin (see Chap. 116). The carboxypeptidase B may also inactivate other vasoactive substances by a similar mechanism. TM also accelerates the proteolytic inactivation of prourokinase by thrombin,53,54 which may affect both fibrinolysis and tissue remodeling.55 Despite these antifibrinolytic affects of TM, many in vivo experiments have demonstrated that soluble TM infusion results in a net antithrombotic and/or anti-inflammatory effect.51 Thus, the physiological effect of thrombin–TM mediated activation of TAFI or inactivation of prourokinase remains unknown at this time.
There is intriguing evidence that, independent of its effect on hemostasis, TM plays a crucial role in development. Thus, when the TM gene is deleted by homologous recombination in mice, embryos die on day 8.5, prior to the development of a functional cardiovascular system,56 implying that TM has functions in addition to its anticoagulant and fibrinolytic properties. In support of this proposal, animals carrying mutations of TM that greatly reduce protein C activation, but leave other regions of the molecule intact, are viable.57 Strong expression of TM has also been observed on neural crest cells of the developing mouse embryo58,59 and on keratinocytes,60,61 indicating other possible roles outside of the cardiovascular system.
Either on the cell surface or after cleavage from the surface, TM may have pro-atherogenic properties. A soluble form has been found to be mitogenic for Swiss 3T3 fibroblasts62 and smooth muscle cells63 in culture. Although the expression of TM is usually limited to vascular endothelial cells, TM has been observed histochemically on vascular smooth muscle cells and monocytes within the vessel wall of atherosclerotic lesions.63 In this milieu, in which anticoagulation may not be a major function, the reported mitogenic properties of TM may dominate, leading to increased smooth muscle cell proliferation and exacerbation of the atherosclerotic lesion. Concurrently, atherogenic stimuli, such as oxidized LDL,64 have been reported to transcriptionally downregulate TM in endothelial cells. Homocysteine, another atherogenic stimulus, has multiple effects on endothelial TM expression, both at the nucleic acid and protein level (reviewed in Lentz65), which overall appear to downregulate TM function. These effects may increase the thrombotic damage in the area of the atheroma, leading to extension of the lesion at the luminal surface.
TM expression on smooth muscle cells can also be protective. In cases of injury in which the endothelial layer is removed, such as in rupture of an atheroma, the underlying smooth muscle cells can act as a site for focal clot formation. However, within a relatively short period of time, the cells undergo passivation, i.e., they no longer support platelet adhesion or fibrin formation.66 Evidence is accumulating that a contributor to this phenomenon may be the induction of TM expression on the smooth muscle cells.66,67 and 68 In this case, in the presence of flowing blood, the anticoagulant properties dominate, limiting thrombus formation until the wound is reendothelialized. Smooth muscle cell TM may also desensitize the smooth muscle cells to the mitogenic properties of thrombin.49
The domain structure of TM, as shown in Fig. 114-4,51,69,70 was deduced from the cloned cDNA for the protein. The gene for TM is intronless and is located on chromosome 20. The amino-terminal 226 residues of the mature protein show weak homology to lectinlike domains, such as the one found in the asialoglycoprotein receptor, and may be involved in the constitutive internalization of the receptor. This region is followed by six epidermal growth factor (EGF)-b type repeats. EGF domains 5 and 6, in particular EGF5, contribute most of the binding affinity for thrombin and can block fibrinogen cleavage by thrombin, although this region is not able to support protein C activation. It has been speculated that EGF5 does not exhibit the canonical disulfide bonding pattern.71 However, this is based on protein expressed in yeast, and the bonding pattern of mammalian expressed protein has not been determined. Since all isomers are capable of binding thrombin, there is uncertainty as to the disulfide bond pattern in native TM. Although EGF45 can promote activation of protein C by thrombin, rates approaching those of the intact TM molecule require the linkage region between EGF3 and EGF4, in addition to the entire fourth, fifth, and sixth EGF modules. EGF3 is required for catalysis of TAFI activation.72 Human TM contains a methionine in position 388 (between the fourth and fifth EGF domains) which is quite sensitive to oxidation, leading to inactivation of the molecule.73

FIGURE 114-4 Structure of the endothelial cell receptors involved in the protein C pathway. The domain structure of thrombomodulin (TM) and the endothelial protein C receptor (EPCR) are illustrated. Structures shown on the Ser-Thr-rich region of TM represent chondroitin sulfate attachment. The zigzag structure at the carboxyl terminus of EPCR represents the palmitic acid modification. See text for detailed descriptions. (Modified figure reprinted with permission from J Biol Chem 264:4743, 1989; copyright the American Society of Biochemistry and Molecular Biology, Inc., 1989.)

