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Thrombin and Fibrinolysis
Thrombin and Fibrinolysis* Michael Nesheim, PhD
When the activities of the coagulation and fibrinolytic cascades are properly regulated, so that fibrin (FN) deposition and removal are properly balanced, the vascular system is protected from catastrophic blood loss at the site of an injury, while its fluidity is ensured elsewhere. When these activities are not properly regulated, however, the organism is subjected to either excessive bleeding or thrombosis. Thrombomodulin on the endothelial cell is very important in this regulation because it converts thrombin to an anticoagulant enzyme by directing it toward the activation of protein C. It also converts thrombin to an antifibrinolytic enzyme by directing it toward the activation of thrombin-activatable fibrinolysis inhibitor (TAFI). By doing so, it creates a direct molecular connection between the coagulation and fibrinolytic cascades, such that activation of the former suppresses the activity of the latter. Recent studies indicate that the TAFI pathway functions in vivo and is likely relevant in maintaining the proper balance between FN deposition and removal. Whether it will be a target for pharmaceutical manipulation of this balance remains to be determined. (CHEST 2003; 124:33S–39S) Key words: bleeding; coagulation; fibrinolysis; plasmin; thrombin; thrombin-activatable fibrinolysis inhibitor; thrombomodulin; thrombosis Abbreviations: FDP ⫽ fibrin degradation product; FN ⫽ fibrin; FPA ⫽ fibrinopeptide A; FPB ⫽ fibrinopeptide B; GPg ⫽ gluplasminogen; GPn ⫽ glu-plasmin; LPg ⫽ lys-plasminogen; LPn ⫽ lys-plasmin; PAI-1 ⫽ plasminogen activator inhibitor type 1; PTCI ⫽ potato tuber carboxypeptidase inhibitor; TAFI ⫽ thrombinactivatable fibrinolysis inhibitor; TAFIa ⫽ carboxypeptidase B-like enzyme; tPA ⫽ tissue-type plasminogen activator
ongoing deposition and removal of fibrin (FN) both T heprotects the organism from untoward blood loss at the
site of injury and maintains the fluidity of the blood elsewhere.1– 4 FN deposition occurs on activation of the coagulation cascade. This results in the ultimate conversion of prothrombin to thrombin, which then catalyzes conversion of soluble fibrinogen to the gel-like substance, FN, the major proteinaceous component of the clot (Fig 1). FN removal occurs on activation of the fibrinolytic cascade. Plasminogen then converts to the enzyme plasmin, which catalyzes digestion of the FN clot to soluble FN degradation products. The activities of the cascades are normally latent. Their *From the Departments of Biochemistry and Medicine, Queen’s University, Kingston, ON, Canada. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Michael Nesheim, PhD, Departments of Biochemistry and Medicine, Botterell Hall, Room A210, Queen’s University, Kingston, ON, Canada, K7L 3N6; e-mail: nesheimm@ post.queensu.ca www.chestjournal.org
Figure 1. The balance between the formation and degradation of FN. The coagulation cascade ultimately generates thrombin, which catalyzes the conversion of fibrinogen to the fibrin clot. The fibrinolytic cascade generates plasmin, which catalyzes solubilization of the FN. The thrombin-thrombomodulin complex promotes down-regulation of thrombin formation by generating activated protein C (APC). It also suppresses fibrinolysis by forming TAFIa. The two cascades are thereby linked through the thrombin, thrombomodulin, and TAFI pathway. PC ⫽ protein C.
potentials, however, have been shown in an animal model (chimpanzee) to be remarkable.5 The coagulation cascade, for example, when acutely stimulated, can convert the entire plasma content of fibrinogen to FN and force the level of circulating platelets to zero in less than a minute. This potentially lethal phenomenon, however, is matched by an equally potent fibrinolytic response, such that within a minute or two, the plasma level of endogenous tissue plasminogen activator increases several hundred fold, the deposited FN is completely solubilized, and the circulating platelet level returns to normal. All of this occurs without long-term negative effects on the animal. Thus, both the coagulation and fibrinolytic cascades have the potential to generate very profound activities in a very acute manner. When the processes of FN deposition and removal are properly regulated, they perform their physiologic functions remarkably well. When they are not properly balanced, however, the pathophysiologic consequences are reflected in tendencies to bleed or thrombose. Prevalent thrombotic events include heart attacks and strokes. The regulation of the function of the cascades is expressed through the endothelium, the blood platelets, and the coagulation and fibrinolytic plasma proteins. One key player, indicated in Figure 1, is thrombomodulin,6 an integral membrane protein found on the endothelial cell. It binds to thrombin and changes its substrate specificity, such that it no longer recognizes fibrinogen as a substrate. Instead, it catalyzes conversion of the zymogen, protein C, to the anticoagulant enzyme, activated-protein C. This enzyme inactivates factors Va and VIIIa of the coagulation CHEST / 124 / 3 / SEPTEMBER, 2003 SUPPLEMENT
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cascade and thereby down-regulates thrombin formation. This subject is discussed in detail in the article by Dr. Esmon in this supplement. In addition, thrombin, particularly when it is bound to thrombomodulin, catalyzes the activation of the zymogen thrombin-activatable fibrinolysis inhibitor (TAFI) to the antifibrinolytic, carboxypeptidase B-like enzyme (TAFIa),7,8 which down-regulates the cofactor activity of FN in plasminogen activation and thereby suppresses fibrinolysis.9 The pathway defined by thrombin, thrombomodulin, and TAFI creates a direct molecular connection between the coagulation and fibrinolytic cascades, such that activation of the former suppresses the activity of the latter.10 Thus, this pathway may play a key role in the regulation of the balance between FN deposition and removal.
