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CHAPTER 116 MOLECULAR MECHANISMS OF FIBRINOLYSIS

CHAPTER 116 MOLECULAR MECHANISMS OF FIBRINOLYSIS
Williams Hematology

CHAPTER 116 MOLECULAR MECHANISMS OF FIBRINOLYSIS

KATHERINE A. HAJJAR

Basic Concepts of Fibrinolysis
Components of the Fibrinolytic System

Plasminogen

Plasminogen Activators

Inhibitors of Fibrinolysis
The Fibrinolytic Actions of Plasmin

Degradation of Fibrinogen and Fibrin

T-PA–Mediated Plasminogen Activation

U-PA–Mediated Plasmin Generation

Thrombin-Activatable Fibrinolysis Inhibitor
The Nonfibrinolytic Actions of Plasmin

Plasmin as a Tissue Remodeler

Angiostatin and Related Plasminogen Fragments
Disorders of Plasmin Generation

Fibrinolytic Deficiency and Thrombosis

Enhanced Fibrinolysis and Bleeding

Developmental Regulation of the Fibrinolytic System

Fibrinolytic Activity During Pregnancy and Puerperium
Chapter References

In recent years, several fundamental advances have enriched our understanding of the molecular mechanisms of fibrinolysis. Molecular characterization of the genes for all the major fibrinolytic proteins has led to an understanding of serine protease stucture and function, a regard for the regulatory role of cellular receptors and other cofactors, and an appreciation of mechanisms regulating transcriptional and posttranscriptional gene expression. The development of genetically engineered animals deficient in one or more fibrinolytic proteins has suggested unexpected roles for these proteins in both intravascular and extravascular settings. In addition, genetic analysis of human deficiency syndromes has revealed specific mutations that result in human disorders reflective of either fibrinolytic deficiency with thrombosis or fibrinolytic excess with hemorrhage. Finally, elucidation of acquired disorders of fibrinolysis, as seen in pregnancy, in the newborn infant, and in certain malignancies, has also served to clarify the role of the fibrinolytic system in both health and disease.

Acronyms and abbreviations that appear in this chapter include: a2-AP, a2-antiplasmin; bFGF, fibroblast growth factor; CRE, cyclic adenosine monophosphate (cAMP)-responsive element; Glu-PLG, glutamic acid plasminogen; GM-CSF, granulocyte-macrophage colony-stimulating factor; HC, homocysteine; IL-6, interleukin-6; LDL, low-density lipoprotein; LRP, LDL receptor–related protein; Lys-PLG, Lys-plasminogen; MMP, matrix metalloproteinase; PAI-1, plasminogen-activator inhibitor-1; TAFI, thrombin-activatable fibrinolysis inhibitor; TGF-b, transforming growth factor b; t-PA, tissue plasminogen activator; u-PA, urokinase plasminogen activator.

BASIC CONCEPTS OF FIBRINOLYSIS
Fibrin, the insoluble end product of the action of thrombin on fibrinogen, is found in both intravascular and extravascular settings. In response to injury, cross-linked fibrin, the final product of the coagulation cascade, is deposited in tissues and blood vessels. Once fibrin is no longer needed, the fibrinolytic system is activated, converting fibrin to its soluble degradation products through the action of the serine protease plasmin (Fig. 116-1a).

FIGURE 116-1 Overview of the fibrinolytic system. (a) Fibrin-based plasminogen activation. The zymogen plasminogen (PLG) is converted to the active serine protease plasmin (PN) through the action of tissue plasminogen activator (t-PA) or urokinase (u-PA). The activity of t-PA is greatly enhanced by its assembly with PLG through lysine residues (K) on a fibrin-containing thrombus. u-PA acts independently of fibrin. Both t-PA and u-PA can be inhibited by plasminogen-activator inhibitor-1 (PAI-1), which is released by endothelial cells and activated platelets. PAI-2, on the other hand, neutralizes u-PA more efficiently than t-PA. By binding to fibrin, plasmin is protected from its major inhibitor, a2-antiplasmin (a2-AP). Bound plasmin degrades cross-linked fibrin, giving rise to soluble fibrin degradation products (FDPs). Streptokinase (SK) is a bacterial protein that forms a complex with plasminogen, allowing it to activate other plasminogen molecules. Minor plasminogen activators include kallikrein (Kal), factor XI (XIa), and factor XII (XIIa). (b) Probable sites of cell surface plasminogen activation. On the blood vessel wall, endothelial cells, like monocytes and macrophages, express the u-PA receptor as well as annexin II, a coreceptor for t-PA and PLG that augments the efficiency of plasmin generation. Circulating monocytes and macrophages have cell surface a-enolase, and resting platelets express the glycoprotein IIb/IIIa complex, both of which represent potential receptors for PLG. (From KA Hajjar1 with permission.)

Under physiologic conditions, fibrinolysis is precisely regulated by the measured participation of activators, inhibitors, and cofactors.1 In addition, cell surface receptors provide specialized, protected environments where plasmin can be generated without compromise by circulating inhibitors (Fig. 116-1b).2 Endothelial cells, platelets, monocytes, macrophages, myeloid cells, and some tumor cells express protein receptor sites for plasminogen, tissue plasminogen activator (t-PA), and/or urokinase. The broad in vitro substrate specificity of plasmin suggests that it may play an important role in such extravascular events as the modification of growth and differentiation factors, matrix proteins, and procoagulant molecules. This chapter reviews the fundamental features of plasmin generation and considers the major clinical disorders resulting from defects in this system.
COMPONENTS OF THE FIBRINOLYTIC SYSTEM
PLASMINOGEN
Synthesized primarily in the liver,3,4 plasminogen is an approximately 92,000-Mr single-chain proenzyme that circulates in plasma at a concentration of approximately 1.5 µM (Table 116-1).5 The plasma half-life of plasminogen in adult males is approximately 2 days.6 Its 791 amino acids are cross-linked by 24 disulfide bridges, 16 of which give rise to 5 homologous triple-loop structures called kringles (Fig. 116-2).7 The first (K1) and fourth (K4) of these 80–amino acid, approximately 10,000-Mr structures impart high- and low-affinity lysine binding, respectively.8 The lysine-binding domains of plasminogen appear to mediate its specific interactions with fibrin, cell surface receptors, and other proteins including its circulating inhibitor a2-antiplasmin.9,10,11,12 and 13

TABLE 116-1 FIBRINOLYTIC PROTEINS

FIGURE 116-2 Structure-function relationships of plasminogen, t-PA, and u-PA. Alignment of the intron-exon structure of plasminogen, t-PA, and u-PA genes with functional protein domains. Protein domains are labeled signal peptide (SP), preactivation peptide (PAP), kringle domains (K), fibronectin-like “finger” (F), epidermal growth factor–like domain (EGF), and protease. The positions of catalytic triad amino acids histidine (H), aspartic acid (D), and serine (S) are shown within individual protease domains. The positions of individual introns relative to amino acid–encoding exons are indicated with inverted triangles (
).

Posttranslational modification of plasminogen results in two glycosylation variants: forms 1 and 2 (see Table 116-1).14,15 O-linked oligosaccharide, consisting of sialic acid, galactose, and galactosamine resident on Thr345, is common to both forms. Only form 2, however, contains N-linked oligosaccharide on Asn288, composed of sialic acid, galactose, glucosamine, and mannose. The carbohydrate portion of plasminogen appears to regulate its affinity for cellular receptors and may also specify its physiologic degradation pathway.
Activation of plasminogen results from cleavage of a single Arg-Val peptide bond at position 560-561,16 giving rise to the active protease plasmin (see Table 116-1). Plasmin contains a typical serine protease catalytic triad (His602, Asp645, and Ser740)5 but exhibits broad substrate specificity compared to other proteases of this class.17 The circulating form of plasminogen, amino-terminal glutamic acid plasminogen (Glu-PLG), is readily converted by limited proteolysis to several modified forms known collectively as Lys-PLG.18,19 Hydrolysis of the Lys77-Lys78 peptide bond gives rise to a conformationally modified form of the zymogen that more readily binds fibrin, displays two- to threefold higher avidity for cellular receptors, and is activated 10 to 20 times more rapidly than is Glu-PLG.10,16,20 Lys-PLG does not normally circulate in plasma16 but has been identified on cell surfaces.21
Spanning 52.5 kb of DNA on chromosome 6q26-27, the plasminogen gene consists of 19 exons22,23 and directs expression of a 2.7-kb mRNA (see Fig. 116-2).7 The 5′ upstream region of the plasminogen gene contains two regulatory elements common to genes for acute-phase reactants (CTGGGA) and six interleukin-6 (IL-6)-responsive elements.23 Plasminogen gene activity, moreover, is stimulated by the acute-phase mediator IL-6 both in vitro and in vivo.24 The gene is closely linked and structurally related to that of apolipoprotein(a), an apoprotein associated with the highly atherogenic low-density lipoprotein–like particle lipoprotein(a)25 and more distantly related to other kringle-containing proteins, such as t-PA, urokinase plasminogen activator (u-PA), hepatocyte growth factor, and macrophage-stimulating protein.26,27,28,29 and 30 The significance of the latter two proteins to the fibrinolytic system remains to be determined.
THE PHYSIOLOGIC FUNCTIONS OF PLASMINOGEN
The development of the plasminogen-deficient mouse has contributed significantly to our understanding of the physiologic function of the serine protease plasmin. Mice made completely plasminogen deficient through gene targeting undergo normal embryogenesis and development, are fertile, and survive to adulthood (Table 116-2).31,32 In addition to runting and ligneous conjunctivitis,33 these animals display a predisposition to thrombosis, with spontaneous thrombi appearing in liver, stomach, colon, rectum, lung, and pancreas; fibrin deposition in the liver; and ulcerative lesions in the gastrointestinal tract and rectum. These results suggest that plasminogen is not strictly required for normal development but does play a crucial role in postnatal intra- and extravascular fibrinolysis.