Following the EGF domains is a 34-residue region rich in serine and threonine. This domain contains several O-linked glycosylation sites as well as two potential chondroitin sulfate attachment sites. This region is apparently elongated and serves the function of a spacer, positioning the binding sites of TM appropriately above the cell surface. The presence of chondroitin sulfate in this region has several functional consequences. It enhances the affinity of TM for thrombin, facilitates inhibition of thrombin by ATIII and PCI, modulates the calcium dependence of protein C activation, and is directly involved in platelet factor 4 modulation of protein C activation.70 Both biochemical74 and electron microscopic75 experiments indicate that the glycosaminoglycan chain can bind a second molecule of thrombin. There was debate for some time as to whether naturally occurring human TM contains chondroitin sulfate.76 However, human placental TM has now been found to contain chondroitin sulfate.77 Addition of chondroitin sulfate can be variable,78 and it has been suggested that this could lead to functionally different TMs in different vascular beds.78,79
The 23-residue hydrophobic region corresponding to the transmembrane domain is the most highly conserved domain of TM among species.80 This suggests it may have an important, specific function, although none has yet been detected. The cytoplasmic domain of 38 residues contains several potential phosphorylation sites, one of which has been observed to be phosphorylated following cell stimulation with phorbol myristate acetate.81
Since total protein C deficiency leads to embryonic or neonatal death, it could be presumed that total TM deficiency, wherein little or no protein C could be activated, would be similarly lethal even if embryos survived to birth. As already stated, TM knockout mice die before the cardiovascular system develops.56 Other strategies have therefore been employed to determine the effect of chronic, decreased TM functional expression. These include heterozygous TM-deficient animals (TM–/+), knock-in mice in which Glu387 is replaced with proline,57 resulting in a molecule with markedly decreased affinity for thrombin and extremely low protein C activation activity (TM–/Pro and TMPro/Pro) and chimeric animals,82 in which focal areas of vessels are null for TM while the surrounding areas are normal. The pattern of fibrin deposition in these animals varied both in terms of amount of fibrin deposited (spontaneously or in response to hypoxia) and the organ distribution of those deposits. A striking observation was that none of the mutant animals, even those with very low expression of TM measured by protein C activation capacity, exhibited generalized thrombosis. Fibrin deposits tended to be organ specific and focal. In addition, although the TMPro/Pro animals showed fibrin deposition in the microcirculation, the chimeric animals did not, even though there were areas devoid of TM. These observations together contribute to the concept that coagulation is controlled by the local microenvironment present in different organs and tissues. Other qualitative and quantitative differences between the models were also observed. However, there was a strong age dependence of the extent of vessel involvement and reactivity in the chimeric animals, supporting the hypothesis that age-related alterations in the vasculature and/or coagulation system can predispose individuals to thrombosis. It is not known whether age played a significant role in the TMPro/Pro or heterozygous null animals. It must be noted that both the direct antithrombin and the protein C activation functions of TM are compromised in all these animals. It is not known whether one or several TM properties are responsible for the observed phenotypes.
The TM gene contains both a cAMP-responsive element in its 3′ untranslated region83 and a retinoic acid response element in its 5′ untranslated region.84,85 Agents that increase cAMP levels intracellularly increase TM expression,83,86,87 as do retinoids. In addition, these same effectors, as well as IL-488 and vascular endothelial growth factor (VEGF),89 will blunt, if not totally block, the effect of suppressors of TM expression.85,90 It is unclear whether protein kinase C-controlled pathways are involved in regulation of TM.87,91,92 The effects of active phorbol esters are biphasic.87 Heat shock also leads to a biphasic response.93 The TM gene contains several tandem heat shock elements in its 5′ untranslated region. Although TM antigen does not change early in response to heat in human umbilical vein endothelial culture, the message decreases significantly for 6 h before it rises dramatically and then continues for at least 48 h. An increase in surface activity can be observed by 18 h of treatment. This is in marked contrast to the normal response of heat-responsive genes, in which the response occurs within 1 h of stress. In addition, the TM response does not attenuate as classic heat shock protein expression does. This would suggest that multiple regulatory mechanisms are operative. This augmentation of TM synthesis may serve to protect the vasculature from further thrombotic damage during an inflammatory response. In line with this concept, histamine,94 an additional inflammatory mediator, has also been reported to enhance TM synthesis.
Inflammatory mediators, in general, tend to decrease the function of TM, thereby sensitizing at least the involved areas of injury to thrombosis. These mediators include endotoxin, IL-1, tumor necrosis factor alpha (TNF-a),87 transforming growth factor beta (TGF-b),95 and viral infection.96 Hypoxia also leads to a decrease in TM expression,97 apparently through the cAMP-responsive element.98 Since hypoxia also increases VEGF production,99 which has been reported to induce TM expression,90 it is difficult to predict which pathway would dominate in any specific microenvironment in vivo.
Another mechanism by which TM expression can be attenuated is through endothelial cell interaction with activated leukocytes, such as those present during inflammation. TM is sensitive to proteolytic release products,100 and neutrophil elastase is the enzyme most commonly implicated in this process. TNF treatment increases the release of TM from endothelial cells by neutrophil elastase.101,102 and 103 Because of the proteolysis of TM in response to inflammation or endothelial injury, measurements of TM in plasma are believed to reflect endothelial injury in various disease states.104,105
Leukocytes may also modulate TM activity by oxidation of the critical methionine in the molecule.73,100 The major basic protein from the granules of eosinophils can also interact with TM to inhibit its protein C activation potential.106 The cationic protein released from platelet granules, platelet factor 4, seems to have opposite effects on TM. At least in vitro, platelet factor 4 can both inhibit the direct anticoagulant functions of TM107 and enhance protein C activation.108 Whether either of these effects are of significance in vivo is unknown.
The EPCR109 is a 220–amino acid, type 1 transmembrane protein.110,111 EPCR has two extracellular domains that show structural homology with the a and b domains of MHC class 1 molecules. Since there are 3 Cys residues in the extracellular domain, the possibility of cross-linking with another protein exists. The transmembrane domain contains two Gly-Gly sequences that are not commonly found in this region of membrane proteins. The cytoplasmic domain of human EPCR is only three amino acids long, Arg-Arg-Cys. The terminal Cys can be acylated with palmitate and this appears to have functional consequences.
The binding of protein C or APC to EPCR is mediated through the Gla domain of the ligands and requires nearly complete carboxylation of the Gla residues.51,70,110,111 Both proteins are bound with similar affinity, about 30 nM.109 Binding requires the presence of calcium and is tightened by the presence of magnesium ions. EPCR augments protein C activation by the thrombin–TM complex, primarily through decreasing the Km for protein C.112,113 Recombinant soluble EPCR binds the ligands with the same affinity as the cellular form and the soluble form can compete for binding by cellular EPCR, thereby inhibiting protein C activation on cells expressing EPCR. The enhanced activation observed on cells can be recapitulated by incorporation of both receptors (TM and EPCR) into phosphatidylcholine vesicles, indicating that additional cellular proteins or architecture are not required.77
When APC is bound to soluble EPCR, it is no longer capable of inactivating factor Va.114 Due to technical difficulties, it is not known whether APC bound to cellular or membrane-bound EPCR can still bind protein S or function as an anticoagulant. The APC–EPCR complex can still be inhibited by its protein inhibitors, PCI and a1-antitrypsin.114 Based on analogy with the thrombin–TM complex, it is tantalizing to hypothesize that a similar switch in substrate specificity occurs in the APC–EPCR complex, changing APC from an anticoagulant into an anti-inflammatory agent.115,116 The new substrates remain to be identified.