The Fibrinolytic Cascade The formation of FN from fibrinogen is depicted in Figure 2. The fibrinogen molecule comprises two globular D domains and a central, smaller, globular E domain. Thrombin catalyzes removal of fibrinopeptide A (FPA) and fibrinopeptide B (FPB) from the molecule. These events expose polymerization sites within the E domain such that E domains associate specifically, tightly, and noncovalently with sites on the D domains of neighboring molecules to initiate the polymerization. Protofibrils con-
Figure 2. The conversion of fibrinogen to FN. When thrombin acts on the soluble fibrinogen monomer, it catalyzes release of FPA and FPB from the amino termini of the ␣ and  chains of fibrinogen. This exposes polymerization sites in the E domains of fibrinogen such that noncovalent associations occur between the E and D domains of neighboring molecules, thereby producing double-stranded protofibrils. These laterally associate to form bundles, which continue to grow and branch, ultimately forming a three-dimensional web, which causes the solution to gel, thereby forming the familiar blood clot. The web is stabilized by covalent crosslinkages between adjacent D domains, which are formed by factor XIIIa. A carboxypeptidase B (CPB)-like enzyme also removes the COOH terminal arginine from FPB. 34S
sisting of two linear polymers form in a half-staggered arrangement. Simultaneously, factor XIII is activated to factor XIIIa, and it catalyzes covalent isopeptide bonds between ␥ chains in the D domains of adjacent FN monomers within the growing polymer. With further liberation of FPA and FPB, double-stranded protomers laterally associate to make FN bundles, which extend in length and occasionally form branches. As a result, a FN mesh develops that resembles a “three-dimensional spider web.” The solution in which this occurs becomes gel-like and forms, along with entrained cells, the familiar blood clot. In addition, a carboxyl terminal arginine is removed from FPB by a carboxypeptidase B-like enzyme. The FN clot is subsequently digested when the fibrinolytic cascade is triggered. These events are depicted in Figure 3.11 When FN forms, it serves as a cofactor for the conversion of glu-plasminogen (GPg) to glu-plasmin (GPn). This process begins digestion of FN by catalyzing cleavage after specific arginine and lysine residues in the ␣, , and ␥ chains of FN. The FN, while still clotted, is modified to a form designated FN⬘ in Figure 3. This modified form has in its structure carboxyl terminal lysine residues, which promote the binding of tissue-type plasminogen activator (tPA) and GPg to FN. This, in effect, up-regulates the
Figure 3. The fibrinolytic cascade. The cascade responds to FN generated by the action of thrombin on fibrinogen. tPA is released from the endothelium and, with FN as a cofactor, catalyzes conversion of GPg to GPn, which begins to digest FN to a modified form (FN⬘) with increased cofactor activity. FN⬘ also serves as a cofactor for the plasmin-catalyzed conversion of GPg to LPg, which is a better substrate than GPg for tPA. This reaction and the conversion of FN to FN⬘ represent positive feedback in the fibrinolytic cascade. When TAFIa is formed, it further modifies FN⬘ to FN⬙ and thereby eliminates positive feedback and attenuates fibrinolysis. The cascade is also downregulated by the serine protease inhibitors, PAI-1, and antiplasmin (AP), which irreversibly inactivate tPA and plasmin, respectively. TM ⫽ thrombomodulin. Thrombin: Physiology and Pathophysiology
cofactor activity of FN⬘ to a level threefold higher than that of intact FN. FN⬘ also serves as a cofactor for the proteolytic conversions of GPg and GPn to their lysine counterparts, lys-plasminogen (LPg) and lys-plasmin (LPn). These reactions are catalyzed by GPn and LPn. LPg is approximately 20-fold better than GPg as a substrate for tPA-catalyzed formation of plasmin (LPn). Thus, modification of FN to FN⬘ represents positive feedback in the fibrinolytic cascade, somewhat akin to that represented by thrombin-catalyzed activation of factors V and VIII in the coagulation cascade by thrombin. In response to thrombin, and especially thrombin bound to thrombomodulin, TAFI is activated to TAFIa. TAFIa then catalyzes removal of the carboxyl terminal lysine (and arginine) residues present in FN⬘, thereby producing a form of modified FN designated FN⬙.10 This down-regulates the cofactor activity of FN with respect to both the activation of GPg and the conversion of GPg to LPg. Thus, TAFIa, while not eliminating plasminogen activation, eliminates the positive feedback steps and thereby substantially attenuates plasminogen activation and fibrinolysis. The fibrinolytic cascade is also down-regulated by two fastacting serine protease inhibitors. One of these is plasminogen activator inhibitor type 1 (PAI-1), which targets tPA. The other is antiplasmin, which targets GPn and LPn. Interestingly, FN reduces the rate of inhibition of plasmin by antiplasmin by a factor of approximately 100. The fibrinolytic process terminates in the digestion of FN⬘ and FN⬙ to a family of soluble FN degradation products (FDPs), the smallest of which (DDE) is shown in Figure 3. The coagulation and fibrinolytic cascades have similar general features in common. They both respond to an activator liberated from the tissues (tissue factor in coagulation) or the endothelium (tPA in fibrinolysis), they both proceed by zymogen to protease conversions, they both have positive feedback steps and therefore generate explosive activity following an initiation phase, and they are down-regulated by both protease inhibitors and reactions mediated by the thrombin-thrombomodulin complex.