TABLE 116-2 GENETIC MOUSE MODELS RELEVANT TO FIBRINOLYSIS

PLASMINOGEN ACTIVATORS
TISSUE PLASMINOGEN ACTIVATOR
One of two major endogenous plasminogen activators, t-PA consists of 527 amino acids comprising a glycoprotein of Mr approximately 72,000 (see Table 116-1).34 t-PA contains five structural domains, including a fibronectin-like “finger,” an epidermal growth factor–like domain, two kringle structures homologous to those of plasminogen, and a serine protease domain (see Fig. 116-2). Cleavage of the Arg275-Ile276 peptide bond by plasmin converts t-PA to a disulfide-linked, two-chain form.34 While single-chain t-PA is less active than two-chain t-PA in the fluid phase, both forms demonstrate equivalent activity when fibrin bound.35
The two glycosylation forms of t-PA are distinguishable by the presence (type 1) or absence (type 2) of a complex N-linked oligosaccharide moiety on Asn18436,37 (see Table 116-1). Both types, however, contain high mannose carbohydrate on Asn117, complex oligosaccharide on Asn448, and an O-linked a-fucose residue on Thr61.38 The carbohydrate moieties of t-PA may modulate its functional activity, regulate its binding to cell surface receptors, and specify degradation pathways. Located on chromosome 8p12-q11.2, the gene for human t-PA is encoded by 14 exons spanning a total of 36.6 kb39,40 and 41 (see Fig. 116-2). Although exon 1 encodes a 58-nucleotide mRNA leader sequence, each of the structural domains of t-PA is encoded by 1 or 2 of the remaining 13 exons. This suggests that the t-PA gene arose by an evolutionary process called exon shuffling, whereby functionally related genes evolved through rearrangement of exons encoding autonomous domains. Consistent with this hypothesis, deletion of exons encoding the fibronectin-like finger or kringle 2, but not kringle 1, domains of t-PA results in expression of mutants resistant to the cofactor activity of fibrin, while catalytic activity in the absence of fibrin remains intact.42
The proximal promoter of the human t-PA gene contains binding sequences for potentially important transcriptional factors, including AP1, NF1, SP1, and AP243,44 as well as a potential cyclic adenosine monophosphate (cAMP)-responsive element (CRE).45 In vitro, many agents have been shown to exert small effects on the expression of t-PA mRNA, but relatively few enhance t-PA synthesis without augmenting plasminogen-activator inhibitor-1 (PAI-1) synthesis as well. Agents that regulate t-PA gene expression independently of PAI-1 include histamine, butyrate, retinoids, arterial levels of shear stress, and dexamethasone.46,47,48 and 49 Forskolin, which increases intracellular cAMP levels, has been reported to decrease synthesis of both t-PA and PAI-1.44,50
t-PA is synthesized and secreted primarily by endothelial cells, and its release is governed by a variety of stimuli, such as thrombin, histamine, bradykinin, epinephrine, acetylcholine, arginine vasopressin, gonadotropins, exercise, venous occlusion, and shear stress.47,51 Its circulating half-life is exceedingly short (»5 min). Functionally, t-PA is a poor activator of plasminogen. However, in the presence of fibrin, the catalytic efficiency of t-PA–dependent plasmin generation (kcat/Km) increases by at least two orders of magnitude.20 This is due to a dramatic increase in affinity (decreased Km) between t-PA and its substrate plasminogen in the presence of fibrin. Although it is expressed by endothelial cells, t-PA appears to represent the major circulating activator of plasminogen.17
UROKINASE
The second endogenous plasminogen activator, single-chain u-PA or prourokinase, is an approximately 54,000-Mr glycoprotein consisting of 411 amino acids (see Table 116-1). u-PA contains an epidermal growth factor–like domain and a single plasminogen-like kringle and possesses a classical catalytic triad (His204, Asp255, and Ser356) within its serine protease domain52 (see Fig. 116-2). Cleavage of the Lys158-Ile159 peptide bond by plasmin or kallikrein converts single-chain u-PA to a disulfide-linked two-chain derivative.53 Located on chromosome 10, the human u-PA gene is encoded by 11 exons spanning 6.4 kb and expressed by endothelial cells, macrophages, renal epithelial cells, and some tumor cells.54,55 Its intron-exon structure is closely related to that of the t-PA gene.
There is circumstantial evidence that u-PA may be induced during neoplastic transformation, possibly through a mechanism involving transcription factors AP1 and AP2.56 Other agents that appear to induce expression of u-PA in vitro include hormones, growth factors, and cAMP.49 Inflammatory cytokines such as interleukin-1 and lipopolysaccharide induce only small increments in u-PA expression, while tumor necrosis factor and transforming growth factor b (TGF-b) have a more dramatic (five- to thirtyfold) effect.57,58 and 59
Two-chain u-PA occurs in both high- (Mr 54,000) and low-molecular-weight (Mr 33,000) forms, which differ by the presence or absence, respectively, of a 135-residue amino-terminal fragment released by plasmin cleavage between Lys135 and Lys136.60,61 Although both forms are capable of activating plasminogen, only the high-molecular-weight form binds to the u-PA receptor. u-PA has much lower affinity for fibrin than does t-PA and is an effective plasminogen activator both in the presence and in the absence of fibrin.62,63 The extent to which prourokinase possesses intrinsic plasminogen-activating capacity is controversial.64,65
ACCESSORY PLASMINOGEN ACTIVATORS
Under certain conditions, proteases traditionally classified within the intrinsic arm of the coagulation cascade have been shown to be capable of activating plasminogen directly. These include kallikrein, factor XIa, and factor XIIa.66,67 These proteases, however, normally account for no more than 15 percent of total plasmin-generating activity in plasma.68
PHYSIOLOGIC FUNCTION OF THE PLASMINOGEN ACTIVATORS
Although abnormalities of the t-PA release mechanism have been reported,69 there are no clinical examples of complete deficiency of t-PA or u-PA in humans. Thus, the most compelling studies of the physiologic functions of these proteins come from gene-disruption analyses (see Table 116-2).70 Both u-PA and t-PA null deletion mice exhibit normal fertility and embryonic development. However, u-PA –/– mice developed rectal prolapse, nonhealing ulcerations of the face and eyelids, and occasional fibrin deposition in tissues. Although they show normal rates of pulmonary clot lysis, endotoxin-induced thrombus formation is significantly enhanced. t-PA–deficient mice display a normal spontaneous phenotype. However, these animals have a decreased rate of lysis of artificially induced pulmonary thrombi as well as enhanced thrombus formation in response to injection of endotoxin. Doubly deficient (t-PA–/–, u-PA–/–) mice exhibit rectal prolapse, nonhealing ulceration, runting, and cachexia, with extensive fibrin deposition in liver, intestine, gonads, and lung. It is not surprising that clot lysis is markedly impaired. These findings demonstrate that t-PA and u-PA are not essential for normal embryologic development but do play crucial roles in lysis of artificially induced thrombi and in fibrinolytic surveillance.
INHIBITORS OF FIBRINOLYSIS
PLASMIN INHIBITORS
The action of plasmin is negatively modulated by a family of serine protease inhibitors, called serpins (see Table 116-1).71 All serpins have a common mechanism of action by forming an irreversible complex with the active-site serine of the target protease following proteolytic cleavage of the inhibitor by the target protease. Within such a complex, both protease and inhibitor lose their activity.
A single-chain glycoprotein with a Mr of approximately 70,000, a2-antiplasmin (a2-AP) circulates in plasma at relatively high concentrations (»0.9 µM) and enjoys a plasma half-life of 2.4 days72 (see Table 116-1). This serpin contains about 1 percent carbohydrate by mass and consists of 452 amino acids with two disulfide bridges.73 In humans, the gene is located on chromosome 18 and contains 10 exons distributed over 16 kb of DNA.74 The promoter region of the a2-AP gene contains a hepatitis B–like enhancer element that directs tissue-specific expression in the liver.73 a2-AP is also a constituent of platelet a granules.75 Plasmin released into flowing blood or in the vicinity of a platelet-rich thrombus is immediately neutralized upon forming an irreversible 1:1 stoichiometric, lysine binding site–dependent complex with a2-AP. Interaction with plasmin is accompanied by cleavage of the Arg364-Met365 peptide bond, and the resulting covalent complexes are cleared in the liver.
Several additional proteins can act as plasmin inhibitors (see Table 116-1). a2-Macroglobulin is a 725,000-Mr dimeric protein synthesized by endothelial cells and macrophages and found in platelet a granules. This nonserpin inhibits plasmin with approximately 10 percent of the efficiency exhibited by a2-AP76 by forming noncovalent complexes with several distinct serine proteases. C1-esterase inhibitor can also serve as an inhibitor of t-PA in plasma.69 Protease nexin may function as a noncirculating cell surface inhibitor of trypsin,77 thrombin, factor Xa, urokinase, or plasmin, resulting in protease-inhibitor complexes that are endocytosed via a specific nexin receptor.78
PLASMINOGEN-ACTIVATOR INHIBITORS>
Plasminogen-Activator Inhibitor-1 Of the two major plasminogen-activator inhibitors79 (see Table 116-1), PAI-1 is the most abundant. This approximately 52,000-Mr, single-chain, cysteine-less glycoprotein is released by endothelial cells, monocytes, macrophages, hepatocytes, adipocytes, and platelets.80,81 and 82 Release of PAI-1 is stimulated by many cytokines, growth factors, and lipoproteins common to the global inflammatory response.58,83,84 The PAI-1 gene consists of 9 exons, spanning 12.2 kb on chromosome 7q21.3-q22.85 The serpin-reactive site is located at Arg346-Met347, and activity of this labile serpin is stabilized upon complex formation with vitronectin, a component of plasma and pericellular matrix.86,87