Control of the expression of EPCR is quite complex and is only beginning to be understood. The gene structure has recently been reported,117 and, although the entire promoter region has not yet been detailed, some of the regulatory elements in the 5′-flanking region have been described.48,110,111 Reporter genes driven by the proximal 220 bases of the promoter region show essentially endothelial-cell–specific expression. A potential thrombin response element is present at –337 to –343. When the region from –1080 to –700 is included in the reporter construct, expression is observed only in cells derived from large-vessel endothelium, not in those derived from microvascular or capillary beds. This is consistent with the observation that EPCR is expressed primarily on the endothelium of large vessels.
Inflammatory mediators affect EPCR expression and can result in the generation of soluble forms of the receptor.110,111 Endotoxin increases EPCR mRNA when given to rats, and the increase can be blocked by co-infusion of hirudin, indicating that the rise is most likely due to stimulation through the thrombin response element. Paradoxically, tissue culture experiments showed that although EPCR message was increased in response to thrombin, cellular EPCR protein was not. Instead, soluble EPCR produced by the action of a metalloproteinase could be recovered from the cell supernatants. When the rat system was reexamined, endotoxin was found to increase EPCR levels in the plasma, and this rise was blocked by hirudin. Soluble EPCR has also been found in significant levels in humans, and the concentration appears to be increased in certain disease states.118 The solubilization of EPCR could potentially decrease protein C function by: (1) decreasing the rate of protein C activation in large vessels because of the loss of endothelial cell TM enhancement and (2) competing for protein C binding to the surface of all endothelium. It is still possible that protein C activation is maintained by the very high concentration of TM in the microcirculation. Ectodomain shedding of membrane proteins is being recognized as a mechanism for the regulated release of bioactive agents,119 and it is possible that the released EPCR–APC complex may serve other functions.
Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor synthesized by microvascular endothelial cells.120 Three pools of TFPI exist in vivo—about 3 percent is platelet associated, about 10 percent circulates in plasma in association with lipoproteins, and about 85 percent remains associated with the endothelial cell surface.121 TFPI has a heterogeneous molecular mass (Mr of 34,000 to 42,000) owing to its susceptibility to proteolytic cleavage by neutrophil elastase. The protein consists of three tandem Kunitz-type inhibitory domains, and its major function appears to be to inhibit factor Xa as well as factor VIIa/tissue factor. Inherited deficiency states of TFPI have not been reported in humans, although low levels of heparin-releasable TFPI have been reported in young individuals with thrombosis.122 Absence of TFPI in genetically engineered mice results in embryonic lethality between days E9.5 and E11.5 due to yolk sac hemorrhage.123 Interestingly, this lethality is overcome in mice who simultaneously lack tissue factor.124 Although cytokines and endotoxin upregulate tissue factor in endothelial cells, these agents have only a slight stimulatory effect upon TFPI so that the prothrombotic effect of elevated tissue factor expression remains relatively unopposed.121
Plasmin, the major clot-dissolving protease, is formed upon the cleavage of a single peptide bond within the zymogen plasminogen (see Chap. 116). This tightly regulated reaction is strongly influenced by endothelial cells that produce plasminogen activators, plasminogen activator inhibitors, and fibrinolytic receptors. In this section, we will consider the role of the blood vessel wall in the regulation of plasmin generation and examine the possible roles that the endothelial cell fibrinolytic system may play in maintaining the patency of blood vessels.
In 1958, Todd demonstrated that fibrinolytic activity in human tissues is focally distributed, relating consistently to blood vessels, especially veins and venous sinusoids and, to a lesser extent, arteries.125 Todd later showed that this activity was localized to the wall of the blood vessel rather than its contents.126 Pandolfi later showed that plasminogen activator activity can be associated with certain individual extravascular cells.127
Cultured endothelial cells derived from umbilical vein, umbilical artery, pulmonary artery, and vena cava all synthesize tissue plasminogen activator (tPA), and the endothelial cell appears to be the principal source of tPA in blood.128 However, the pattern of tPA expression in vivo appears to be highly restricted to specific types of vessels and anatomic locations. This pattern of expression likely reflects the extreme heterogeneity of endothelial cells in vivo.129 In the baboon, neither tPA antigen nor tPA mRNA were detected in femoral artery or vein, carotid artery, or aorta, whereas positive signals were apparent in precapillary arterioles, postcapillary venules, and the vasa vasora ranging in diameter from 7 to 30 µm.130 In the mouse lung, similarly, all bronchial blood vessels displayed endothelial-cell–associated tPA antigen, whereas pulmonary blood vessels were uniformly negative.131 Expression of tPA at branch points of pulmonary blood vessels may reflect stimulation by laminar shear stress.132
Although in vitro studies suggest that tPA expression in cultured endothelial cells is regulated by a wide array of factors, only a few of these have been evaluated in vivo. Thrombin,133 histamine,134,135 oxygen radicals,136 phorbol myristate acetate,137 DDAVP,138 and butyric acid liberated from dibutyryl cAMP139 all increase tPA mRNA in the cultured endothelial cell. Both thrombin and histamine appear to act via a receptor-mediated activation of the protein kinase C pathway.128 Laminar shear stress stimulates both tPA secretion140 and steady-state mRNA levels.141 Hyperosmotic stress and repetitive stretch also enhance tPA expression.142,143 In addition, differentiating agents such as retinoids144,145 and butyrate139 stimulate transcription of tPA in endothelial cells in vitro.