dependent carboxypeptidase and one molecule of zinc ion has been found per molecule of the enzyme.16 The protein was described by several groups at approximately the same time, and therefore has acquired several monikers. It is known as TAFI,14 plasma procarboxypeptidase B,17 procarboxy-peptidase U (for unstable),18 and procarboxypeptidase R (for arginine).19 The chromosomal location for TAFI is 13q14.11.20 The gene comprises 11 exons distributed on approximately 48 kilobase pairs of genomic DNA.21 It is expressed in the liver. It also has been found in platelets, and the messenger RNA has been found in the megakaryocyte cell lines DAMI and CHRF.22
Activation of TAFI and the Inactivation of TAFIa TAFI is activated by thrombin alone at a rate and extent sufficient to influence fibrinolysis, provided the thrombin level is very high, such as that immediately following clot formation in normal blood. In addition, it is very effectively activated by thrombin at low levels in the presence of thrombomodulin. Plasmin at high levels will also activate TAFI and the process is stimulated by heparin.23 The physiologic activator, however, is presumed to be thrombin, and particularly the thrombin-thrombomodulin complex.7 The kinetic mechanism for thrombin-thrombomodulin dependent activation is depicted in Figure 4. It can be described as a parallel assembly, enzyme central mechanism. In this mechanism, the central enzyme, thrombin, can interact with either TAFI or thrombomodulin to form the corresponding binary species. These then further interact with the third component to form the ternary thrombin-thrombomodulin-TAFI complex, from which TAFIa is generated. TAFIa can also be generated from the binary thrombin-TAFI complex, but the catalytic efficiency of the ternary complex is 1,250-fold greater than that of the binary complex. The reaction with thrombomodulin is Ca2⫹ dependent.14 This mechanism is formally very similar to that described for protein C activation, but numerous differences exist. More of the structure of
TAFI Protein and TAFI Gene TAFI is a single-chain plasma protein of 401 amino acids.12 Its peptide molecular mass is 45,999 d. In gel electrophoresis in dodecyl sulfate, it migrates with an apparent molecular weight of 60,000 d, a feature that can be attributed to four glycosaminoglycan chains in the amino-terminal region of the molecule.13 It circulates in plasma at a concentration of approximately 100 nmol (6 g/mL), a value similar to that of protein C.14 The plasma level of TAFI appears to be strongly determined by polymorphisms in the promoter and 3⬘ untranslated region of the gene.15 It is cleaved after arginine 92 by thrombin, thrombin-thrombomodulin, and plasmin to yield a 92amino acid activation fragment from the amino terminus and the carboxypeptidase B-like enzyme, TAFIa, from the carboxyl terminus. The enzyme comprises 309 amino acids and migrates in gel electrophoresis in dodecyl sulfate with an apparent molecular weight of 35,000 d. The enzyme is highly homologous to the pancreatic carboxypeptidaseB.12 The enzyme, like that of the pancreas, is a Zn2⫹www.chestjournal.org
Figure 4. Mechanism of TAFI activation. TAFI can be generated from the binary thrombin-TAFI complex (T-TAFI) or the ternary thrombin-thrombomodulin-TAFI complex (T-TMTAFI). Kd and Km values are the dissociation constants for the corresponding interactions, and k1 and k2 are turnover numbers for TAFIa generation. The catalytic efficiency for the thrombomodulin-dependent reaction is 1,250-fold greater than that for the thrombomodulin-independent reaction. CHEST / 124 / 3 / SEPTEMBER, 2003 SUPPLEMENT
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thrombomodulin is required for TAFI activation.24,25 In addition, regions of thrombin that are sufficient for the two reactions differ.26,27 Furthermore, the thrombomodulin dependence of TAFI activation is not determined by the amino acids surrounding the cleavage site.28 One particularly notable difference involves methionine 388 of thrombomodulin. This residue is highly susceptible to oxidation, a reaction that can be accomplished by inflammatory cells.29 Oxidation of this residue reduces the effectiveness of thrombomodulin in protein C activation by approximately 90% but does not affect TAFI activation.24,29 This observation prompts the speculation that the balance between FN deposition and FN removal might be tipped strongly toward thrombosis in an inflammatory milieu due to the loss of the anticoagulant but not antifibrinolytic properties of thrombomodulin. The enzyme, TAFIa, is intrinsically unstable.30,31 The kinetics of decay are highly dependent on temperature. At body temperature the enzyme has a half-life of approximately 8 min. The antifibrinolytic potential is directly related to the half-life. Since no naturally occurring inhibitors of TAFIa have been found in the plasma, the intrinsic instability appears to reflect the physiologic mechanism for down-regulation. The region of the molecule associated with instability has been shown to encompass amino acids 302–330.30,32 A common polymorphism in the human population has been found at position 325. One form exhibits threonine at this position and the other isoleucine. The latter form is twice as stable and 60% more potent as an antifibrinolytic. Whether this fact has pathophysiologic consequences, however, has yet to be determined, although a recent report indicates that it is not associated with a tendency for myocardial infarction.33
deficiencies in the initiation phase. Rather, they seem to correlate more obviously with the failure to produce the propagation phase. A logical conundrum still exists, however, because clotting is associated with the initiation phase, which is nearly normal in hemophiliac blood. Studies36 –38 have shown, however, that plasmas deficient in components of the intrinsic pathway (factors VIII, IX, and XI, but not XII), when clotted via the extrinsic pathway, in the presence of tPA, exhibit subsequent fibrinolysis three times earlier than normal plasmas. The phenomenon has been designated “premature lysis.” The mechanism behind this phenomenon has been worked out. In normal plasma, the burst of thrombin following clotting is sufficient, even in the absence of thrombomodulin, to activate sufficient TAFI to suppress subsequent plasminogen activation and fibrinolysis. This does not happen in the hemophiliac plasma, however, because of the lack of the thrombin burst in the propagation phase. Consequently, the clots formed in the initiation phase dissolve more quickly. The concepts are presented in Figure 5. This premature lysis to date has only been demonstrated in vitro. Its existence, however, has led to the hypothesis that bleeding in patients with defects in the intrinsic pathway may be as much due to the failure to suppress fibrinolysis as it is to a failure to form a clot in the first place.