The upstream regulatory region of the human PAI-1 gene contains a strong endothelial cell- or fibroblast-specific element within the first 187 bp of the 5′-flanking region,88,89 a glucocorticoid-responsive enhancer between positions –90 and +75,89 and TGF-b–responsive elements between bases –791 and –546 and bases –328 and –186.90 TGF-b is known to stimulate fos and jun, the two components of the AP1 complex, and an AP1 binding site (GGAGTCA) is located at –672 to –666 upstream of the PAI-1 cap site.91 Agents that have been shown to enhance expression of PAI-1 at the message level, the protein level, or both, without affecting t-PA synthesis include the inflammatory cytokines lipopolysaccharide, interleukin-1, tumor necrosis factor-a,57,58,83,92,93 and 94 TGF-b and basic fibroblast growth factor,59,90,93,95 very low-density lipoprotein and lipoprotein(a),96,97 angiotensin II,98 thrombin,99,100 and phorbol esters.101 In addition, endothelial cell PAI-1 is down-regulated by forskolin43,50 and by endothelial cell growth factor in the presence of heparin.102
PAI-1 is the most important and rapidly acting physiologic inhibitor of both t-PA and u-PA. Transgenic mice that overexpress PAI-1 exhibit thrombotic occlusion of tail veins and swelling of hindlimbs within 2 weeks of birth (see Table 116-2).103 Mice deficient in PAI-1, on the other hand, exhibit normal fertility, viability, tissue histology, and development, and show no evidence of hemorrhage.104 These observations contrast with the moderately severe bleeding disorder observed in a human patient with complete PAI-1 deficiency.105
Plasminogen-Activator Inhibitor-2 Originally purified from human placenta,79,106 plasminogen activator inhibitor-2 (PAI-2) is a 393–amino acid member of the serpin family whose reactive site is the Arg358-Thr359 peptide bond106 (see Table 116-1). The gene encoding PAI-2 is located on chromosome 18q21–23, spans 16.5 kb, and contains 8 exons.107 PAI-2 exists as both a 47,000-Mr nonglycosylated intracellular form and a 60,000-Mr glycosylated form secreted by leukocytes and fibrosarcoma cells. Functionally, PAI-2 inhibits both two-chain t-PA and two-chain u-PA with comparable efficiency (second-order rate constant 105 M–1s–1). However, it is less effective toward single-chain t-PA (second-order rate constant 103 M–1s–1) and does not inhibit prourokinase.
Significant levels of PAI-2 are found in human plasma only during pregnancy. The 5′-untranslated region of the gene has not yet been characterized.106 The 3′-downstream sequences include the TTATTTAT motif, which has been identified with inflammatory mediators.108,109 In macrophages in vitro, secretion of PAI-2 is enhanced by endotoxin and phorbol esters,109,110 and dexamethasone decreases PAI-2 expression in HT-1080 cells.49
CELLULAR RECEPTORS
Although structurally diverse, cell surface fibrinolytic receptors can be classified into two groups whose integrated actions are likely to be essential for homeostatic control of plasmin activity2 (see Table 116-1). “Activation” receptors localize and potentiate plasminogen activation, while “clearance” receptors eliminate plasmin and plasminogen activators from the blood or focal microenvironments.
Activation Receptors There are three types of activation receptors: plasminogen receptors, the u-PA receptor (u-PAR), and annexin II.
Plasminogen receptors are a diverse group of proteins expressed on a wide array of cell types.2 Reported receptors include a-enolase, glycoprotein IIb/IIIa complex, the Heymann nephritis antigen, amphoterin, and annexin II, which are expressed primarily on monocytoid cells,111 platelets,112 renal epithelial cells,113 neuroblastoma cells,114 and endothelial cells,115,116 respectively. These binding proteins commonly interact with the kringle structures of plasminogen through carboxyl-terminal lysine residues.111
u-PAR is expressed on monocytes, macrophages, fibroblasts, endothelial cells, and a variety of tumor cells2 (see Table 116-1). u-PAR cDNA was cloned and sequenced from a human fibroblast cDNA library117 and encodes a protein of 313 amino acids with a 21-residue signal peptide. The gene consists of 7 exons distributed over 23 kb of genomic DNA and places this glycoprotein within the Ly-1/elapid venom toxin superfamily of cysteine-rich proteins.118,119 u-PAR is anchored to the plasma membrane through glycosylphosphatidylinositol linkages.120 u-PA bound to its receptor maintains its activity and susceptibility to the physiologic inhibitor PAI-1.121 Formation of u-PA–PAI-1 complexes appears to hasten clearance of u-PA by hepatic or monocytoid cells.121,122 and 123
u-PAR appears to play a novel role in cellular signaling and adhesion events.124 u-PAR binds the adhesive glycoprotein vitronectin at a site distinct from the u-PA binding domain,125,126 and u-PA transfected renal epithelial cells acquire enhanced adhesion to vitronectin while they lose their adhesion to fibronectin.127 u-PAR, furthermore, colocalizes with integrins in focal contacts and at the leading edge of migrating cells128 and also associates with caveolin, a major component of caveolae, structures abundant in endothelial cells and thought to participate in signaling events.129,130 and 131 Thus, integrin function may be regulated by u-PAR, signifying an integrated relationship between cellular adhesion and proteolysis.
Annexin II is a widely distributed, highly conserved, 36,000-Mr peripheral membrane protein expressed abundantly on endothelial cells,132,133,134 and 135 macrophages,136 myeloid cells,137 and some tumor cells.138,139 and 140 It belongs to a 20-member superfamily of calcium-dependent, phospholipid-binding proteins141 that have in common a conserved C-terminal “core” region preceded by a more variable N-terminal “tail.”142 The human annexin II gene consists of 13 exons distributed over 40 kb of genomic DNA on chromosome 15 (15q21).143
Annexin II possesses the unique property of binding both plasminogen (Kd 114 nM)115 and t-PA (Kd 30 nM),116 but not u-PA.116 Purified native human annexin II stimulates the catalytic efficiency of t-PA–dependent plasminogen activation by sixtyfold in the fluid phase.144 This effect is completely inhibited in the presence of lysine analogs or upon treatment of annexin II with carboxypeptidase B, an agent that removes basic carboxyl-terminal amino acids. Although it lacks a classical signal peptide, within 16 h of its biosynthesis, annexin II is constitutively translocated to the endothelial cell surface, where it binds phospholipid via core repeat 2 containing the linear sequence KGLGT and downstream aspartate residue (Asp161).145 Annexin II heterotetramer, composed of two annexin monomers and two p11 subunits, may have even greater stimulatory effects on t-PA–dependent plasmin generation.134
Lys307 appears to be crucial for the effective interaction of plasminogen with annexin II. “Activation” of the receptor with respect to plasminogen binding apparently requires cleavage at Lys307-Arg308 by a plasmin-like protease and subsequent exposure of the carboxyl-terminal Lys307 residue.144 Lipoprotein(a), an atherogenic low-density lipoprotein (LDL)–like particle, competes with plasminogen for binding to annexin II146 and reduces cell surface plasmin generation. This mechanism may contribute to atherogenesis by reducing fibrinolytic surveillance at the blood vessel wall.
t-PA binding to annexin II requires a separate domain consisting of residues 8 through 13 (LCKLSL) within the amino-terminal “tail” domain of the receptor.147 This sequence is a target for homocysteine (HC), a thiol-containing amino acid that accumulates in association with nutritional deficiencies of vitamin B6, vitamin B12, or folic acid, or in inherited abnormalities of cystathionine b-synthase, methylenetetrahydrofolate reductase, or methionine synthase,148 and is associated with atherothrombotic disease.148,149 and 150 In vitro, HC impairs the intrinsic fibrinolytic system of the endothelial cell by approximately 50 percent151 by forming a covalent derivative with Cys9, thus preventing its interaction with t-PA.147 The half-maximal dose of HC for inhibition of t-PA binding to annexin II is approximately 11 µM HC, a value close to the upper limit of normal for HC in plasma (14 µM).
CLEARANCE RECEPTORS
Both u-PA and t-PA are cleared from the circulation via the liver.152 In vitro, clearance of t-PA–PAI-1 complexes appears also to be mediated by a large two-chain receptor called the LDL-receptor–related protein (LRP).153,154 and 155 This complex interaction requires both growth factor and finger domains of t-PA. An additional 39,000-Mr “receptor-associated protein” copurifies with LRP and may regulate the binding and uptake of LRP ligands.156 It is interesting to note that LRP knockout embryos undergo developmental arrest by 13.5 days after conception, suggesting that regulation of serine protease activity may be crucial for early embryogenesis.157,158
Although several PAI-1–independent clearance pathways for t-PA have been proposed,152 involving the large LRP subunit,159 the mannose receptor,160 or an a-fucose–specific receptor,161 in vivo studies in mice suggest that LRP and the mannose receptor play a dominant role in t-PA clearance.162
THE FIBRINOLYTIC ACTIONS OF PLASMIN
DEGRADATION OF FIBRINOGEN AND FIBRIN
FIBRINOGEN
Fibrinogen possesses distinct proteolytic cleavage sites for plasmin and thrombin (Fig. 116-3). While plasmin cleaves carboxyl-terminal Aa and N-terminal fibrinopeptide B moieties, thrombin primarily releases fibrinopeptide A, exposing the Gly-Pro-Arg tripeptide sequence and allowing fibrinogen to polymerize and form insoluble fibrin.163 Plasmin cleavage of fibrinogen (Mr 340,000) initially produces carboxyl-terminal fragments from the a chain within the D domain of fibrinogen (Aa fragment).164,165 Simultaneously but more slowly, the N-terminal segments of the b chains are cleaved, releasing a peptide containing fibrinopeptide B. The resulting approximately 250,000-Mr molecule is termed fragment X and represents a clottable form of fibrinogen. Additional cleavage events may release the Bb fragment from the a chain carboxyl terminus, and, in a series of subsequent reactions, plasmin cleaves the three polypeptide chains that connect the D and E domains, giving rise to free D domain (Mr »100,000) plus the binodular D-E fragment known as fragment Y (Mr »150,000). Finally, domains D and E are separated from each other, and some of the N-terminal fibrinopeptide A sites on domain E are also modified. Although fragment X can be converted to fibrin by thrombin, the fragments Y, D, and E are all nonclottable and in fact may inhibit the spontaneous polymerization of fibrinogen166 (see Chap. 124).