In vivo, the circulating half-life of tPA is approximately 5 min. Infusion of DDAVP, bradykinin, platelet activating factor (PAF), endothelin, or thrombin is associated with an acute release of tPA, and a burst of fibrinolytic activity can be detected within minutes.128 In the mouse lung, exposure to hyperoxia leads to 4.5-fold up-regulation of tPA mRNA in small-vessel endothelial cells.131 In humans, infusion of TNF into patients with malignancy is associated with an increase in tPA,146 while treatment of cultured endothelial cells with TNF either has no effect or decreases tPA production.147 Deficient release of tPA in response to venous occlusion in humans has been associated with deep venous thrombotic vascular disease,148 as well as atrophie blanche and other cutaneous vasculitides.149
In vivo, urokinase plasminogen activator (uPA) is not a product of resting endothelium,150 but is produced primarily by renal tubular epithelium.151 Expression of uPA mRNA in endothelium, however, is strongly stimulated during wound repair and physiologic angiogenesis within ovarian follicles, corpus luteum, and maternal decidua.152 Endothelial cells passaged in culture do synthesize uPA,153 and expression of its mRNA is stimulated by TNF by 5- to 30-fold.154 Small increases in uPA have also been observed in vitro in response to IL-1 and lipopolysaccharide (LPS).155,156 and 157
The association of uPA with the blood vessel wall may be at least partly reflective of uPA’s association with the uPA receptor, uPAR. In the adult mouse, uPAR mRNA is not normally detected by in situ hybridization in the endothelium of either large or small blood vessels.158 However, upon stimulation with endotoxin, expression is detected in endothelium lining aorta, arteries, veins, and capillaries of a variety of organs including heart, kidney, brain, and liver,158 whereas the same stimulus leads to a dramatic decrease in expression in the renal tubules.151
Plasminogen activator inhibitor (PAI)-1 is likely to function as a major regulator of plasmin generation in the vicinity of the endothelial cell. In vitro, PAI-1 appears to be associated mainly with the substratum of cultured human umbilical vein endothelial cells, rather than the external face of the plasma membrane.159,160 Thrombin, IL-1, TGF-b, TNF, and endotoxin all induce dramatic increases in steady-state PAI-1 message levels.133,155,156,161 In addition, the low-density lipoprotein-like particle, lipoprotein(a) [Lp(a)], which contains an apoprotein homologous to plasminogen, also induces a two- to fourfold increase in PAI-1 mRNA without affecting mRNA for tPA.162 Heparin-binding growth factor 1 (endothelial cell growth factor) is recognized as a down-regulator of PAI-1 mRNA production by cultured endothelial cells; this agent has no effect on tPA.163 These studies suggest that in vitro synthesis and secretion of PAI-1 by the endothelial cell may be regulated independently of tPA.
In vivo, quiescent endothelial cells express little or no PAI-1, the liver being the major source of plasma PAI-1. During in vivo decidual neovascularization in the ovary, PAI-1 seems to be expressed near capillary sprouts that also express uPA.152 In addition, inflammatory cytokines are powerful stimuli for induction of PAI-1 in a variety of tissues, including liver.164 In both rats and humans with active malignancy, injection of TNF results in a striking increase in plasma concentrations of PAI-1.128,146
In contrast, the endothelial cell coreceptor for tPA and plasminogen, annexin II, appears to be expressed constitutively in vivo in association with blood vessels in a wide variety of tissues. In the adult chicken, endothelial cells of vessels in the dermis, lung, renal glomeruli, pancreas, liver, and meninges stain intensely positive by immunohistology.165 Blood vessels of the developing mouse brain are also strongly cross-reactive,166 and in both rats167 and humans,168 vascular endothelial cells are positive for annexin II in all tissues studied so far.
Accumulating evidence suggests a potential anticoagulant role for plasmin based on its ability to specifically modify cofactors important in the thrombin-generating cascade. Plasmin has been shown to inactivate bovine factor Va in vitro by cleaving both the heavy and light chains of this Mr 168,000 protein.169 This lipid-dependent inactivation results in a series of plasmin-specific cleavages that are distinct from those produced by activated protein C.170 The inactivation of human factor V by plasmin may be preceded by transient generation of procoagulant fragments that are subsequently degraded to inactive form.171 Plasmin can also inactivate factor VIIIa, another coagulant cofactor that is structurally homologous to factor Va.172 Factor X, finally, is subject to a well-defined pattern of cleavage events, some of the products of which may stimulate tPA-dependent plasminogen activation.173
The effects of plasmin on in vitro platelet function are complex. Platelet glycoproteins IIb/IIIa and Ib, the cell surface receptors for fibrinogen and von Willebrand factor respectively, are both plasmin substrates.174,175 Thus, plasmin formation in the vicinity of a hemostatic plug could lead to impaired adhesion and poor aggregation in response to agonists. Plasmin generation has been shown to be associated with both platelet activation176,177 and platelet inhibition178 or disaggregation.179 The ultimate effect of plasmin appears to depend upon the incubation conditions, particularly the dose and duration of plasmin treatment. These findings are of potential significance in view of reports that plasminogen can interact with platelets in a manner that is enhanced upon thrombin-mediated conversion of platelet fibrinogen to fibrin.180,181 In vivo, prolonged bleeding times were found in patients 90 min after tPA infusion for thrombolysis, suggesting early impairment of platelet function upon plasmin generation.182 However, there is also evidence that platelets may play a role in thrombotic reocclusion following successful thrombolytic therapy.183
Several types of evidence suggest that dysfunction of the fibrinolytic system may be associated with atherosclerotic vascular disease. Elevated levels of circulating PAI-1, for example, have been epidemiologically connected to risk for myocardial infarction.148 uPA receptor expression appears to be significantly up-regulated in the intima versus media of atherosclerotic coronary arteries, suggesting a role for plasmin in smooth muscle cell migration.184 Circulating levels of TGF-b are reduced in individuals with atherosclerosis, possibly reflecting impaired activation by plasmin.185 However, the exact role of plasmin and its activators in occlusive vascular disease remains to be elucidated.