Fibrinolysis and the Intrinsic Pathway of Coagulation A comprehensive analysis has been made of the temporal course of events associated with the clotting of whole blood when the process is initiated through the tissue factor-dependent (extrinsic) pathway.34,35 The process begins with an initiation phase, which lasts approximately 4.5 min. In this phase, thrombin at low levels catalyzes in sequence platelet activation, factor V activation, factor XIII activation, and FPA release. Over this interval, the thrombin level reaches approximately 25 nmol (2.5 U/mL). At the end of this phase, clotting occurs (4.7 min). In blood from normal individuals, the initiation phase is immediately followed by a propagation phase in which the intrinsic pathway is triggered and a relatively enormous burst of thrombin occurs within the clot, peaking at a concentration of approximately 850 nmol. This is then consumed fairly rapidly over the next few minutes by antithrombin and other protease inhibitors. In blood from people with deficiencies in the intrinsic pathway (eg, hemophilia A or B), the initiation phase is similar to that of blood from normal individuals, although clotting is delayed modestly. Strikingly, however, the large burst of thrombin following clotting is extremely diminished or absent. The dire bleeding tendencies in hemophilia A and B do not seem to be rationalized very well by modest 36S
Figure 5. Suppression of fibrinolysis through the intrinsic pathway. Initiation of coagulation via the extrinsic pathway generates small amounts of thrombin (IIa) from prothrombin (II) in the initiation phase. This is sufficient to form a clot by converting fibrinogen (FGN) to FN. It is also sufficient to initiate the intrinsic pathway through the thrombin-catalyzed activation of factor XI. This leads to a large burst of thrombin formation within the clot. The thrombin level is sufficiently high in this phase to activate enough TAFI to suppress plasminogen (Pg) activation to plasmin (Pn) and thereby attenuate digestion of the clot to soluble FDPs. Thrombin: Physiology and Pathophysiology
Some Potential Clinical Implications of the TAFI System Insufficient time has passed since the discovery of the TAFI system to have acquired enough information to determine definitively whether it will be relevant with respect to pathophysiology, diagnosis, or treatment. Epidemiologic studies and work in animals, however, suggest that it might. For example, elevated levels of plasma TAFI have been associated with a mildly elevated risk for venous thrombosis,39 and it is significantly elevated (50%) in patients with type 2 diabetes.40 In a large cohort studied in France, an increase in the TAFI antigen level was found to be a risk factor for angina pectoris; interestingly, the same result was not seen in a study in Northern Ireland.41 TAFI levels measured both by antigen detection and functional assays were found to be significantly lower than normal in disseminated intravascular coagulation.42 Similar measurements in acute promyelocytic leukemia showed that antigen levels were normal, but levels measured on the basis of function were reduced approximately 60%.43 The suggestion made in the report of this study was that the acquired TAFI deficiency in acute promyelocytic leukemia may contribute to the severity of the hemorrhagic diathesis in this disease because of an impairment in the capacity of the coagulation system to protect the clot from the fibrinolytic system. Levels of TAFI were found to be very low in patients with liver cirrhosis and even undetectable in patients with advanced hepatocellular disease.44,45 Recombinant factor VIIa, used for therapy of hemophiliacs with inhibitors to factor VIII, was shown to prolong clot lysis in vitro.46 The effect was shown to be TAFI dependent. The authors concluded that recombinant factor VIIa both accelerates clot formation and inhibits fibrinolysis in factor VIII-deficient plasma. They also reported a large individual variability in antifibrinolytic potential of recombinant factor VIIa in the population studied. These studies and others like them all suggest that TAFI may be linked to certain pathologic conditions, but to date no pathologic condition has been identified that can be linked directly to a defect in the TAFI system. Studies in animal models show that TAFIa functions as an antifibrinolytic agent in vivo. Early work by Redlitz et al47 showed that a carboxypeptidase B-like activity is induced when thrombolysis is initiated in a canine thrombolysis model. The activation of TAFI in another dog model of coronary artery thrombosis was shown to be inhibited by the thrombin inhibitor melagatran.48 Klement et al49 investigated the impact of a TAFIa inhibitor on tPA-induced clot lysis in a rabbit model of arterial thrombolysis. The inhibitor was a peptide isolated from the potato called potato tuber carboxypeptidase inhibitor (PTCI). They found that when the inhibitor was administered along with tPA, numerous parameters associated with thrombolysis were enhanced. The time to reperfusion was reduced by an approximate factor of three. Vessel patency was similarly improved. In their experiments, they measured both clot accretion and lysis. The TAFIa inhibitor did not influence accretion but it substantially promoted clot lysis. Similar studies were carried out by Nagashima et al50 in a rabbit model of venous thrombolwww.chestjournal.org
ysis, where the impact of PTCI on tPA-mediated clot lysis was determined. Indwelling clots were removed and weighted 90 min after initiation of the thrombolytic treatment, which consisted of a bolus of tPA, with or without PTCI, followed by continuous infusion of tPA, with or without PTCI. They found that the weight of the thrombus was not affected by PTCI when it was administered along with a saline solution control. When it was administered along with tPA, however, the clot weight was reduced to approximately one half of the control weight. tPA alone at the same dose reduced clot weight only 26%. This combined effect of tPA and PTCI could be obtained with three times the dose of tPA in the absence of PTCI. Thus, the TAFIa inhibitor effectively tripled the effect of a given dose of tPA. In addition, when PTCI was administered along with the higher dose of tPA, the clots were completely digested. A similar apparent tripling of the effectiveness of tPA was reported by Refino et al51 in another animal model of thrombolysis. These studies suggest that TAFIa inhibitors could be fruitfully employed as adjuvants in thrombolytic therapy. Whether drugs will inhibit TAFIa or suppress TAFI activation in order to promote endogenous fibrinolysis and thereby offer long-term protection from undesirable thrombotic events is not yet known. To date, no work in animals along these lines has been reported. Most work in vitro, however, indicates that TAFIa modulation affects the balance between FN deposition and removal, and thereby provides solid rationale for pursuing the appropriate investigations. The TAFI knockout mouse has been obtained.52 The mice are viable, and the absence of TAFI in this species does not create a phenotype, which suggests that TAFIa inhibition would not be unacceptably dangerous. In the case of hemophilia, a reasonable argument can be made that an agent that would enhance TAFI activation might help alleviate bleeding. An obvious choice for such an agent would be a soluble recombinant thrombomodulin engineered to promote TAFI activation but not protein C activation. In vitro, soluble thrombomodulin has been shown to correct the premature lysis of clots in hemophiliac plasma,36 because in the presence of thrombomodulin the thrombin generated in the initiation phase is adequate to activate TAFI, even in the absence of the thrombin burst, which occurs in normal plasma during the propagation phase. Since the elements of thrombomodulin structure needed to activate TAFI and protein C differ somewhat, the acquisition of a form of thrombomodulin selective for TAFI activation is feasible. In fact, two potential variants are currently known. One with oxidized methionine 388 is effective with TAFIa but not with protein C.29 In addition, a construct with tyrosine 348 replaced with alanine functions normally in TAFI activation but not protein C activation.24 Whether measurements of TAFI, TAFIa, the inactivated form of TAFIa, or other proteolytic fragments will prove useful for diagnosis, prognosis, or risk assessment is not yet known. This is also true for analyzing numerous polymorphisms in both the translated and untranslated portions of the TAFI gene. Several enzyme-linked immunosorbent assays are available commercially, however. In CHEST / 124 / 3 / SEPTEMBER, 2003 SUPPLEMENT
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addition, commercial assays exist that are based on the measurement of carboxypeptidase B-like activity after intentional activation of TAFI in the sample. A specific assay for endogenous TAFIa, however, has proven difficult to obtain. This is due, in large part, to the existence of a constitutively active carboxypeptidase B-like enzyme known as carboxypeptidase N.53 It is present at a relatively high concentration in plasma and provides an unacceptably high background activity for measuring TAFIa with small and not sufficiently selective substrates. Our laboratory has devised an assay based on attenuation by TAFIa of the plasminogen activator cofactor activity of high-molecular-soluble FN degradation products. Preliminary work has shown that endogenous TAFIa levels in normal plasma are remarkably low, on the order of 5 to 10 pmol (the TAFI zymogen level is approximately 100 nmol or 100,000 pmol). In collaboration with Dr. Fletcher Taylor, we have found that continuous infusion of the baboon with thrombin at a low level over a period of 1 h leads to a progressive increase in the plasma TAFIa concentration up to a level of 2,000 pmol. On termination of the thrombin infusion, the TAFIa level decreases approximately exponentially, with a half-life of approximately 10 min. Thus, TAFI is activated in vivo in response to thrombin, and the concentration of TAFIa achieved is sufficient to seriously attenuate fibrinolysis.14,31 Its relatively short half-life would suggest that elevated TAFIa would reflect events that are currently happening, rather than those that happened in the past.