FIGURE 116-3 Degradation of fibrinogen and cross-linked fibrin by plasmin. (Top panel) Plasmin initially cleaves the C-terminal regions of the a and b chains within the D domain of fibrinogen, releasing the Aa and Bb fragments. In addition, a fragment containing fibrinopeptide B (FPB) from the N-terminal region of the fibrinogen b-chain is also released, giving rise to the intermediate fragment known as fragment X. Subsequently, plasmin cleaves the three polypeptide chains connecting the D and E domains, giving rise to fragments D, E, and Y. (Bottom panel) Fibrinogen can also be polymerized by thrombin to form fibrin. When degrading cross-linked fibrin, plasmin initially cleaves the C-terminal region of the a and b chains within the D domain. Subsequently, some of the connecting regions between the D and E domains are severed. Fibrin is ultimately solubilized upon hydrolysis of additional peptide bonds within the central portions of the coiled-coil connectors, giving rise to fibrin degradation products such as D-dimer. (From KA Hajjar1 with permission).

FIBRIN
Plasmin degradation of fibrin leads to a distinct set of molecular products.167 Species similar to fragments Y, D, and E but lacking fibrinopeptide sites are released from non–cross-linked fibrin. If fibrin has been extensively cross-linked by factor XIII, however, the resulting D fragments are cross-linked to an E domain fragment. Assay of cross-linked D-dimer fragments is employed clinically to identify disseminated intravascular coagulation states associated with excessive plasmin-mediated fibrinolysis (see Chap. 126). Several biologic activities, including inhibition of platelet function,168 potentiation of the hypotensive effects of bradykinin,169 chemotaxis,170 and immune modulation have been ascribed to fibrin breakdown products.171
T-PA–MEDIATED PLASMINOGEN ACTIVATION
With or without fibrin, t-PA–mediated activation of plasminogen follows Michaelis-Menten kinetics.20 In the absence of fibrin, t-PA is a weak activator of plasminogen. However, in the presence of fibrin, the catalytic efficiency (kcat/Km) of t-PA–dependent plasminogen activation is enhanced by at least two orders of magnitude. This is the basis for its specificity as a lytic agent in the treatment of thrombosis (see Chap. 134). The affinity between t-PA and plasminogen in the absence of fibrin is low (Km 65 µM) but increases significantly in its presence (Km 0.16 µM), even though the catalytic rate constant remains essentially unchanged (kcat »0.05 s–1). When plasmin forms on the fibrin surface, both its lysine-binding sites and its active site are occupied. Thus, it is relatively protected from its physiologic inhibitor, a2-AP.172 The interaction of t-PA with fibrin is probably initiated by its “finger” domain. However, once fibrin is modified by plasmin, carboxyl-terminal lysine residues are generated, and these become binding sites for kringle 2 of t-PA and kringles 1 and 4 of plasminogen.173 Therefore, fibrin accelerates its own destruction by (1) enhancing the catalytic efficiency of plasmin formation by t-PA, (2) protecting plasmin from its physiologic inhibitor, a2-AP, and (3) providing new binding sites for plasminogen and t-PA once its degradation has begun.
U-PA–MEDIATED PLASMIN GENERATION
For the activation of Glu-plasminogen by u-PA in a fibrin-free system, reported Michaelis constants (Km) vary from 1.4 to 200 µM, while catalytic rate constants (kcat) range from 0.26 to 1.48 s–1.1 It is interesting to note that activation of Glu-plasminogen by two-chain u-PA is increased in the presence of fibrin by about tenfold even though u-PA does not bind to fibrin.174 In contrast, single-chain u-PA has considerable fibrin specificity. This may reflect neutralization by fibrin of components in plasma that impair plasminogen activation.175 It may also reflect a conformational change in plasminogen upon binding to fibrin.176 It is important to recognize, however, that the intrinsic plasminogen-activating potential of single-chain u-PA is less than 1 percent of that of two-chain u-PA.1 Two-chain u-PA has been used effectively as a thrombolytic agent for many years.177
THROMBIN-ACTIVATABLE FIBRINOLYSIS INHIBITOR
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase with specificity for carboxyl-terminal arginine and lysine residues that acts as a potent inhibitor of fibrinolysis.178 Identical to the previously cloned carboxypeptidase B179 and the previously isolated carboxypeptidase U,180 this single-chain 60,000-Mr polypeptide circulates in plasma at concentrations of about 75 nM and undergoes limited proteolysis in the presence of thrombin, which leads to its activation.181 The profibrinolytic effect of activated protein C in plasma is due to its ability to inactivate coagulation factors Va and VIIIa, thereby preventing activation of prothrombin and inhibiting activation of TAFI.178 The profibrinolytic effect of activated protein C in an in vitro plasma-based system was TAFI dependent,182 and, in a system of purified components, TAFI has been shown to down-regulate t-PA–induced fibrinolysis half-maximally at a concentration of approximately 1 nM, which is 2 percent of its concentration in plasma.183 Inhibition of the intrinsic pathway of coagulation and inhibition of TAFI activity both result in a doubling of endogenous clot lysis in an in vivo rabbit jugular vein model of thrombolysis.184 Carboxypeptidases in plasma may regulate plasminogen binding to both cell surface receptors and fibrin.185
THE NONFIBRINOLYTIC ACTIONS OF PLASMIN
PLASMIN AS A TISSUE REMODELER
A large number of in vitro studies suggest a role for plasmin in tissue remodeling. Basement membrane proteins, such as thrombospondin,186 laminin,187 fibronectin,188 and fibrinogen,189 are readily degraded by plasmin in vitro, suggesting possible roles in inflammation,190 tumor cell invasion,191 embryogenesis,192 ovulation,193 neurodevelopment,194,195 and prohormone activation.196,197 Plasmin also activates matrix metalloproteinases (MMPs) types 1, 3, 7, and 10,198 thereby facilitating the degradation of matrix proteins, such as the collagens, laminin, fibronectin, vitronectin, elastin, aggrecan, and tenascin C.198 On the other hand, MMP activation can apparently proceed in the absence of plasminogen, possibly providing the basis for the mild phenotype observed in plasminogen null homozygote animals.199
A role for plasmin in tissue remodeling is further supported by in vivo observations in plasminogen-deficient mice (see Table 116-2). Impaired wound healing is observed in the plasminogen knockout200 and is reversed upon simultaneous deletion of fibrinogen.201 Plasminogen-deficient mice also display diminished recruitment of monocytes in response to intraperitoneal thioglycolate202 and impaired neointima formation following electrical injury to blood vessels.203 In studies involving Borrelia burgdorferi, the agent of Lyme disease, dissemination of the spirochete within its arthropod vector Ixodes dammini is absolutely dependent upon host plasminogen even though the deer tick contains no fibrin.204 Further, kainate-induced excitotoxicity and attendant neuronal cell dropout in the hippocampus are not observed in plasminogen knockout mice but do occur in fibrinogen-deficient animals.205 The latter two studies may define new roles for plasmin that appear to be unrelated to degradation of fibrin.
Plasmin may play a role in the activation of growth factors and in the proliferative response of the blood vessel to injury. TGF-b is a 25,000-Mr homodimeric polypeptide whose effects on vascular cell growth and differentiation are pleiomorphic.206 In culture, cell-associated plasmin appears to convert latent TGF-b to its physiologically relevant active state. Inhibition of wound healing in this system was dependent upon active TGF-b, and activation of this agent could be blocked in the presence of plasmin inhibitors, such as aprotinin or a2-plasmin inhibitor. Activation of TGF-b by plasmin may reflect alteration of its tertiary structure upon cleavage of an amino-terminal glycopeptide.207 Once activated by plasmin, TGF-b can stimulate production of PAI-1, thus impairing further activation of plasminogen.
ANGIOSTATIN AND RELATED PLASMINOGEN FRAGMENTS
Angiostatin is a circulating inhibitor of angiogenesis originally isolated from the urine of Lewis lung carcinoma–bearing mice.208 This approximately 38,000 Mr fragment of plasminogen is identical to kringles 1 through 4 and inhibits bFGF-stimulated endothelial cell proliferation in vitro, possibly by inducing apoptosis,209 and blocks new blood vessel formation in both the chick chorioallantoic membrane and mouse cornea assays. In several experimental animal models of metastasis, exogenous angiostatin induces dormancy of tumors critically dependent upon an intact blood supply.210 Inhibition of primary and metastatic tumor growth is also seen upon implantation of tumor cells stably transfected with an angiostatin gene in a murine fibrosarcoma model.211 The cellular target or receptor for angiostatin is unknown, although an endothelial cell–binding site distinct from annexin II has been proposed.212 In other studies, kringle 5 of plasminogen was found to be an even more potent inhibitor of growth factor–stimulated endothelial cell proliferation.213 Angiostatin may represent a promising new approach to antitumor therapy.214
The mechanism of angiostatin formation is a topic of intense investigation. Lewis lung carcinoma–associated macrophages stimulated with tumor-derived GM-CSF express high levels of metalloelastase, which can produce angiostatin from the parent molecule plasminogen.215 Alternatively, angiostatin can form in vitro upon exposure of plasmin to a plasmin reductase followed by an unidentified serine protease secreted by cultured CHO or HT1080 cells.216 Matrix metalloproteinases 7 and 9,217 as well as urokinase in the presence of free sulfhydryl donors,218 have also been proposed as angiostatin-generating agents. These studies suggest the possibility of multiple pathways for the generation of angiostatin.
DISORDERS OF PLASMIN GENERATION
FIBRINOLYTIC DEFICIENCY AND THROMBOSIS
Partial human plasminogen deficiency was first described in a 31-year-old male with a history of repeated episodes of thrombophlebitis, intracranial and mesenteric venous thrombosis, and pulmonary embolism.219 Reduced plasminogen activity (50% of normal) in his plasma was traced to a Ala601Thr point mutation, and several additional patients with this defect or related substitutions have now been described.220 Acquired plasminogen deficiency, as may occur in liver disease, sepsis, and Argentine hemorrhagic fever due to decreased synthesis and/or increased catabolism, has frequently been associated with thrombotic vascular occlusion.221
Congenital plasminogen deficiency has been classified into two types.222 In type I, the concentration of immunoreactive plasminogen is reduced in parallel with functional activity. Although no examples of complete aplasminogenemia have been reported in humans, type I mutations giving rise to reduced synthesis of plasminogen are well defined (e.g., Ser572Pro).223 In a study of consecutive patients with thrombophilia, the prevalence of plasminogen deficiency was 1.9 percent.224 Approximately half of these individuals had other risk factors, such as deficiency of antithrombin III, protein C, or protein S, or resistance to activated protein C. Among 93 patients with type I plasminogen deficiency, the prevalence of thrombosis was 24 percent, or 9 percent when the propositi were excluded.225 These data suggest that, compared with other thrombophilic conditions, congenital plasminogen deficiency is associated with a lower risk of thrombosis.225
In one well-documented case of type I plasminogen deficiency, an infant with less than 1 percent of normal plasminogen antigen and activity presented with hydrocephalus, central nervous system malformations, poor wound healing, recurrent respiratory infections, but no family history of thrombosis. His severe ligneous conjunctivitis (i.e., the development of fibrinous membrane over the eyes) resolved completely upon infusion of Lys-plasminogen.226 This case illustrates the importance of plasminogen in extravascular fibrinolysis and underscores the role of plasminogen deficiency as a relatively weak predisposing risk factor for thrombosis.222
In type II plasminogen deficiency, immunoreactive protein is normal, while functional activity is reduced.220 In a study of a Japanese cohort, 94 percent of 129 families with type II dysplasminogenemia had the Ala601Thr mutation, while 3 percent and 1 percent had the Val355Phe and Asp676Asn, respectively.227 In this study, approximately 27 percent of individuals with dysplasminogenemia had a clinical history of thrombosis. A number of additional plasminogen polymorphisms228,229 and clinically significant dysplasminogenemias230 have also been reported.
Mutations in tissue plasminogen activator or urokinase have not been clinically linked to thrombophilia. However, defects in plasminogen activator release from the vessel wall as well as increased inhibition of t-PA by PAI-1 have both been associated with a thrombotic diathesis.231,232 Increased circulating PAI-1 appears to represent an independent risk factor for vascular reocclusion in young survivors of myocardial infarction.233 In addition, increased levels of PAI-1 have been associated with deep vein thrombosis in patients undergoing hip replacement surgery234 and in individuals with insulin resistance.235 With regard to the latter studies, however, one should bear in mind that PAI-1 is itself an acute-phase reactant and thus may not be directly responsible for the observed prothrombotic tendency.236
ENHANCED FIBRINOLYSIS AND BLEEDING
Enhanced fibrinolysis due to congenital or acquired loss of fibrinolytic inhibitor activity is associated with a bleeding diathesis.237 Patients with congenital deficiency of a2-AP may present with a severe hemorrhagic disorder due to impaired inactivation of plasmin and premature lysis of the hemostatic plug.238 Acquired a2-AP deficiency may be seen in patients with severe liver disease due to decreased synthesis, disseminated intravascular coagulation due to consumption, or nephrotic syndrome due to urinary loss, or during thrombolytic therapy, which induces excessive utilization of the inhibitor.238
Patients with acute promyelocytic leukemia demonstrate excessive expression of annexin II on their developmentally arrested promyelocytes. Bleeding in this disorder is accompanied by evidence of high levels of plasmin generation and depletion of a2-antiplasmin. Bleeding resolves upon initiation of all-trans-retinoic acid therapy, which eliminates expression of promyelocyte annexin II, probably through a transcriptional mechanism.137
Complete loss of PAI-1 expression, resulting in hemorrhage in a 9-year-old child, was associated with severe hemorrhage in the setting of trauma or surgery.105 This autosomal recessive trait reflected a frameshift mutation within exon 4 that induced a premature stop codon. This case demonstrates that the function of PAI-1 in humans is apparently limited to the regulation of fibrinolysis.
DEVELOPMENTAL REGULATION OF THE FIBRINOLYTIC SYSTEM
In the resting, nonstressed state, the plasmin-generating potential in the newborn is significantly less than that of the adult.239 Although the amino acid composition and apparent molecular mass of neonatal plasminogen are indistinguishable from those of the adult protein,240,241 plasma concentrations of plasminogen in the neonate are approximately 50 percent of those observed in adults.240,242,243 On the other hand, levels of histidine-rich glycoprotein, a carrier protein that may limit the interaction of plasminogen with fibrin, are reduced by 50 to 80 percent in healthy, term newborns.244 Finally, plasminogen in the neonate is heavily glycosylated, less readily activated by tissue plasminogen activator, and only weakly bound to the endothelial cell surface.241
Although t-PA antigen and activity levels in the healthy newborn are reduced by 63 and 75 percent, respectively, compared with adult values,243 stressed infants, such as those with severe congenital heart disease or respiratory distress syndrome, may have t-PA antigen levels that are increased by up to eightfold.245,246 In contrast, the principal plasmin inhibitors undergo only minimal change from birth to adulthood.242,247,248 Thus, reduced fibrinolytic activity may contribute to the thrombogenic state commonly observed in the newborn,249 but this predilection may be reversed under conditions of physiologic stress.
FIBRINOLYTIC ACTIVITY DURING PREGNANCY AND PUERPERIUM
Pregnancy is a hypofibrinolytic state.250 Both plasminogen and fibrinogen levels in plasma increase by 50 to 60 percent in the third trimester. Overall fibrinolytic activity, as reflected by euglobulin lysis activity, is reduced, and increased fibrin deposition is suggested by increasing D-dimer levels throughout pregnancy.251 Between the twentieth week of pregnancy and term, PAI-1 levels increase to three times their normal level, while PAI-2 levels rise to 25 times their level in early pregnancy.250 Less dramatic increases in both u-PA and t-PA levels are also observed. Within 1 h of delivery, concentrations of both PAI-1 and PAI-2 begin to decrease, and they return to normal within 3 to 5 days.250
In preeclampsia, the hemostatic and fibrinolytic imbalances seen in pregnancy are further exaggerated.252 Circulating PAI-1 levels exceed those in normal pregnancy, and fibrin deposition is seen in glomerular capillaries and spiral arteries of the placenta. It is interesting to note that levels of PAI-2, a marker of placental function, are reduced during preeclampsia compared with normal pregnancy, and this decrease correlates with intrauterine growth retardation of the fetus.
CHAPTER REFERENCES

1.
Hajjar KA: The molecular basis of fibrinolysis, in Hematology of Infancy and Childhood, 5th ed, edited by DG Nathan, SH Orkin, p 1557. Saunders, Philadelphia, 1998.

2.
Hajjar KA: Cellular receptors in the regulation of plasmin generation. Thromb Haemost 74:294, 1995.

3.
Raum D, Marcus D, Alper CA, Levey R, Taylor PD, Starzl TE: Synthesis of human plasminogen by the liver. Science 208:1036, 1980.

4.
Bohmfalk J, Fuller G: Plasminogen is synthesized by primary cultures of rat hepatocytes. Science 209:408, 1980.

5.
Castellino FJ: Biochemistry of human plasminogen. Sem Thromb Hemost 10:18, 1984.

6.
Collen D, Tytgat G, Claeys H, Verstraete M, Wallen P: Metabolism of plasminogen in healthy subjects: effect of tranexamic acid. J Clin Invest 51:1310, 1972.

7.
Forsgren M, Raden B, Israelsson M, Larsson K, Heden LO: Molecular cloning and characterization of a full-length cDNA clone for human plasminogen. FEBS Lett 213:254, 1987.

8.
Miles LA, Dahlberg CM, Plow EF: The cell-binding domains of plasminogen and their function in plasma. J Biol Chem 263:11656, 1988.

9.
Markus G, De Pasquale JL, Wissler FC: Quantitative determination of the binding of epsilon-aminocaproic acid to native plasminogen. J Biol Chem 253:727, 1978.

10.
Markus G, Priore RL, Wissler FC: The binding of tranexamic acid to native (glu) and modified (lys) human plasminogen and its effect on conformation. J Biol Chem 254:1211, 1979.

11.
Hajjar KA, Harpel PC, Jaffe EA, Nachman RL: Binding of plasminogen to cultured human endothelial cells. J Biol Chem 261:11656, 1986.

12.
Miles LA, Plow EF: Cellular regulation of fibrinolysis. Thromb Haemost 66:32, 1991.

13.
Rakoczi I, Wiman B, Collen D: On the biologic significance of the specific interaction between fibrin, plasminogen, and antiplasmin. Biochim Biophys Acta 540:295, 1978.

14.
Hayes ML, Castellino FJ: Carbohydrate of the human plasminogen variants: I. Carbohydrate composition, glycopeptide isolation, and characterization. J Biol Chem 254:8768, 1979.