The role of plasmin in the evolution of the atherosclerotic lesion is presently unclear. Plasminogen deficiency in mice (PLG –/–) is associated with impaired healing of cutaneous wounds186 and reduced migration of monocytes to sites of inflammation.187 Such wound healing responses appear to depend largely on the fibrinolytic action of plasmin, since loss of fibrinogen rescues this defect.188 Upon electrical injury to the femoral artery of wild-type mice, a wound-healing response is initiated such that a neointima forms consisting mainly of smooth muscle cells that migrate to the intima from the media. In PLG –/– mice, this response is significantly attenuated. These results lead to the conclusion that plasminogen plays a significant role in vascular wound healing and arterial neointima formation, possibly by mediating cellular migration.189 On the other hand, mice doubly deficient in plasminogen and apolipoprotein(e) [ApoE] show a predisposition to atherosclerosis compared to animals deficient in either ApoE or plasminogen alone.190 Thus, plasmin may also play an important protective role, possibly related to degradation of fibrin. Thus, the role of plasmin in either accelerating or preventing vascular disease appears to be both complex and highly context-dependent.
As discussed in Chap. 116, there is abundant in vitro evidence to support the concept that plasminogen and plasminogen activators can assemble on cell surfaces and that assembly enhances the potential for plasmin activation. The major endothelial cell fibrinolytic receptors are uPAR and annexin II (Fig. 114-5); uPA bound to uPAR on endothelial cells is relatively protected from PAI-1 and PAI-2. However, there are as yet no known human states of deficiency or dysfunction of uPAR that relate to vascular disease. On the other hand, the profibrinolytic function of annexin II, an endothelial cell coreceptor for plasminogen and tPA, is attenuated by two atherothrombotic agents, Lp(a) and homocysteine. The assembly of tPA and plasminogen on annexin II is reviewed in detail in Chap. 116.

FIGURE 114-5 Two-dimensional representation of the major endothelial cell fibrinolytic receptors. Annexin II consists of an amino-terminal tail domain (~3 kDa) and a carboxyl-terminal core domain (~33 kDa).285,286 The tail domain contains a binding site for tPA. The core domain is composed of four homologous annexin repeats, each consisting of five a-helical regions (A through E) that contribute to potential calcium-dependent phospholipid binding sites. Current evidence indicates that repeat 2 is important for the interaction of annexin II with the endothelial cell surface while plasminogen binds to lysine residue 307 within helix C of repeat 4. UPAR is a 55- to 60-kDa, glycosylphosphatidylinositol-linked protein that consists of three disulfide-linked domains.287 Domain I contains the uPA-binding sequences, while domains II and III appear to mediate the receptor’s interaction with matrix proteins such as vitronectin.

Lp(a) is a low-density lipoprotein (LDL)-like particle that is an independent risk factor for atherosclerosis.191,192,193 and 194 Lp(a) contains, in addition to apolipoprotein B-100, a disulfide-linked moiety called apolipoprotein (a) [Apo(a)]. Apo(a) shares a remarkable degree of homology with plasminogen, including multiple tandem repeats of domains similar to kringle IV, a single region resembling kringle V, and a pseudoprotease segment.195 Plasminogen and Apo(a), furthermore, are genetically linked on chromosome 6 and may have arisen from a common ancestral gene.196
While Lp(a) levels are, at best, only transiently responsive to diet, heredity may play a more important role in regulating fibrinolytic potential at the endothelial cell surface.197,198,199,200,201 and 202 In general, plasma Lp(a) concentrations seem to correlate inversely with the ratio of kringle IV to kringle V encoding domains within the Apo(a) gene.203 Thus, the larger the Apo(a) gene product, reflected in a greater number of kringle IV domains, the lower the concentration of Apo(a) in plasma. In addition, Lp(a) appears to represent an acute phase reactant in the postsurgical and postmyocardial infarction setting200 and in patients with cancer,201 suggesting a role for soluble inflammatory mediators in regulating its synthesis or assembly. The high-affinity lysine-binding site within kringle 1 of plasminogen possesses a crucial tetrad of amino acids consisting of anionic Asp55 and Asp57 plus cationic Arg34 and Arg71.204 Kringle 4 of plasminogen, which lacks one of the four key residues (Arg34),205 contains a lysine-binding site of intermediate affinity.204,206,207 and 208 Kringle 37 of the originally cloned ApoA resembles plasminogen kringle 4 in that it possesses amino acids corresponding to three of the four lysine binding site residues (Asp55, Asp57, and Arg71), and many isoforms of Lp(a) have been found to have moderate lysine-binding affinity.209 In vitro, Lp(a) binds to plasmin-treated fibrin210 and colocalizes histologically with fibrin in atheromatous tissue.211 Since the cell-binding activity of plasminogen is lysine-binding-site-related, kringle 37 may play a role in the interaction of Lp(a) with cell surfaces as well.