Summary The coagulation and fibrinolytic cascades are generally thought of as separate and independent entities. Recent observations, however, suggest that they are directly linked to one another. This occurs not only through the TAFI pathway, but also through reactions whereby plasmin, for example, can both activate and/or inactivate coagulation factors V and IX.54,55 Consequently, methods for more efficacious control of bleeding or thrombosis will likely be obtained from insights gained through studies aimed at acquiring a more global appreciation of the mechanisms involved in regulating not just coagulation or fibrinolysis, but rather in regulating the balance between the deposition and removal of FN.
References 1 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991; 30:10363–10370 2 Mann KG, Bovill EG, Krishnaswamy S. Surface-dependent reactions in the propagation phase of blood coagulation. Ann N Y Acad Sci 1991; 614:63–75 3 Collen D, Lijnen HR. Basic and clinical aspects of fibrinolysis and thrombolysis. Blood 1991; 78:3114 –3124 4 Irigoyen JP, Munoz-Canoves P, Montero L, et al. The plasminogen activator system: biology and regulation. Cell Mol Life Sci 1999; 56:104 –132 5 Giles AR, Nesheim ME, Herring SW, et al. The fibrinolytic potential of the normal primate following the generation of thrombin in vivo. Thromb Haemost 1990; 63:476 – 481 6 Esmon CT. Regulation of blood coagulation. Biochim Biophys Acta 2000; 1477:349 –360 38S
7 Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. J Biol Chem 1996; 271:16603–16608 8 Bajzar L. Thrombin activatable fibrinolysis inhibitor and an antifibrinolytic pathway. Arterioscler Thromb Vasc Biol 2000; 20:2511–2518 9 Wang W, Boffa MB, Bajzar L, et al. A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor. J Biol Chem 1998; 273:27176 –27181 10 Nesheim M, Wang W, Boffa M, et al. Thrombin, thrombomodulin and TAFI in the molecular link between coagulation and fibrinolysis. Thromb Haemost 1997; 78:386 –391 11 Collen D, Lijnen HR. Molecular and cellular basis of fibrinolysis. In: Hoffman R, Benz E, Shattil SJ, et al, eds. Hematology: basic principles and practice. New York, NY: Churchill Livingstone, 1991; 1232–1242 12 Eaton DL, Malloy BE, Tsai SP, et al. Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma. J Biol Chem 1991; 266:21833–21838 13 Boffa MB, Wang W, Bajzar L, et al. Plasma and recombinant thrombin-activable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/ thrombomodulin-dependent activation, thermal stability, and enzymatic properties. J Biol Chem 1998; 273:2127–2135 14 Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. J Biol Chem 1995; 270:14477–14484 15 Henry M, Aubert H, Morange PE, et al. Identification of polymorphisms in the promoter and the 3⬘ region of the TAFI gene: evidence that plasma TAFI antigen levels are strongly genetically controlled. Blood 2001; 97:2053–2058 16 Marx PF, Bouma BN, Meijers JC. Role of zinc ions in activation and inactivation of thrombin-activatable fibrinolysis inhibitor. Biochemistry 2002; 41:1211–1216 17 Tan AK, Eaton DL. Activation and characterization of procarboxypeptidase B from human plasma. Biochemistry 1995; 34:5811–5816 18 Wang W, Hendriks DF, Scharpe SS. Carboxypeptidase U, a plasma carboxypeptidase with high affinity for plasminogen. J Biol Chem 1994; 269:15937–15944 19 Campbell W, Okada H. An arginine specific carboxypeptidase generated in blood during coagulation or inflammation which is unrelated to carboxypeptidase N or its subunits. Biochem Biophys Res Commun 1989; 162:933–939 20 Vanhoof G, Wauters J, Schatteman K, et al. The gene for human carboxypeptidase U (CPU): a proposed novel regulator of plasminogen activation; maps to 13q14.11. Genomics 1996; 38:454 – 455 21 Boffa MB, Reid TS, Joo E, et al. Characterization of the gene encoding human TAFI (thrombin-activable fibrinolysis inhibitor; plasma procarboxypeptidase B). Biochemistry 1999; 38:6547– 6558 22 Mosnier LO, Buijtenhuijs P, Marx PF, et al. Identification of thrombin activatable fibrinolysis inhibitor (TAFI) in human platelets. Blood 2003; 101:4844 – 4846 23 Anderson GM, Shaw AR, Shafer JA. Functional characterization of promoter elements involved in regulation of human B beta-fibrinogen expression: evidence for binding of novel activator and repressor proteins. J Biol Chem 1993; 268: 22650 –22655 24 Wang W, Nagashima M, Schneider M, et al. Elements of the primary structure of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation. J Biol Chem 2000; 275:22942–22947 25 Kokame K, Zheng X, Sadler JE. Activation of thrombin-activable fibrinolysis inhibitor requires epidermal growth factor-like doThrombin: Physiology and Pathophysiology
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30
31
32