15.
Hayes ML, Castellino FJ: Carbohydrate of the human plasminogen variants: III. Structure of the O-glycosidically-linked oligosacchraide unit. J Biol Chem 254:8777, 1979.

16.
Holvoet P, Lijnen HR, Collen D: A monoclonal antibody specific for lys-plasminogen. J Biol Chem 260:12106, 1985.

17.
Saksela O: Plasminogen activation and regulation of proteolysis. Biochim Biophys Acta 823:35, 1985.

18.
Wallen P, Wiman B: Characterization of human plasminogen: I. On the relationship between different molecular forms of plasminogen demonstrated in plasma and found in purified preparations. Biochim Biophys Acta 221:20, 1970.

19.
Wallen P, Wiman B: Characterization of human plasminogen: II. Separation and partial characterization of different molecular forms of human plasminogen. Biochim Biophys Acta 157:122, 1972.

20.
Hoylaerts M, Rijken DC, Lijnen HR, Collen D: Kinetics of the activation of plasminogen by human tissue plasminogen activator: Role of fibrin. J Biol Chem 257:2912, 1982.

21.
Hajjar KA, Nachman RL: Endothelial cell-mediated conversion of glu-plasminogen to lys-plasminogen: further evidence for assembly of the fibrinolytic system on the endothelial cell surface. J Clin Invest 82:1769, 1988.

22.
Murray JC, Buetow KH, Donovan M, et al: Linkage disequilibrium of plasminogen polymorphisms and assignment of the gene to human chromosome 6q26-6q27. Am J Hum Genet 40:338, 1987.

23.
Petersen TE, Martzen MR, Ichinose A, Davie EW: Characterization of the gene for human plasminogen, a key proenzyme in the fibrinolytic system. J Biol Chem 265:6104, 1990.

24.
Jenkins GR, Seiffert D, Parmer RJ, Miles LA: Regulation of plasminogen gene expression by interleukin-6. Blood 89:2394, 1997.

25.
McLean JW, Tomlinson JE, Kuang WJ, et al: cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature 330:132, 1987.

26.
Nakamura T, Nishizawa T, Hagiya M, et al: Molecular cloning and expression of human hepatocyte growth factor. Nature 342:440, 1989.

27.
Weissbach L, Treadwell BV: A plasminogen-related gene is expressed in cancer cells. Biochem Biophys Res Commun 186:1108, 1992.

28.
Yoshimura T, Yuhki N, Wang MH, Skeel A, Leonard EJ: Cloning, sequencing, and expression of human macrophage stimulating protein (MSP, MST 1) confirms MSP as a member of the family of kringle proteins and locates the MSP gene on chromosome 3. J Biol Chem 268:15461, 1993.

29.
Byrne CD, Schwartz K, Meer K, Cheng JF, Lawn RM: The human apolipoprotein(a)/plasminogen gene cluster contains a novel homologue transcribed in liver. Arterioscler Thromb 14:534, 1994.

30.
Ichinose A: Multiple members of the plasminogen-apolipoprotein(a) gene family associated with thrombosis. Biochemistry 31:3113, 1992.

31.
Bugge TH, Flick MJ, Daugherty CC, Degen JL: Plasminogen deficiency causes severe thrombosis but is compatible with development and reproduction. Genes Develop 9:794, 1995.

32.
Carmeliet P, Collen D: Gene targeting and gene transfer studies of the plasminogen/plasmin system: Implications in thrombosis, hemostasis, neointima formation, and atherosclerosis. FASEB J 9:934, 1995.

33.
Drew AF, Kaufman AH, Kombrinck KW, Danton MJS, Degen JL, Bugge TH: Ligneous conjunctivitis in plasminogen-deficient mice. Blood 91:1616, 1998.

34.
Pennica D, Holmes WE, Kohr WJ, et al: Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature 301:214, 1983.

35.
Tate KM, Higgins DL, Holmes WE, Winkler ME, Heyneker HL, Vehar GL: Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis. Biochemistry 26:338, 1987.

36.
Pohl G, Kenne L, Nilsson B, Einarsson M: Isolation and characterization of three different carbohydrate chains from melanoma tissue tissue plasminogen activator. Eur J Biochem 170:69, 1987.

37.
Spellman MW, Basa LJ, Leonard CK, Chakel JA: Carbohydrate structures of tissue plasminogen activator expressed in Chinese hamster ovary cells. J Biol Chem 264:14100, 1989.

38.
Harris RJ, Leonard CK, Guzzetta AW: Tissue plasminogen activator has an O-linked fucose attached to threonine-61 in the epidermal growth factor domain. Biochemistry 30:2311, 1991.

39.
Ny T, Elgh F, Lund B: Structure of the human tissue-type plasminogen activator gene: Correlation of intron and exon structures to functional and structural domains. Proc Natl Acad Sci USA 81:5355, 1984.

40.
Browne MJ, Tyrrell AWR, Chapman CG, et al: Isolation of a human tissue-type plasminogen activator genomic clone and its expression in mouse L cells. Gene 33:279, 1985.

41.
Degen SJF, Rajput B, Reich E: The human tissue plasminogen activator gene. J Biol Chem 261:6872, 1986.

42.
Van Zonnefeld A, Veerman H, Pannekoek H: Autonomous functions of structural domains on human tissue-type plasminogen activator. Proc Natl Acad Sci USA 83:4670, 1986.

43.
Feng P, Ohlsson M, Ny T: The structure of the TATA-less rat tissue-type plasminogen activator gene. J Biol Chem 265:2022, 1990.

44.
Kooistra T, Bosma PJ, Toet K, et al: Role of protein kinase C and cyclic adenosine monophosphate in the regulation of tissue-type plasminogen activator, plasminogen activator inhibitor-1, and platelet-derived growth factor mRNA levels in human endothelial cells: Possible involvement of proto-oncogenes c-jun and c-fos. Arterioscler Thromb 11:1042, 1991.

45.
Medcalf RL, Ruegg M, Schleuning WD: A DNA motif related to the cAMP-responsive element and an exon-located activator protein-2 binding site in the human tissue-type plasminogen activator gene promoter cooperate in basal expression and convey activation by phorbol ester and cAMP. J Biol Chem 265:14618, 1990.

46.
Kooistra T, Van den Berg J, Tons A, Platenburg G, Rijken DC, Van den Berg E: Butyrate stimulates tissue type plasminogen activator synthesis in cultured human endothelial cells. Biochem J 247:605, 1987.

47.
Diamond SL, Eskin SG, McIntire LV: Fluid flow stimulates tissue plasminogen activator secretion by cultured human endothelial cells. Science 243:1483, 1989.

48.
Hanss M, Collen D: Secretion of tissue-type plasminogen activator and plasminogen activator inhibitor by cultured human endothelial cells: Modulation by thrombin, endotoxin, and histamine. J Lab Clin Med 109:97, 1987.

49.
Medcalf RL, Van den Berg E, Schleuning WD: Glucocorticoidmodulated gene expression of tissue- and urinary-type plasminogen activator and plasminogen activator inhibitor-1 and -2. J Cell Biol 106:971, 1988.

50.
Santell L, Levin EG: Cyclic AMP potentiates phorbol ester stimulation of tissue plasminogen activator release and inhibits secretion of plasminogen activator inhibitor-1 from human endothelial cells. J Biol Chem 263:16802, 1988.

51.
Dichek D, Quertermous T: Thrombin regulation of mRNA levels of tissue plasminogen activator inhibitor-1 in cultured human umbilical vein endothelial cells. Blood 74:222, 1989.

52.
Kasai S, Arimura H, Nishida M, Suyama T: Primary structure of single-chain pro-urokinase. J Biol Chem 260:12382, 1985.

53.
Gunzler WA, Steffens GJ, Otting F, Buse G, Flohe L: Structural relationship between high and low molecular mass urokinase. Physiol Chem 363:133, 1982.

54.
Riccio A, Grimaldi G, Verde P, Sebastio G, Boast S, Blasi F: The human urokinase-plasminogen activator gene and its promoter. Nucleic Acids Res 13:2759, 1985.

55.
Holmes WE, Pennica D, Blaber M, et al: Cloning and expression of the gene for pro-urokinase in Escherichia coli. Biotechnology 3:923, 1985.

56.
Schmitt M, Wilhelm O, Janicke F, et al: Urokinase-type plasminogen activator (uPA) and its receptor (CD87): A new target in tumor invasion and metastasis. J Obstet Gynaecol 21:151, 1995.

57.
Van Hinsbergh VWM, Van den Berg EA, Fiers W, Dooijewaard G: Tumor necrosis factor induces the production of urokinase-type plasminogen activator by human endothelial cells. Blood 10:1991, 1990.

58.
Medina R, Socher SH, Han JH, Friedman PA: Interleukin-1, endotoxin, or tumor necrosis factor/cachectin enhance the level of plasminogen activator inhibitor messenger RNA in bovine aortic endothelial cells. Thromb Res 54:41, 1989.

59.
Gerwin BI, Keski-Oja J, Seddon M, Lechner JF, Harris CC: TGF beta 1 modulation of urokinase and PAI-1 expression in human bronchial epithelial cells. Am J Pathol 259:262, 1990.

60.
Stump DC, Lijnen HR, Collen D: Purification and characterization of a novel low molecular weight form of single-chain urokinase-type plasminogen activator. J Biol Chem 261:17120, 1986.

61.
Steffens GJ, Gunzler WA, Olting F, Frankus E, Flohe L: The complete amino acid sequence of low molecular mass urokinase from human urine. Physiol Chem 363:1043, 1982.

62.
Lijnen HR, Zamarron C, Blaber M, Winkler M, Collen D: Activation of plasminogen by pro-urokinase. J Biol Chem 261:1253, 1986.

63.
Gurewich V, Pannell R, Louie S, Kelley P, Suddith RL, Greenlee R: Effective and fibrin-specific clot lysis by a zymogen precursor from urokinase (pro-urokinase): A study in vitro and in two animal species. J Clin Invest 73:1731, 1984.

64.
Lijnen HR, Van Hoef B, DeCock F, Collen D: The mechanism of plasminogen activation and fibrin dissolution by single chain urokinase-type plasminogen activator in a plasma milieu in vitro. Blood 73:1864, 1989.

65.
Petersen LC, Lund LR, Nielsen LS, Dano K, Skriver L: One-chain urokinase-type plasminogen activator from human sarcoma cells is a precursor with little or no intrinsic activity. J Biol Chem 263:11189, 1988.

66.
Colman RW: Activation of plasminogen by human plasma kallikrein. Biochem Biophys Res Commun 35:273, 1968.

67.
Goldsmith GH, Saito H, Ratnoff OD: The activation of plasminogen by Hageman factor (factor XII) and Hageman factor fragments. J Clin Invest 62:54, 1978.

68.
Ouimet H, Loscalzo J: Fibrinolysis, in Thrombosis and Hemorrhage, edited by J Loscalzo, AI Schafer, p 127. Blackwell Scientific, Boston, 1994.

69.
Huisman LG, Van Griensven JM, Kluft C: On the role of C1-inhibitor as inhibitor of tissue-type plasminogen activator in human plasma. Thromb Haemost 73:466, 1995.

70.
Carmeliet P, Schoonjans L, Kieckens L, et al: Physiological consequences of loss of plasminogen activator gene function in mice. Nature 368:419, 1994.

71.
Travis J, Salvesan GS: Human plasma proteinase inhibitors. Annu Rev Biochem 52:655, 1983.

72.
Aoki N: Genetic abnormalities of the fibrinolytic system. Sem Thromb Haemost 10:42, 1984.

73.
Holmes WE, Nelles L, Lijnen HR: Primary structure of human alpha2-antiplasmin, a serine protease inhibitor (serpin). J Biol Chem 262:1659, 1987.

74.
Hirosawa S, Nakamura Y, Miura O, Sumi Y, Aoki N: Organization of the human alpha2-antiplasmin inhibitor gene. Proc Natl Acad Sci USA 85:6836, 1988.

75.
Plow EF, Collen D: The presence and release of alpha-2-antiplasmin from human platelets. Blood 58:1069, 1981.

76.
Aoki N, Moroi M, Tachiya K: Effects of alpha-2-plasmin inhibitor on fibrin clot lysis: Its comparison with alpha-2-macroglobulin. Thromb Haemost 39:22, 1978.