There are three potential mechanisms whereby Lp(a) may exert a prothrombotic effect by inhibiting generation of plasmin: First, both Lp(a) and Apo(a) inhibit the binding of lysine-plasminogen (Lys-PLG) to endothelial cells (ID50 = 36-fold excess).212 Lp(a) also binds to annexin II in vitro213 and can inhibit 95 percent of plasminogen activation by tPA at the endothelial cell surface (Fig. 114-6). The estimated dissociation constants for Apo(a) and plasminogen with respect to the endothelial cell surface are comparable. This finding suggests that receptor occupancy in vivo is largely determined by the ambient level of Lp(a), since plasminogen concentrations do not appear to change significantly.212,214,215 Furthermore, anti-Lp(a) cross-reactive material can be detected within atherosclerotic lesions.212

FIGURE 114-6 Schematic representation of annexin II–mediated fibrinolytic assembly. Annexin II interacts with tPA and plasminogen (PLG) through specific domains located in the tail and core domains, respectively. A. Lipoprotein(a) [Lp(a)] competes with plasminogen for binding to annexin II, thereby serving as a potential modulator of plasmin generation. B. Homocysteine (HC) forms an adduct with cysteine 9 (C) within the tail domain, thus impairing tPA binding and reducing plasmin generation.

Second, treatment of cultured endothelial cells with Lp(a) is associated with enhanced functional, antigenic, and transcript levels of PAI-1 without a concomitant change in plasminogen activator activity or steady-state mRNA levels for tPA. Increases in PAI-1 were not found with LDL, Lp(-), or plasminogen162 but have been reported for very low density lipoprotein (VLDL).216
Third, Lp(a) may act as an inhibitor of plasminogen activators. In vitro, Lp(a) attenuates the plasmin-generating activity of streptokinase, a frequently employed thrombolytic agent, by a mechanism that involves direct competition with plasminogen for binding to streptokinase.217,218 In addition, Lp(a) can impair plasminogen activation by tPA by acting as a competitive inhibitor of tPA in the presence of fibrinogen,219 or as an uncompetitive inhibitor of the fibrin-dependent enhancement of tPA-induced plasmin generation.220 Experiments in transgenic mice suggest that cell-associated plasmin activity is reduced when Apo(a) is overexpressed221 and that these mice are resistant to lysis of an artificial thrombus by tPA.222
Lp(a) is not expressed in mammals other than primates and hedgehogs. However, when Lp(a) was overexpressed in mice receiving a high-fat diet, atherosclerotic lesions containing both lipid and anti-Apo(a) cross-reactive material were observed.223 Deposition of both lipid and Apo(a) was reduced in mice expressing ApoA in which lysine binding sites had been mutated.224 These data indicate that lysine binding sites of ApoA play a role in its atherogenicity in vivo, possibly by competing with plasminogen for cell surface binding sites.
Homocysteine is a thiol-containing metabolic intermediate that can accumulate in association with nutritional deficiencies of vitamin B6, vitamin B12, or folic acid, or in inherited abnormalities of cystathionine b-synthase, methylene tetrahydrofolate reductase, or methionine synthase.225 A meta-analysis of 27 studies including approximately 4000 patients showed homocysteine to be an independent risk factor for atherosclerosis of coronary, cerebral, and peripheral arteries.226 Of 10 subsequent prospective studies, 8 demonstrated an increased risk of coronary heart disease, venous thromboembolism, cardiovascular complications, and death in individuals with elevated homocysteine levels.227
Several in vitro studies suggest that homocysteine induces dysfunction of the endothelial cell, which is highly susceptible to oxidative stress. Evidence that homocysteine impairs the intrinsic fibrinolytic system of the endothelial cell comes from experiments in which homocysteine-treated endothelial cells bound about 50 percent less tPA than untreated cells, and activated about 50 percent less plasminogen.228 Electrospray ionization mass spectrometry studies indicate that homocysteine directly disables the tPA-binding domain of annexin II by forming a covalent adduction product with cysteine 9 within the tail domain of purified annexin II.229 Direct disulfide-mediated complex formation between homocysteine and annexin II can also be demonstrated in cultured endothelial cells. Homocysteine treatment of annexin II further inhibits its ability to bind tPA with half-maximal effect observed at about 11 µM, a value close to the upper limit of normal for homocysteine in plasma (14 µM). Thus, inhibition of tPA–annexin II assembly on the endothelial cell may contribute to the prothrombotic effect of homocysteine in vivo.
A proinflammatory environment is also prothrombotic. Endothelial cells express molecules that regulate binding of leukocytes to their surface during inflammation. These interactions have both direct and indirect roles in hemostasis and thrombosis, as many of the cytokines and bioactive molecules that promote the inflammatory response also trigger the former. Moreover, the inflammatory response itself results in the expression of adhesion molecules and mediators that secondarily promote hemostasis.
Histamine produced locally at the site of inflammation by degranulation of resident tissue mast cells stimulates the overlying endothelial cells to express P-selectin on their surfaces. This change occurs within minutes and is due to the rapid fusion of Weibel-Palade bodies with the plasma membrane bringing P-selectin to the surface. Along with P-selectin expression, fusion of the Weibel-Palade bodies with the membrane also results in the release of von Willebrand factor (vWF) into the local environment. In addition to its ability to induce exocytosis of platelet a granules and expression of P-selectin on the surface of platelets, thrombin can also trigger the release of P-selectin on the endothelial cell surface at sites of inflammation.
P-selectin serves as a receptor for P-selectin glycoprotein ligand 1 [PSGL-1] and probably other unidentified ligands located on leukocytes. PSGL-1 is a specific sialomucin containing sialylated, fucosylated O-linked oligosaccharides as well as an unusual sulfated tyrosine residue motif.230 Dimerization of PSGL-1 may be required for optimal recognition of P-selectin.231 Adhesive interactions between P-selectin and its ligands result in the tethering of passing leukocytes to, and rolling on, the surface of the endothelial cell—the first step in leukocyte emigration. L-selectin, another member of the selectin family of adhesion molecules, is constitutively expressed on the surfaces of most leukocytes. It binds to sialylated, fucosylated glycoprotein ligands expressed by endothelial cells in response to inflammation, as well as to CD34 constitutively expressed by cells of the high endothelial venules in peripheral lymph nodes.