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main 3 of thrombomodulin and is inhibited competitively by protein C. J Biol Chem 1998; 273:12135–12139 Hall SW, Nagashima M, Zhao L, et al. Thrombin interacts with thrombomodulin, protein C, and thrombin-activatable fibrinolysis inhibitor via specific and distinct domains. J Biol Chem 1999; 274:25510 –25516 Bell R, Stevens WK, Jia Z, et al. Fluorescence properties and functional roles of tryptophan residues 60d, 96, 148, 207, and 215 of thrombin. J Biol Chem 2000; 275:29513–29520 Schneider M, Nagashima M, Knappe S, et al. Amino acid residues in the P6-P’3 region of thrombin-activable fibrinolysis inhibitor (TAFI) do not determine the thrombomodulin dependence of TAFI activation. J Biol Chem 2002; 277: 9944 –9951 Glaser CB, Morser J, Clarke JH, et al. Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity: a potential rapid mechanism for modulation of coagulation. J Clin Invest 1992; 90:2565–2573 Boffa MB, Bell R, Stevens WK, et al. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor. J Biol Chem 2000; 275:12868 –12878 Schneider M, Boffa M, Stewart R, et al. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme. J Biol Chem 2002; 277:1021–1030 Marx PF, Hackeng TM, Dawson PE, et al. Inactivation of active thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage. J Biol Chem 2000; 275:12410 –12415 Morange PE, Henry M, Frere C, et al. Thr325IIe polymorphism of the TAFI gene does not influence the risk of myocardial infection. Blood 2002; 99:1878 –1879 Brummel KE, Paradis SG, Butenas S, et al. Thrombin functions during tissue factor-induced blood coagulation. Blood 2002; 100:148 –152 Butenas S, van ’t Veer C, Cawthern K, et al. Models of blood coagulation. Blood Coagul Fibrinolysis 2000; 11(suppl 1):S9– S13 Broze GJ Jr, Higuchi DA. Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma. Blood 1996; 88:3815– 3823 Mosnier LO, Lisman T, van den Berg HM, et al. The defective down regulation of fibrinolysis in haemophilia A can be restored by increasing the TAFI plasma concentration. Thromb Haemost 2001; 86:1035–1039 Von dem Borne PA, Bajzar L, Meijers JC, et al. Thrombinmediated activation of factor XI results in a thrombinactivatable fibrinolysis inhibitor-dependent inhibition of fibrinolysis. J Clin Invest 1997; 99:2323–2327 van Tilburg NH, Rosendaal FR, Bertina RM. Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis. Blood 2000; 95:2855–2859
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40 Yano Y, Kitagawa N, Gabazza EC, et al. Increased plasma thrombin-activatable fibrinolysis inhibitor levels in normotensive type 2 diabetic patients with microalbuminuria. J Clin Endocrinol Metab 2003; 88:736 –741 41 Morange PE, Juhan-Vague I, Scarabin PY, et al. Association between TAFI antigen and Ala147Thr polymorphism of the TAFI gene and the angina pectoris incidence. Thromb Haemost 2003; 89:554 –560 42 Watanabe R, Wada H, Watanabe Y, et al. Activity and antigen levels of thrombin-activatable fibrinolysis inhibitor in plasma of patients with disseminated intravascular coagulation. Thromb Res 2001; 104:1– 6 43 Meijers JC, Oudijk EJ, Mosnier LO, et al. Reduced activity of TAFI (thrombin-activatable fibrinolysis inhibitor) in acute promyelocytic leukaemia. Br J Haematol 2000; 108:518 –523 44 Van Thiel DH, George M, Fareed J. Low levels of thrombin activatable fibrinolysis inhibitor (TAFI) in patients with chronic liver disease. Thromb Haemost 2001; 85:667– 670 45 Lisman T, Leebeek FW, Mosnier LO, et al. Thrombinactivatable fibrinolysis inhibitor deficiency in cirrhosis is not associated with increased plasma fibrinolysis. Gastroenterology 2001; 121:131–139 46 Lisman T, Mosnier LO, Lambert T, et al. Inhibition of fibrinolysis by recombinant factor VIIa in plasma from patients with severe hemophilia A. Blood 2002; 99:175–179 47 Redlitz A, Nicolini FA, Malycky JL, et al. Inducible carboxypeptidase activity: a role in clot lysis in vivo. Circulation 1996; 93:1328 –1330 48 Mattsson C, Bjorkman JA, Abrahamsson T, et al. Local proCPU (TAFI) activation during thrombolytic treatment in a dog model of coronary artery thrombosis can be inhibited with a direct, small molecule thrombin inhibitor (melagatran). Thromb Haemost 2002; 87:557–562 49 Klement P, Liao P, Bajzar L. An inhibitor of activated TAFI enhances thrombolysis. Blood 1998; 92:1967–1975 50 Nagashima M, Werner M, Wang M, et al. An inhibitor of activated thrombin-activatable fibrinolysis inhibitor potentiates tissue-type plasminogen activator-induced thrombolysis in a rabbit jugular vein thrombolysis model. Thromb Res 2000; 98:333–342 51 Refino CJ, Schmitt D, Pater C, et al. Carboxypeptidase inhibitor markedly improves the potency of tPA in vivo. Fibrinolysis Proteolysis 1998; 12:29 52 Nagashima M, Yin ZF, Zhao L, et al. Thrombin-activatable fibrinolysis inhibitor (TAFI) deficiency is compatible with murine life. J Clin Invest 2002; 109:101–110 53 Skidgel RA. Human carboxypeptidase N: lysine carboxypeptidase. Methods Enzymol 1995; 248:653– 663 54 Lee CD, Mann KG. Activation/inactivation of human factor V by plasmin. Blood 1989; 73:185–190 55 Samis JA, Ramsey GD, Walker JB, et al. Proteolytic processing of human coagulation factor IX by plasmin. Blood 2000; 95:943–951