77.
Scott RW, Bergman BL, Bajpai A, et al: Protease nexin: Properties and a modified purification procedure. J Biol Chem 260:7029, 1985.

78.
Cunningham DD, Van Nostrand WE, Farrell DH, Campbell CH: Interactions of serine proteases with cultured fibroblasts. J Cell Biochem 32:281, 1986.

79.
Sprengers ED, Kluft D: Plasminogen activator inhibitors. Blood 69:381, 1987.

80.
Ny T, Sawdey M, Lawrence D, Millan JL, Loskutoff DJ: Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor. Proc Natl Acad Sci USA 83:6776, 1986.

81.
Kruithof EKO: Plasminogen activator inhibitor type 1: Biochemical, biological, and cinical aspects. Fibrinolysis 2:59, 1988.

82.
Samad F, Yamamoto K, Loskutoff DJ: Distribution and regulation of plasminogen activator inhibitor-1 in murine adipose tissue in vivo. J Clin Invest 97:37, 1996.

83.
Van Hinsbergh VWM, Kooistra T, Van den Berg EA, et al: Tumor necrosis factor increases the production of plasminogen activator inhibitor in human endothelial cells in vitro and in rats in vivo. Blood 72:1467, 1988.

84.
Van den Berg EA, Sprengers ED, Jaye M, Burgess W, Maciag T, Van Hinsbergh VW: Regulation of plasminogen activator inhibitor-1 mRNA in human endothelial cells. Thromb Haemost 60:63, 1988.

85.
Loskutoff DJ, Linders M, Keijer J, Veerman H, Van Heerikhauizen H, Pannekoek H: Structure of the human plasminogen activator inhibitor-1 gene: Non-random distribution of introns. Biochemistry 26:3763, 1987.

86.
Mottonen J, Strand A, Symersky J, et al: Structural basis of latency in plasminogen activator inhibitor-1. Nature 355:270, 1992.

87.
Declerck PJ, De Mol M, Alessi MC, et al: Purification and characterization of a plasminogen activator inhibitor-1 binding protein from human plasma: Identification as multimeric form of S protein (vitronectin). J Biol Chem 263:15454, 1988.

88.
Bosma PJ, Van den Berg EA, Kooistra T, Siemieniak DR, Slightom JL: Human plasminogen activator inhibitor-1 gene: Promoter and structural nucleotide sequences. J Biol Chem 263:9129, 1988.

89.
Van Zonnefeld AJ, Curriden SA, Loskutoff DJ: Type 1 plasminogen activator inhibitor gene: Functional analysis and glucocorticoid regulation of its promoter. Proc Natl Acad Sci USA 85:5525, 1988.

90.
Westerhausen DR, Hopkins WE, Billadello JJ: Multiple transforming growth factor beta-inducible elements regulate expression of the plasminogen activator inhibitor type-1 gene in HepG2 cells. J Biol Chem 266:1092, 1991.

91.
Keeton MR, Curriden SA, Van Zonneveld AJ, Loskutoff DJ: Identification of regulatory sequences in the type 1 plasminogen activator inhibitor gene responsive to transforming growth factor. J Biol Chem 266:23048, 1991.

92.
Emeis JJ, Kooistra T: Interleukin 1 and lipopolysaccharide induce an inhibitor of tissue-type plasminogen activator in vivo and in cultured endothelial cells. J Exp Med 163:1260, 1986.

93.
Sawdey M, Podor TJ, Loskutoff DJ: Regulation of type-1 plasminogen activator inhibitor gene expression in cultured bovine aortic endothelial cells. J Biol Chem 264:10396, 1989.

94.
Schleef RR, Bevilaqua MP, Sawdey M, Gimbrone MA, Loskutoff DJ: Cytokine activation of vascular endothelium: Effects on tissue-type plasminogen activator and type 1 plasminogen activator inhibitor. J Biol Chem 263:5797, 1988.

95.
Craik CS, Rutter WJ, Fletternick R: Splice junctions: Association with variation in protein structure. Science 220:1125, 1983.

96.
Stiko-Rahm A, Wiman B, Hamsten A, Nilsson J: Secretion of plasminogen activator inhibitor-1 from cultured human umbilical vein endothelial cells is induced by very low density lipoprotein. Arteriosclerosis 10:1067, 1990.

97.
Etingin OR, Hajjar DP, Hajjar KA, Harpel PC, Nachman RL: Lipoprotein(a) regulates plasminogen activator inhibitor-1 expression in endothelial cells. J Biol Chem 266:2459, 1990.

98.
Vaughan DE, Shen C, Lazo S: Angiotensin II induces plasminogen activator inhibitor synthesis in vitro. Circulation 86:I-557, 1992.

99.
Gelehrter TD, Scyncer-Laszuk R: Thrombin induction of plasminogen activator-inhibitor synthesis in vitro. J Clin Invest 77:165, 1986.

100.
Van Hinsbergh VWM, Sprengers ED, Kooistra T: Effect of thrombin on the production of plasminogen activators and PA inhibitor-1 by human foreskin microvascular endothelial cells. Thromb Haemost 57:148, 1987.

101.
Scarpati EM, Sadler JE: Regulation of endothelial cell coagulant properties: Modulation of tissue factor, plasminogen activator inhibitors, and thrombomodulin by phorbol 12-myristate 13-acetate and tumor necrosis factor. J Biol Chem 264:20705, 1989.

102.
Konkle BA, Kollros PR, Kelly MD: Heparin-binding growth factor-1 modulation of plasminogen activator inhibitor-1 expression. J Biol Chem 265:21867, 1990.

103.
Erickson LA, Fici GJ, Lund JE, Boyle TP, Polites HG, Marotti KR: Development of venous occlusions in transgenic mice for the plasminogen activator inhibitor-1 gene. Nature 346:74, 1990.

104.
Carmeliet P, Kieckens L, Schoonjans L, et al: Plasminogen activator inhibitor-1 gene-deficient mice: I. Generation by homologous recombination and characterization. J Clin Invest 92:2746, 1993.

105.
Fay WP, Shapiro AD, Shih JL, Schleef RR, Ginsburg D: Complete deficiency of plasminogen activator inhibitor type 1 due to a frame-shift mutation. N Engl J Med 327:1729, 1992.

106.
Ye RD, Wun T, Sadler JE: cDNA cloning and expression in Escherichia coli of a plasminogen activator inhibitor from human placenta. J Biol Chem 262:3718, 1987.

107.
Ye RD, Aherns SM, Le Beau MM, Lebo RV, Sadler JE: Structure of the gene for human plasminogen activator inhibitor-2: The nearest mammalian homologue of chicken ovalbumin. J Biol Chem 264:5495, 1989.

108.
Antalis TM, Clok MA, Barnes T, et al: Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor. Proc Natl Acad Sci USA 85:985, 1988.

109.
Schleuning WD, Medcalf RL, Hession C, Rothenbuhler R, Shaw A: Plasminogen activator inhibitor 2: Regulation of gene transcription during phorbol ester-mediated differentiation of U-937 human histiocytic lymphoma cells. Mol Cell Biol 7:4564, 1987.

110.
Chapman HA, Stone OL: A fibrinolytic inhibitor of human alveolar macrophages: Induction with endotoxin. Am Rev Respir Dis 132:569, 1985.

111.
Miles LA, Dahlberg CM, Plescia J, Felez J, Kato K, Plow EF: Role of cell surface lysines in plasminogen binding to cells: Identification of alpha-enolase as a candidate plasminogen receptor. Biochemistry 30:1682, 1991.

112.
Miles LA, Ginsberg MA, White JG, Plow EF: Plasminogen interacts with platelets through two distinct mechanisms. J Clin Invest 77:2001, 1986.

113.
Kanalas JJ, Makker SP: Identification of the rat Heymann nephritis autoantigen (GP330) as a receptor site for plasminogen. J Biol Chem 266:10825, 1991.

114.
Barnathan ES, Kuo A, Van der Keyl H, McCrae KR, Larsen GR, Cines DB: Tissue-type plasminogen activator binding to human endothelial cells: Evidence for two distinct binding sites. J Biol Chem 263:7792, 1988.

115.
Hajjar KA: The endothelial cell tissue plasminogen activator receptor: Specific interaction with plasminogen. J Biol Chem 266:21962, 1991.

116.
Hajjar KA, Hamel NM: Identification and characterization of human endothelial cell membrane binding sites for tissue plasminogen activator and urokinase. J Biol Chem 265:2908, 1990.

117.
Roldan AL, Cubellis MV, Masucci MT, et al: Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin-dependent proteolysis. EMBO J 9:467, 1990.

118.
Casey JR, Petranka JG, Kottra J, Fleenor DE, Rosse WF: The structure of the urokinase-type plasminogen activator receptor gene. Blood 84:1151, 1994.

119.
Behrendt N, Ronne E, Ploug M, et al: The human receptor for urokinase plasminogen receptor. J Biol Chem 265:6453, 1990.

120.
Ploug M, Ronne E, Behrendt N, Jensen AL, Blasi F, Dano K: Cellular receptor for urokinase plasminogen activator: Carboxyl-terminal processing and membrane anchoring by glycosylphosphatidylinositol. J Biol Chem 266:1926, 1991.

121.
Cubellis MV, Andreasson P, Ragno P, Mayer M, Dano K, Blasi F: Accessibility of receptor-bound urokinase to type-1 plasminogen activator inhibitor. Proc Natl Acad Sci USA 86:4828, 1989.

122.
Cubellis MV, Wun TC, Blasi F: Receptor-mediated internalization and degradation of urokinase is caused by its specific inhibitor PAI-1. EMBO J 9:1079, 1990.

123.
Ellis V, Behrendt N, Dano K: Plasminogen activation by receptor-bound urokinase. J Biol Chem 266:12752, 1991.

124.
Chapman HA: Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration. Curr Opin Cell Biol 9:714, 1997.

125.
Waltz DA, Chapman HA: Reversible cellular adhesion to vitronectin linked to urokinase receptor occupancy. J Biol Chem 269:14746, 1994.

126.
Wei Y, Waltz DA, Rao N, Drummond RJ, Rosenberg S, Chapman HA: Identification of the urokinase receptor as an adhesion receptor for vitronectin. J Biol Chem 269:32380, 1994.

127.
Wei Y, Lukashev M, Simon DI, et al: Regulation of integrin function by the urokinase receptor. Science 273:1551, 1996.

128.
Xue W, Kindzelskii AL, Todd RF, Petty HR: Physical association of complement receptor type 3 and urokinase-type plasminogen activator in neutrophil membranes. J Immunol 152:4630, 1994.

129.
Stahl A, Mueller BM: The urokinase-type plasminogen activator receptor, a GPI-linked protein, is localized in caveolae. J Cell Biol 129:335, 1995.

130.
Anderson RG: Caveolae: Where incoming and outgoing messengers meet. Proc Natl Acad Sci USA 90:10909, 1993.

131.
Okamoto T, Schlegel A, Scherer PE, Lisanti MP: Caveolins, a family of scaffolding proteins for organizing “preassembled signaling complexes” at the plasma membrane. J Biol Chem 273:5419, 1998.

132.
Chung CY, Erickson HP: Cell surface annexin II is a high affinity receptor for the alternatively spliced segment of tenascin-C. J Cell Biol 126:539, 1994.

133.
Wright JF, Kurosky A, Wasi S: An endothelial cell-surface form of annexin II binds human cytomegalovirus. Biochem Biophys Res Commun 198:983, 1994.

134.
Kassam G, Choi KS, Ghuman J, et al: The role of annexin II tetramer in the activation of plasminogen. J Biol Chem 273:4790, 1998.

135.
Siever DA, Erickson HP: Extracellular annexin II. Int J Biochem Cell Biol 29:1219, 1997.

136.
Falcone DJ, Borth W, Faisal Khan KM, Layne T, Hajjar KA: Annexin II, a constitutively expressed plasminogen receptor, mediates matrix invasion and degradation by macrophages [abstr]. FASEB J 1997.

137.
Menell JS, Cesarman GM, Jacovina AT, McLaughlin MA, Lev EA, Hajjar KA: Annexin II and bleeding in acute promyelocytic leukemia. N Engl J Med 340:994, 1999.