The low-affinity reversible adhesions of leukocytes to the endothelium at the site of inflammation result in their rolling along the luminal surface. This stage serves to slow down the movement of leukocytes and bring them into contact with a variety of chemical mediators that trigger the next stage of leukocyte emigration—tight adhesion to the endothelial surface. These mediators include surface-bound chemokines,232 new adhesion molecules expressed by the endothelium in response to inflammatory cytokines,233 PAF,234 soluble chemokines,235 and ligands that cross-link leukocyte CD31.236,237 and 238 The variety of chemical signals that can trigger tight adhesion is large239 and may vary according to the nature of the inflammatory stimulus, the tissue involved, and the chronology of the response. However, they all seem to work by stimulating the activation of leukocyte integrin adhesion molecules by so-called inside-out signaling. This involves a conformational change and/or clustering of the two chains of these heterodimeric surface molecules such that the affinity or avidity respectively for their ligands on the surfaces of endothelial cells is increased.240 Where these ligands have been identified, they are members of a third family of adhesion molecules, the immunoglobulin gene superfamily.241 While immunoglobulin gene superfamily members have not been shown to undergo conformational change, there is evidence that the active form of intercellular adhesion molecule 1 (ICAM-1) is dimerized.242,243
Table 114-2 lists some of the more common leukocyte/endothelial cell adhesion molecule pairs participating in the inflammatory response. It is interesting to note that the mucosal addressin MAdCAM-1, a unique molecule expressed by endothelial cells of high endothelial venules of mesenteric lymph nodes and Peyer’s patches, has structural features of both a mucin and an Ig superfamily molecule. It can bind both L-selectin and the leukocyte integrin a4b7, expressed by a subset of memory T cells. It is believed to interact with L-selectin through its mucin [carbohydrate] domain and with a4b7 through its Ig domains. However, identified protein ligands for L-selectin [MAdCAM-1 and CD34] have been demonstrated to bind to L-selectin only in the context of lymphocyte homing. Their role in leukocyte rolling and adhesion in postcapillary venules during an inflammatory response has not been demonstrated.


In addition to being made and secreted acutely by leukocytes and mast cells at the site of inflammation, PAF is rapidly made and expressed on the surfaces of stimulated endothelial cells. PAF [1-alkyl-2-acetyl-sn-glycero-3-phosphocholine] is produced enzymatically from phosphatidyl choline in the plasma membrane. While its role in this environment as an activator of neutrophils has been established,234 it appears to be a relatively weak agonist of platelet activation in this location.
Examination of the rolling phenomenon in vivo by intravital microscopy shows that leukocytes may roll on other leukocytes that are already tightly adherent. These interactions, which are promoted through L-selectin and PSGL-1 on the leukocytes, amplify the inflammatory process.243,244
Adherent leukocytes migrate to nearby interendothelial junctions by repeated cycles of adhesion in the front and disadhesion in the rear.239,245 At the junction, yet another distinct molecular interaction between leukocytes and endothelial cells regulates transendothelial migration for the vast majority of neutrophils, monocytes, and natural killer (NK) cells. Platelet/endothelial cell adhesion molecule-1 (PECAM/CD31) on the leukocyte contacts the same molecule concentrated at the endothelial junctions in a homophilic manner.246,247 and 248 The relevant signals transduced by this interaction have not been worked out. However, a transient rise in the intracellular calcium ion content of the endothelial cell cytoplasm accompanies transmigration and is required for the process to proceed.249 Blocking the function of either leukocyte or endothelial cell PECAM-1 arrests the leukocyte poised over the junction, tightly adherent to the apical side of the endothelial cell,246,250,251 a phenotype very similar to that seen when the rise in endothelial cell intracellular calcium was blocked by the intracellular chelator, bis(2-amino-5-methylphenoxy)ethane-N,N,N’,N’-tetraacetic acid tetraacetoxymethyl ester (MAPTAM).249
Anti-PECAM reagents never block diapedesis completely, however, and PECAM-1-deficient mice do not have a significant defect in inflammation.252 Therefore, PECAM-1 independent pathways of transendothelial migration must exist. The leukocyte integrin a4b1 (VLA-4) and the aLb2/aMb2 (LFA1/Mac-1), and their endothelial counterreceptors, vascular adhesion molecule 1 (VCAM-1) and ICAM-1, have been implicated in transmigration.239 In addition, under certain specialized conditions, there appear to be pathways across the endothelial cell that bypass the intercellular junction.253
At the onset of most acute inflammatory responses there is a transient increase in vascular permeability due to histamine release. The endothelial junctions are soon reestablished, and the leukocytes that arrive at the scene over the next hour find the junctions closed. During diapedesis, leukocytes migrate in ameboid fashion across the junction between tightly apposed endothelial cells. Studies performed both in vivo and in vitro indicate that during diapedesis leukocytes penetrate the vessel wall without breaching the vascular permeability barrier.249,254 This prevents exposure of subendothelial collagen and vWF deposits to circulating platelets. While there is no known role for PECAM-1 in binding platelets to endothelial cells, it has been hypothesized to maintain the tight apposition of endothelial cells and leukocytes during diapedesis.248
In addition to stimulating immediate responses by endothelial cells, cytokines and inflammatory mediators released at the site of inflammation activate the surrounding endothelial cells to initiate new genetic programs. De novo synthesis of mRNA and protein leads to the establishment of an inflammatory phenotype within several hours of exposure to adequate levels of the mediator. The changes result in the endothelial cell developing a procoagulant and proadhesive phenotype.