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When the activities of the coagulation and fibrinolytic cascades are properly regulated, so that fibrin (FN) deposition and removal are properly balanced, the vascular system is protected from catastrophic blood loss at the site of an injury, while its fluidity is ensured elsewhere. When these activities are not properly regulated, however, the organism is subjected to either excessive bleeding or thrombosis. Thrombomodulin on the endothelial cell is very important in this regulation because it converts thrombin to an anticoagulant enzyme by directing it toward the activation of protein C. It also converts thrombin to an antifibrinolytic enzyme by directing it toward the activation of thrombin-activatable fibrinolysis inhibitor (TAFI). By doing so, it creates a direct molecular connection between the coagulation and fibrinolytic cascades, such that activation of the former suppresses the activity of the latter. Recent studies indicate that the TAFI pathway functions in vivo and is likely relevant in maintaining the proper balance between FN deposition and removal. Whether it will be a target for pharmaceutical manipulation of this balance remains to be determined. (CHEST 2003; 124:33S–39S) Key words: bleeding; coagulation; fibrinolysis; plasmin; thrombin; thrombin-activatable fibrinolysis inhibitor; thrombomodulin; thrombosis Abbreviations: FDP ⫽ fibrin degradation product; FN ⫽ fibrin; FPA ⫽ fibrinopeptide A; FPB ⫽ fibrinopeptide B; GPg ⫽ gluplasminogen; GPn ⫽ glu-plasmin; LPg ⫽ lys-plasminogen; LPn ⫽ lys-plasmin; PAI-1 ⫽ plasminogen activator inhibitor type 1; PTCI ⫽ potato tuber carboxypeptidase inhibitor; TAFI ⫽ thrombinactivatable fibrinolysis inhibitor; TAFIa ⫽ carboxypeptidase B-like enzyme; tPA ⫽ tissue-type plasminogen activator
ongoing deposition and removal of fibrin (FN) both T heprotects the organism from untoward blood loss at the
site of injury and maintains the fluidity of the blood elsewhere.1– 4 FN deposition occurs on activation of the coagulation cascade. This results in the ultimate conversion of prothrombin to thrombin, which then catalyzes conversion of soluble fibrinogen to the gel-like substance, FN, the major proteinaceous component of the clot (Fig 1). FN removal occurs on activation of the fibrinolytic cascade. Plasminogen then converts to the enzyme plasmin, which catalyzes digestion of the FN clot to soluble FN degradation products. The activities of the cascades are normally latent. Their *From the Departments of Biochemistry and Medicine, Queen’s University, Kingston, ON, Canada. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Michael Nesheim, PhD, Departments of Biochemistry and Medicine, Botterell Hall, Room A210, Queen’s University, Kingston, ON, Canada, K7L 3N6; e-mail: nesheimm@ post.queensu.ca www.chestjournal.org
Figure 1. The balance between the formation and degradation of FN. The coagulation cascade ultimately generates thrombin, which catalyzes the conversion of fibrinogen to the fibrin clot. The fibrinolytic cascade generates plasmin, which catalyzes solubilization of the FN. The thrombin-thrombomodulin complex promotes down-regulation of thrombin formation by generating activated protein C (APC). It also suppresses fibrinolysis by forming TAFIa. The two cascades are thereby linked through the thrombin, thrombomodulin, and TAFI pathway. PC ⫽ protein C.
potentials, however, have been shown in an animal model (chimpanzee) to be remarkable.5 The coagulation cascade, for example, when acutely stimulated, can convert the entire plasma content of fibrinogen to FN and force the level of circulating platelets to zero in less than a minute. This potentially lethal phenomenon, however, is matched by an equally potent fibrinolytic response, such that within a minute or two, the plasma level of endogenous tissue plasminogen activator increases several hundred fold, the deposited FN is completely solubilized, and the circulating platelet level returns to normal. All of this occurs without long-term negative effects on the animal. Thus, both the coagulation and fibrinolytic cascades have the potential to generate very profound activities in a very acute manner. When the processes of FN deposition and removal are properly regulated, they perform their physiologic functions remarkably well. When they are not properly balanced, however, the pathophysiologic consequences are reflected in tendencies to bleed or thrombose. Prevalent thrombotic events include heart attacks and strokes. The regulation of the function of the cascades is expressed through the endothelium, the blood platelets, and the coagulation and fibrinolytic plasma proteins. One key player, indicated in Figure 1, is thrombomodulin,6 an integral membrane protein found on the endothelial cell. It binds to thrombin and changes its substrate specificity, such that it no longer recognizes fibrinogen as a substrate. Instead, it catalyzes conversion of the zymogen, protein C, to the anticoagulant enzyme, activated-protein C. This enzyme inactivates factors Va and VIIIa of the coagulation CHEST / 124 / 3 / SEPTEMBER, 2003 SUPPLEMENT
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cascade and thereby down-regulates thrombin formation. This subject is discussed in detail in the article by Dr. Esmon in this supplement. In addition, thrombin, particularly when it is bound to thrombomodulin, catalyzes the activation of the zymogen thrombin-activatable fibrinolysis inhibitor (TAFI) to the antifibrinolytic, carboxypeptidase B-like enzyme (TAFIa),7,8 which down-regulates the cofactor activity of FN in plasminogen activation and thereby suppresses fibrinolysis.9 The pathway defined by thrombin, thrombomodulin, and TAFI creates a direct molecular connection between the coagulation and fibrinolytic cascades, such that activation of the former suppresses the activity of the latter.10 Thus, this pathway may play a key role in the regulation of the balance between FN deposition and removal.