138.
Tressler RJ, Updyke TV, Yeatman TJ, Nicolson GL: Extracellular annexin is associated with divalent cation-dependent tumor cell adhesion of metastatic RAW 117 large-cell lymphoma cells. J Cell Biochem 53:265, 1993.

139.
Yeatman TJ, Updyke TV, Kaetzel MA, Dedman JR, Nicolson GL: Expression of annexins on the surfaces of non-metastatic human and rodent tumor cells. Clin Exp Metastasis 11:37, 1993.

140.
Tressler RJ, Nicolson GL: Butanol-extractable and detergent-solubilized cell surface components from murine large cell lymphoma cells associated with adhesion to organ microvessel endothelial cells. J Cell Biochem 48:162, 1992.

141.
Raynal P, Pollard HB: Annexins: The problem of assessing the biologic role for a gene family of multifunctional calcium- and phospholipid-binding proeins. Biochim Biophys Acta 1197:63, 1994.

142.
Swairjo MA, Seaton BA: Annexin structure and membrane interactions: A molecular perspective. Ann Rev Biophys Biomol Struct 23:193, 1994.

143.
Spano F, Raugei G, Palla E, Colella C, Melli M: Characterization of the human lipocortin-2-encoding multigene family: Its structure suggests the existence of a short amino acid unit undergoing duplication. Gene 95:243, 1990.

144.
Cesarman GM, Guevara CA, Hajjar KA: An endothelial cell receptor for plasminogen/tissue plasminogen activator: II. Annexin II-mediated enhancement of t-PA-dependent plasminogen activation. J Biol Chem 269:21198, 1994.

145.
Hajjar KA, Guevara CA, Lev E, Dowling K, Chacko J: Interaction of the fibrinolytic receptor, annexin II, with the endothelial cell surface: Essential role of endonexin repeat 2. J Biol Chem 271:21652, 1996.

146.
Hajjar KA, Gavish D, Breslow J, Nachman RL: Lipoprotein(a) modulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis. Nature 339:303, 1989.

147.
Hajjar KA, Mauri L, Jacovina AT, et al: Tissue plasminogen activator binding to the annexin II tail domain: Direct modulation by homocysteine. J Biol Chem 273:9987, 1998.

148.
Kraus JP: Molecular basis of phenotype expression in homocystinuria. J Inher Metab Dis 17:383, 1994.

149.
Boushey CJ, Beresford SAA, Omenn GS, Motulsky AG: A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. JAMA 274:1049, 1995.

150.
Refsum H, Ueland PM, Nygard O, Vollset SE: Homocysteine and cardiovascular disease. Ann Rev Med 49:31, 1998.

151.
Hajjar KA: Homocysteine-induced modulation of tissue plasminogen activator to its endothelial cell membrane receptor. J Clin Invest 91:2873, 1993.

152.
Bu G, Warshawsky I, Schwartz AL: Cellular receptors for the plasminogen activators. Blood 83:3427, 1994.

153.
Beiseigel U, Weber W, Ihrke G, Herz J, Stanley KK: The LDL-receptor-related protein, LRP, is an apolipoprotein E-binding protein. Nature 341:162, 1989.

154.
Brown MS, Herz J, Kowal RC: The low-density lipoprotein receptor-related protein: Double agent or decoy? Curr Opin Lipidol 2:65, 1991.

155.
Orth K, Madison EL, Gething MJ, Sambrook JF: Complexes of tissue-type plasminogen activator and its serpin inhibitor plasminogen-activator inhibitor type 1 are internalized by means of the low-density lipoprotein receptor–related protein/alpha-2-macroglobulin receptor. Proc Natl Acad Sci USA 89:7422, 1992.

156.
Herz J, Goldstein JL, Strickland DK, Ho YK, Brown MS: 39-kDa protein modulates binding of ligands to low-density lipoprotein receptor–related protein/alpha-2-macroglobulin receptor. J Biol Chem 266:21232, 1991.

157.
Herz J, Clouthier DE, Hammer RE: LDL receptor–related protein internalizes and degrades uPA–PAI-1 complexes and is essential for embryo implantation. Cell 71:411, 1992.

158.
Herz J, Clouthier DE, Hammer RE: Correction: LDL receptor–related protein internalizes and degrades uPA–PAI-1 complexes and is essential for embryo implantation. Cell 73:428, 1993.

159.
Bu G, Morton PA, Schwartz AL: Identification and partial characterization by chemical cross-linking of a binding protein for tissue-type plasminogen activator (t-PA) on rat hepatoma cells. J Biol Chem 267:15595, 1992.

160.
Otter M, Barrett-Bergshoeff MM, Rijken DC: Binding of tissue type plasminogen activator by the mannose receptor. J Biol Chem 266:13931, 1991.

161.
Hajjar KA, Reynolds CM: a-Fucose-mediated binding and degradation of tissue plasminogen activator by HepG2 cells. J Clin Invest 93:703, 1994.

162.
Narita M, Bu G, Herz J, Schwartz AL: Two receptor systems are involved in the plasma clearance of tissue-type plasminogen activator (t-PA) in vivo. J Clin Invest 96:1164, 1995.

163.
Bailey K, Bettelheim FR, Lorand L, Middlebrook WR: Action of thrombin in the clotting of fibrinogen. Nature 167:233, 1951.

164.
Doolittle RF: The molecular biology of fibrin, in The Molecular Basis of Blood Diseases, edited by G Stamatoyannopoulos, AW Nienhuis, PW Majerus, H Varmus, p 701. Saunders, Philadelphia, 1994.

165.
Gaffney PJ, Dobos P: A structural aspect of human fibrinogen suggested by its plasmin degradation. FEBS Lett 15:13, 1971.

166.
Latallo ZS, Flether AP, Alkjaersig N, Sherry S: Inhibition of fibrin polymerization by fibrinogen proteolysis products. Am J Physiol 202:681, 1962.

167.
Pizzo SV, Schwartz ML, Hill RL, McKee PA: The effect of plasmin on the subunit structure of human fibrin. J Biol Chem 248:4574, 1973.

168.
Culasso DE, Donati MB, DeGaetano G, Vermylen J, Verstraete M: Inhibition of human platelet aggregation by plasmin digests of human and bovine preparations: Role of contaminating factor VIII–related material. Blood 44:169, 1974.

169.
Buluk K, Malofiegen M: The pharmacologic properties of fibrinogen degradation products. Br J Pharmacol 35:79, 1969.

170.
Richardson DL, Pepper DS, Kay AB: Chemotaxis for human monocytes by fibrinogen degradation products. Br J Haematol 32:507, 1976.

171.
Girmann G, Pees H, Schwarze G, Scheulen PG: Immunosuppression by micromolecular fibrin-fibrinogen degradation products in cancer. Nature 259:399, 1976.

172.
Wiman B, Collen D: On the kinetics of the reaction between human antiplasmin and plasmin. Eur J Biochem 84:573, 1978.

173.
Van Zonnefeld AJ, Veerman H, Pannekoek H: On the interaction of the finger and the kringle-2 domain of tissue-type plasminogen activator with fibrin: Iinhibition of kringle-1 binding to fibrin by epsilon-aminocaproic acid. J Biol Chem 261:14214, 1986.

174.
Camiolo SM, Thorsen S, Astrup T: Fibrinogenolysis and fibrinolysis with tissue plasminogen activator, urokinase, streptokinase-activated human globulin and plasmin. Proc Soc Exp Biol Med 138:277, 1971.

175.
Lijnen HR, Zamarron C, Blaber M, Winkler ME, Collen D: Activation of plasminogen by prourokinase: I. Mechanism. J Biol Chem 261:1253, 1986.

176.
Pannell R, Black J, Gurewich V: Complementary modes of action of tissue-type plasminogen activator and pro-urokinase by which their synergistic effect on clot lysis may be explained. J Clin Invest 81:853, 1988.

177.
Bell W: Fibrinolytic therapy: Indications and management, in Hematology: Basic Principles and Practice, edited by R Hoffman, EJ Benz, SJ Shattil, B Furie, HJ Cohen, LE Silberstein, p 1814. Churchill Livingstone, New York.

178.
Nesheim M, Wang W, Boffa M, Nagashima M, Morser J: Thrombin, thrombomodulin and TAFI in the molecular link between coagulation and fibrinolysis. Thromb Haemost 78:386, 1997.

179.
Eaton DL, Malloy BE, Tsai SP, Henzel W, Drayna D: Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from plasma. J Biol Chem 269:21833, 1991.

180.
Wang W, Hendriks DF, Scharpe SS: Carboxypeptidase U, a plasma carboxypeptidase with high affinity for plasminogen. J Biol Chem 269:15937, 1994.

181.
Bajzar L, Manuel R, Nesheim M: Purification and characterization of TAFI, a thrombin activatable fibrinolysis inhibitor. J Biol Chem 270:14477, 1995.

182.
Bajzar L, Nesheim ME, Tracy PB: The profibrinolytic effect of activated protein C in clots formed from plasma is TAFI-dependent. Blood 88:2093, 1996.

183.
Bajzar L, Morser J, Nesheim M: TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades thorugh the thrombin-thrombomodulin complex. J Biol Chem 271:16603, 1996.

184.
Minnema MC, Friederich PW, Levi M, et al: Enhancement of rabbit jugular vein thrombolysis by neutralization of factor XI: In vivo evidence for a role of factor XI as an anti-fibrinolytic factor. J Clin Invest 101:10, 1998.

185.
Redlitz A, Tan AK, Eaton D, Plow EF: Plasma carboxypeptidases as regulators of the plasminogen system. J Clin Invest 96:2534, 1995.

186.
Coligan JE, Slayter HS: Structure of thrombospondin. J Biol Chem 259:3944, 1984.

187.
Ott U, Odermatt E, Engel J, Furthmayr H, Timpl R: Protease resistance and conformation of laminin. Eur J Biochem 123:63, 1982.

188.
Aplin JD, Hughes RC: Complex carbohydrates of the extracellular matrix structures, interactions, and biologic roles. Biochim Biophys Acta 694:375, 1982.

189.
Marder VJ, Sherry S: Thrombolytic therapy: Current status. N Engl J Med 318:1512, 1988.

190.
Unkeless JC, Gordon S, Reich E: Secretion of plasminogen activator by stimulated macrophages. J Exp Med 834, 1974.

191.
Ossowski L, Reich E: Antibodies to plasminogen activator inhibit human tumor metastasis. Cell 35:611, 1983.

192.
Strickland SE, Reich E, Sherman MI: Plasminogen activator in early embryogenesis: Enzyme production by trophoblast and parietal endoderm. Cell 9:231, 1976.

193.
Strickland SE, Beers WH: Studies on the role of plasmingen activator in ovulation. J Biol Chem 254:5694, 1976.

194.
Moonen G, Grau-Wagemans MP, Selak I: Plasminogen activator-plasmin system and neuronal migration. Nature 298:753, 1982.

195.
Pittman RN, Ivins JK, Buettner HM: Neuronal plasminogen activators: Cell surface binding sites and involvement in neurite outgrowth. J Neurosci 9:4269, 1989.

196.
Virji MA, Vassalli JD, Estensen D, Reich E: Plasminogen activator of islets of Langerhans: Modulation by glucose and correlation with insulin production. Proc Natl Acad Sci USA 77:875, 1980.

197.
Russell J, Schneider AB, Katzhendler J, Kowalski K, Sherwood LM: Modification of human placental lactogen with plasmin. J Biol Chem 254:2296, 1979.

198.
Nagase H: Activation mechanisms of matrix metalloproteinases. Biol Chem 378:151, 1997.

199.
Hiraoka N, Allen E, Apel IJ, Gyetko MR, Weiss SJ: Matrix metalloproteinases regulate neovascularization by acting as pericellular fibrinolysins. Cell 95:365, 1998.

200.
Romer J, Bugge TH, Pyke C, et al: Impaired wound healing in mice with a disrupted plasminogen gene. Nature Med 2:287, 1996.

201.
Bugge TH, Kombrinck KW, Flick MJ, Daugherty CC, Danton MJS, Degen JL: Loss of fibrinogen rescues mice from the pleiotropic effects of plasminogen deficiency. Cell 87:709, 1996.