Stimulated by inflammatory cytokines like TNF-a or IL-1, vascular endothelial cells express several important cell adhesion molecules on their surface. E-selectin expression is induced within hours of cytokine stimulation. Expression peaks at 4 to 6 h in vitro, but in the presence of interferon gamma (IFNg), and in vivo, expression is maintained over several days.255,256 E-selectin mediates rolling of leukocytes bearing sialylated, fucosylated carbohydrate receptors similar to sialylated Lewisx antigen. This molecule is important for the slow rolling seen in some vascular beds.257 While P-selectin expression on the endothelial cell surface stimulated by thrombin or histamine is transient, expression of P-selectin can be prolonged by IL-3,258 IL-4, or oncostatin M stimulation259 of human endothelium, and by TNF-a stimulation of murine, but not human, endothelium.260,261 Expression is often seen to last for hours to days. This prolonged expression requires de novo message and protein synthesis.
In general, expression of the immunoglobulin superfamily members ICAM-1 and VCAM-1 is induced by the same stimuli that induce E-selectin. Some specializations exist, at least in vitro. For example, IL-4 induces VCAM-1 but not E-selectin or ICAM-1 in microvascular endothelial cells.262,263 These molecules serve as counterreceptors for the leukocyte integrins in the tight adhesion step, as discussed above.
Prolonged stimulation of endothelial cells with interferon g leads to expression of MHC class II molecules (HLA-DR and DQ) on their surfaces. This takes several days in vitro. In human tissues such as skin and gut, class II is commonly seen even in the absence of overt inflammation and is thought to be due to chronic exposure of these sites to subclinical inflammation and antigenic stimulation. Cytokines can also induce the expression of CD40 ligand on endothelial cells. The significance of class II expression on endothelial cells is that when costimulatory molecules such as CD40, ICAM-1, or lymphocyte-function-associated antigen-3 (LFA-3) are also induced by inflammatory stimuli, the endothelial cell becomes capable (at least in vitro) of acting as an antigen-presenting cell that can stimulate CD4+ memory T cells. While this may not be a major threat in the normal host, when the endothelium belongs to an organ graft with foreign MHC class II, this mechanism may well stimulate graft rejection by the host.264,265 and 266
The expression of the adhesion molecule ICAM-2 does not change in response to inflammatory mediators. PECAM-1 shows a unique expression pattern in response to IFNg in vitro267 (also Muller, unpublished) and in vivo.268 The distribution, but not absolute amount, of PECAM on the surface changes as the molecule is no longer concentrated at intercellular borders but becomes expressed diffusely over the surface of the cell. In vitro chronic exposure of human umbilical vein endothelial cells to a combination of IFN-g and TNF-a at relatively high doses leads to a decrease in total PECAM-1 expression.269 It is possible that such a cytokine milieu could exist in vivo, but a similar phenotype has not been described to date.
In addition to the adhesive interactions germane to thrombosis and hemostasis, such an environment exposes leukocytes to ligands that promote their adhesion and recruitment to the vessel wall. For example, in vitro thrombin has been shown to induce E-selectin expression and IL-8 secretion by human umbilical vein endothelial cells.270 These changes are classically induced by inflammatory cytokines such as IL-1 and TNF-a. Table 114-3 lists some mediators that could have dual roles in inflammation and hemostasis/thrombosis.


Activated platelets bind to circulating lymphocytes in a P-selectin-dependent manner. This interaction can facilitate rolling on the endothelium271 and can even allow homing of lymphocytes to peripheral lymph nodes in the absence of L-selectin, since P-selectin on the adherent platelets will interact with the peripheral lymph node addressin.272 In vitro, neutrophils have been shown to be capable of rolling on immobilized platelets that have undergone the release reaction via PSGL-1 on the leukocyte interacting with P-selectin on the platelet surface.273 Moreover, following P-selectin-dependent rolling, aMb2 (CD11b/CD18)-dependent arrest and tight adhesion of neutrophils to bound platelets has been described.273,274 The platelet ligand for this is not known. ICAM-2 has been found on the surface of activated platelets,274 but it is not a ligand for aMb2, and in fact, antibodies against neither ICAM-2 nor its neutrophil receptor aL (CD11) blocked this adhesion.275 On the other hand, neutrophil aMb2 has been reported to bind to fibrinogen, which may be present on the surfaces of activated platelets bound to aIIbb3 (GPIIb/IIIa).
The same proinflammatory stimuli that stimulate de novo expression of E-selectin and VCAM-1, and augment expression of ICAM-1 for the recruitment of leukocytes, stimulate synthesis and expression of tissue factor (TF) by the endothelial cells.276 Furthermore, interaction of monocytes with endothelium stimulates production of TF in monocytes. Adhesion of monocytic cell lines to cytokine-activated endothelial cells in culture leads to a rapid increase in procoagulant activity due to induction of TF. This effect is partially blocked by a monoclonal antibody directed against E-selectin on endothelium and is mimicked by cross-linking LeX on the monocyte cell lines.277 A similar increase in TF gene expression can be induced by cross-linking a4 or b1 integrin chains, the components of VLA-4 on monocytic cell lines.278
A study of prolonged interaction of peripheral blood monocytes with human endothelial cells showed that within a few hours of transendothelial migration, monocytes in the collagen matrix expressed functional TF on their surfaces.279 Furthermore, over the next several days, approximately half of these monocytes had differentiated into immature dendritic cells, bearing even higher levels of TF, migrated back across the intact endothelial monolayer in the abluminal-to-luminal direction. Tissue factor on the surface of monocytes was involved in this migration, since it could be blocked by soluble fragments of TF. The same TF fragments blocked adhesion of endothelial cells to TF in vitro. Therefore, it was hypothesized that TF expressed by the emigrating dendritic cells is directly involved in an adhesive step of this process in addition to any procoagulant role it may play.279
Leukocytes bound to P-selectin exposed on the surfaces of platelets on adherent thrombi promote the conversion of fibrinogen to fibrin.280 The leukocyte integrin aMb2 has been shown to bind fibrinogen.281 The same integrin has a conformational form that binds coagulation factor X.282 Monocytic cells are capable of activating the bound factor X to Xa when activated,283 defining a pathway for activation of X that is independent of tissue factor.

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Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

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