202.
Ploplis VA, French EL, Carmeliet P, Collen D, Plow EF: Plasminogen deficiency differentially affects recruitment of inflammatory cell populations in mice. Blood 91:2005, 1998.

203.
Carmeliet P, Moons L, Ploplis VA, Plow EF, Collen D: Impaired arterial neointima formation in mice with disruption of the plasminogen gene. J Clin Invest 99:200, 1997.

204.
Coleman JL, Gebbia JA, Piesman J, Degen JL, Bugge TH, Benach JL: Plasminogen is required for efficient dissemination of B. burgdorferi in ticks and for enhancement of spirochetemia in mice. Cell 89:1111, 1997.

205.
Chen ZL, Strickland SE: Neuronal cell death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell 91:917, 1997.

206.
Sporn MB, Roberts AB, Wakefield LM, Assoian RK: Transforming growth factor-beta: Biological function and chemical structure. Science 233:532, 1986.

207.
Lyons RM, Gentry LE, Purchio AF, Moses HL: Mechanism of activation of latent recombinant transforming growth factor beta1 by plasmin. J Cell Biol 110:1361, 1990.

208.
O’Reilly MS, Holmgren L, Shing Y, et al: Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79:315, 1995.

209.
Lucas R, Holmgren L, Garcia I, et al: Multiple forms of angiostatin induce apoptosis in endothelial cells. Blood 92:4730, 1998.

210.
O’Reilly MS, Holmgren L, Chen C, Folkman J: Angiostatin induces and sustains dormancy of human primary tumors in mice. Nature Med 2:689, 1996.

211.
Cao Y, O’Reilly MS, Marshall B, Flynn E, Ji RW, Folkman J: Expression of angiostatin cDNA in a murine fibrosarcoma suppresses primary tumor growth and produces long-term dormancy of metastases. J Clin Invest 101:1055, 1998.

212.
Moser TL, Pizzo SV, Enghild JJ, Hubchak S, Stack MS: Isolation of an angiostatin receptor from the membranes of human umbilical vein endothelial cells. Fibrinol Proteol 11:39, 1997.

213.
Cao Y, Chen A, Seong SSA, Ji RW, Davidson D, Llinas M: Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J Biol Chem 272:22924, 1997.

214.
Griscelli F, Li H, Bennaceur-Griscelli A, et al: Angiostatin gene transfer: Inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest. Proc Natl Acad Sci USA 95:6367, 1998.

215.
Dong Z, Kumar R, Yang X, Fidler I: Macrophage-derived metallo-elastase is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell 88:801, 1997.

216.
Stathakis P, Fitzgerald M, Matthias LJ, Chesterman CN, Hogg PJ: Generation of angiostatin by reduction and proteolysis of plasmin: Catalysis by a plasmin reductase secreted by cultured cells. J Biol Chem 272:20641, 1997.

217.
Patterson BC, Sang QXA: Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9). J Biol Chem 272:28823, 1997.

218.
Gately S, Twardowski P, Stack MS, et al: The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin. Proc Natl Acad Sci USA 94:10868, 1998.

219.
Aoki N, Moroi M, Sakata Y, Yoshida N, Matsuda M: Abnormal plasminogen: A hereditary molecular abnormality found in a patient with recurrent thrombosis. J Clin Invest 61:1186, 1978.

220.
Ichinose A, Espling ES, Takamatsu J, et al: Two types of abnormal genes for plasminogen in families with a predisposition for thrombosis. Proc Natl Acad Sci USA 88:115, 1991.

221.
Lijnen HR, Collen D: Congenital and acquired deficiencies of components of the fibrinolytic system and their relationship to bleeding or thrombosis. Fibrinolysis 3:67, 1989.

222.
Robbins KC: Dysplasminogenemia. Prog Cardiovasc Dis 34:295, 1992.

223.
Azuma H, Mima N, Shirakawa M, et al: Molecular pathogenesis of type I congenital plasminogen deficiency: Expression of recombinant human mutant plasminogens in mammalian cells. Blood 89:183, 1997.

224.
Demarmels Biasiutti F, Sulzer I, Stucki B, Wuillemin WA, Furlan M, Lammle B: Is plasminogen deficiency a thrombotic risk factor? A study on 23 thrombophilic patients and their family members. Thromb Haemost 80:167, 1998.

225.
Sartori MT, Patrassi GM, Theodoridis P, Perin A, Pietrogrande F, Girolami A: Heterozygous type I plasminogen deficiency is associated with an increased risk for thrombosis: A statistical analysis of 20 kindreds. Blood Coagul Fibrinol 5:889, 1994.

226.
Schott D, Dempfle CE, Beck P, et al: Therapy with a purified plasminogen concentrate in an infant with ligneous conjunctivitis and homozygous plasminogen deficiency. N Engl J Med 339:1679, 1998.

227.
Tsutsumi S, Saito T, Sakata T, Miyata T, Ichinose A: Genetic diagnosis of dysplasminogenemia: Detection of an Ala601-Thr mutation in 118 out of 125 families and identification of a new Asp676-Asn mutation. Thromb Haemost 76:135, 1996.

228.
Summaria L, Arzadon L, Bernabe P, Robbins KC: Studies on the isolation of the multiple molecular forms of human plasminogen and plasmin by isoelectric focusing methods. J Biochem Biophys 247:4691, 1972.

229.
Raum D, Marcus D, Alper CA: Genetic polymorphism of human plasminogen. Am J Hum Genet 32:681, 1980.

230.
Robbins KC: Classification of abnormal plasminogens: Dysplasminogenemias. Semin Thromb Haemost 16:217, 1990.

231.
Rakoczi I, Chamone D, Collen D, Verstraete M: Prediction of postoperative leg vein thrombosis in gynaecological patients. Lancet 1:509, 1978.

232.
Juhan-Vague I, Valadier J, Alessi MC, et al: Deficient t-PA release and elevated PA inhibitor levels in patients with spontaneous or recurrent leg thrombosis. Thromb Haemost 57:67, 1987.

233.
Hamsten A, Wiman B, De Faire U, Blomback M: Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction. N Engl J Med 313:1557, 1985.

234.
Paramo JA, Alfaro MJ, Rocha E: Postoperative changes in the plasmatic levels of tissue-type plasminogen activator and its fast-acting inhibitor: Relationship to deep vein thrombosis and influence of prophylaxis. Thromb Haemost 54:713, 1985.

235.
Juhan-Vague I, Roul C, Alessi MC, Ardissone JP, Heim M, Vague P: Increased plasminogen activator inhibitor activity in non-insulin dependent diabetic patients: Relationship with plasma insulin. Thromb Haemost 61:370, 1989.

236.
Juhan-Vague I, Alessi MC, Joly P, et al: Plasma plasminogen activator inhibitor-1 in angina pectoris: Influence of plasma insulin and acute-phase response. Arteriosclerosis 9:362, 1989.

237.
Stump DC, Taylor FB, Nesheim ME, Giles AR, Dzik WH, Bovill EG: Pathologic fibrinolysis as a cause of clinical bleeding. Semin Thromb Hemost 16:260, 1990.

238.
Saito H: Alpha-2-plasmin inhibitor and its deficiency states. J Lab Clin Med 112:671, 1988.

239.
Suarez CR, Walenga J, Mangogna LC, Fareed J: Neonatal and maternal fibrinolysis: Activation at time of birth. Am J Hematol 19:365, 1985.

240.
Summaria L: Comparison of human normal, full-term, fetal and adult plasminogen by physical and clinical analyses. Haemostasis 19:266, 1989.

241.
Edelberg JM, Enghild JJ, Pizzo SV, Gonzales-Gronow M: Neonatal plasminogen displays altered cell surface binding and activation kinetics: Correlation with increased glycosylation of the protein. J Clin Invest 86:107, 1990.

242.
Andrew M, Brooker L, Leaker M, Paes B, Weitz J: Fibrin clot lysis by thrombolytic agents is impaired in newborns due to a low plasminogen concentration. Thromb Haemost 68:325, 1992.

243.
Corrigan JJ, Sleeth JJ, Jeter MA, Lox CD: Newborn’s fibrinolytic mechanism: Components and plasmin generation. Am J Hematol 32:273, 1989.

244.
Corrigan JJ, Jeter MA: Histidine-rich glycoprotein and plasminogen plasma levels in term and preterm newborns. Am J Dis Child 144:825, 1990.

245.
Corrigan JJ, Jeter MA: Tissue-type plasminogen activator, plasminogen activator inhibitor, and histidine-rich glycoprotein in stressed human newborns. Pediatrics 89:43, 1992.

246.
Brus F, Van Oeveren W, Okkern A, Oetomo SB: Activation of the plasma clotting, fibrinolytic, and kinin-kallikrein system in preterm infants with severe idiopathic respiratory distress syndrome. Pediatr Res 36:647, 1994.

247.
Cederholm-Williams SA, Spencer JAD, Wilkerson AR: Plasma levels of selected haemostatic factors in newborn babies. Thromb Res 23:555, 1981.

248.
Andrew M, Massicotte-Nolan PM, Karpatkin M: Plasma protease inhibitors in premature infants: Influence of gestational age, postnatal age, and health status. Proc Soc Exp Biol Med 173:495, 1983.

249.
Corrigan JJ: Thrombosis and thromboembolism, in Hemorrhagic and Thrombotic Disease in Childhood and Adolescence, edited by JJ Corrigan, p 147. Churchill Livingstone, New York, 1985.

250.
Bonnar J, Daly L, Sheppard BL: Changes in the fibrinolytic system during pregnancy. Semin Thromb Hemost 16:221, 1990.

251.
Hellgren M: Hemostasis during pregnancy and puerperium. Haemostasis 26:244, 1996.

252.
Schjetlein R, Haugen G, Wisloff F: Markers of intravascular coagulation and fibrinolysis in preeclampsia: Association with intrauterine growth retardation. Acta Obstet Gynecol Scand 76:541, 1997.

253.
Ploplis VA, Carmeliet P, Vazirzadeh S, et al: Effects of disruption of the plasminogen gene on thrombosis, growth, and health in mice. Circulation 92:2585, 1995.

254.
Carmeliet P, Stassen JM, Schoonjans L, et al: Plasminogen activator inhibitor-1 gene-deficient mice: II. Effects on hemostasis, thrombosis, and thrombolysis. J Clin Invest 92:2756, 1993.

255.
Dewerchin M, Van Nuffelen A, Wallays G, et al: Generation and characterization of urokinase receptor-deficient mice. J Clin Invest 97:870, 1996.

256.
Lawn RM, Wade DP, Hammer RE, Chiesa G, Verstuyft JG, Rubin EM: Atherogenesis in transgenic mice expressing human apolipoprotein(a). Nature 360:670, 1992.

257.
Grainger DJ, Kemp PR, Liu AC, Lawn RM, Metcalfe JC: Activation of transforming growth factor-beta is inhibited in transgenic apolipoprotein(a) mice. Nature 370:460, 1994.

258.
Palabrica TM, Liu AC, Aronovitz MJ, Furie B, Lawn RM, Furie BC: Antifibrinolytic activity of apolipoprotein(a) in vivo: Human apolipoprotein(a) transgenic mice are resistant to tissue plasminogen activator-mediated thrombolysis. Nature Med 1:256, 1995.

259.
Boonmark NW, Lou XJ, Schwartz K, Zhang JL, Rubin EM, Lawn RM: Modification of apolipoprotein(a) lysine binding site reduces atherosclerosis in transgenic mice. J Clin Invest 100:558, 1997.

260.
Heckel JL, Sandgren EP, Degen JL, Palmiter RD, Brinster RL: Neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator. Cell 62:447, 1990.

261.
Meiri N, Masos T, Rosenblum K, Miskin R, Dudai Y: Overexpression of urokinase-type plasminogen activator in transgenic mice is correlated with impaired learning. Proc Natl Aca Sci USA 91:3196, 1994.
Books@Ovid
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology

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One comment on “CHAPTER 116 MOLECULAR MECHANISMS OF FIBRINOLYSIS

  1. […] CHAPTER 116 MOLECULAR MECHANISMS OF FIBRINOLYSIS … […]

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