The Fibrinolytic Cascade The formation of FN from fibrinogen is depicted in Figure 2. The fibrinogen molecule comprises two globular D domains and a central, smaller, globular E domain. Thrombin catalyzes removal of fibrinopeptide A (FPA) and fibrinopeptide B (FPB) from the molecule. These events expose polymerization sites within the E domain such that E domains associate specifically, tightly, and noncovalently with sites on the D domains of neighboring molecules to initiate the polymerization. Protofibrils con-
Figure 2. The conversion of fibrinogen to FN. When thrombin acts on the soluble fibrinogen monomer, it catalyzes release of FPA and FPB from the amino termini of the ␣ and  chains of fibrinogen. This exposes polymerization sites in the E domains of fibrinogen such that noncovalent associations occur between the E and D domains of neighboring molecules, thereby producing double-stranded protofibrils. These laterally associate to form bundles, which continue to grow and branch, ultimately forming a three-dimensional web, which causes the solution to gel, thereby forming the familiar blood clot. The web is stabilized by covalent crosslinkages between adjacent D domains, which are formed by factor XIIIa. A carboxypeptidase B (CPB)-like enzyme also removes the COOH terminal arginine from FPB. 34S
sisting of two linear polymers form in a half-staggered arrangement. Simultaneously, factor XIII is activated to factor XIIIa, and it catalyzes covalent isopeptide bonds between ␥ chains in the D domains of adjacent FN monomers within the growing polymer. With further liberation of FPA and FPB, double-stranded protomers laterally associate to make FN bundles, which extend in length and occasionally form branches. As a result, a FN mesh develops that resembles a “three-dimensional spider web.” The solution in which this occurs becomes gel-like and forms, along with entrained cells, the familiar blood clot. In addition, a carboxyl terminal arginine is removed from FPB by a carboxypeptidase B-like enzyme. The FN clot is subsequently digested when the fibrinolytic cascade is triggered. These events are depicted in Figure 3.11 When FN forms, it serves as a cofactor for the conversion of glu-plasminogen (GPg) to glu-plasmin (GPn). This process begins digestion of FN by catalyzing cleavage after specific arginine and lysine residues in the ␣, , and ␥ chains of FN. The FN, while still clotted, is modified to a form designated FN⬘ in Figure 3. This modified form has in its structure carboxyl terminal lysine residues, which promote the binding of tissue-type plasminogen activator (tPA) and GPg to FN. This, in effect, up-regulates the
Figure 3. The fibrinolytic cascade. The cascade responds to FN generated by the action of thrombin on fibrinogen. tPA is released from the endothelium and, with FN as a cofactor, catalyzes conversion of GPg to GPn, which begins to digest FN to a modified form (FN⬘) with increased cofactor activity. FN⬘ also serves as a cofactor for the plasmin-catalyzed conversion of GPg to LPg, which is a better substrate than GPg for tPA. This reaction and the conversion of FN to FN⬘ represent positive feedback in the fibrinolytic cascade. When TAFIa is formed, it further modifies FN⬘ to FN⬙ and thereby eliminates positive feedback and attenuates fibrinolysis. The cascade is also downregulated by the serine protease inhibitors, PAI-1, and antiplasmin (AP), which irreversibly inactivate tPA and plasmin, respectively. TM ⫽ thrombomodulin. Thrombin: Physiology and Pathophysiology
cofactor activity of FN⬘ to a level threefold higher than that of intact FN. FN⬘ also serves as a cofactor for the proteolytic conversions of GPg and GPn to their lysine counterparts, lys-plasminogen (LPg) and lys-plasmin (LPn). These reactions are catalyzed by GPn and LPn. LPg is approximately 20-fold better than GPg as a substrate for tPA-catalyzed formation of plasmin (LPn). Thus, modification of FN to FN⬘ represents positive feedback in the fibrinolytic cascade, somewhat akin to that represented by thrombin-catalyzed activation of factors V and VIII in the coagulation cascade by thrombin. In response to thrombin, and especially thrombin bound to thrombomodulin, TAFI is activated to TAFIa. TAFIa then catalyzes removal of the carboxyl terminal lysine (and arginine) residues present in FN⬘, thereby producing a form of modified FN designated FN⬙.10 This down-regulates the cofactor activity of FN with respect to both the activation of GPg and the conversion of GPg to LPg. Thus, TAFIa, while not eliminating plasminogen activation, eliminates the positive feedback steps and thereby substantially attenuates plasminogen activation and fibrinolysis. The fibrinolytic cascade is also down-regulated by two fastacting serine protease inhibitors. One of these is plasminogen activator inhibitor type 1 (PAI-1), which targets tPA. The other is antiplasmin, which targets GPn and LPn. Interestingly, FN reduces the rate of inhibition of plasmin by antiplasmin by a factor of approximately 100. The fibrinolytic process terminates in the digestion of FN⬘ and FN⬙ to a family of soluble FN degradation products (FDPs), the smallest of which (DDE) is shown in Figure 3. The coagulation and fibrinolytic cascades have similar general features in common. They both respond to an activator liberated from the tissues (tissue factor in coagulation) or the endothelium (tPA in fibrinolysis), they both proceed by zymogen to protease conversions, they both have positive feedback steps and therefore generate explosive activity following an initiation phase, and they are down-regulated by both protease inhibitors and reactions mediated by the thrombin-thrombomodulin complex.
dependent carboxypeptidase and one molecule of zinc ion has been found per molecule of the enzyme.16 The protein was described by several groups at approximately the same time, and therefore has acquired several monikers. It is known as TAFI,14 plasma procarboxypeptidase B,17 procarboxy-peptidase U (for unstable),18 and procarboxypeptidase R (for arginine).19 The chromosomal location for TAFI is 13q14.11.20 The gene comprises 11 exons distributed on approximately 48 kilobase pairs of genomic DNA.21 It is expressed in the liver. It also has been found in platelets, and the messenger RNA has been found in the megakaryocyte cell lines DAMI and CHRF.22
Activation of TAFI and the Inactivation of TAFIa TAFI is activated by thrombin alone at a rate and extent sufficient to influence fibrinolysis, provided the thrombin level is very high, such as that immediately following clot formation in normal blood. In addition, it is very effectively activated by thrombin at low levels in the presence of thrombomodulin. Plasmin at high levels will also activate TAFI and the process is stimulated by heparin.23 The physiologic activator, however, is presumed to be thrombin, and particularly the thrombin-thrombomodulin complex.7 The kinetic mechanism for thrombin-thrombomodulin dependent activation is depicted in Figure 4. It can be described as a parallel assembly, enzyme central mechanism. In this mechanism, the central enzyme, thrombin, can interact with either TAFI or thrombomodulin to form the corresponding binary species. These then further interact with the third component to form the ternary thrombin-thrombomodulin-TAFI complex, from which TAFIa is generated. TAFIa can also be generated from the binary thrombin-TAFI complex, but the catalytic efficiency of the ternary complex is 1,250-fold greater than that of the binary complex. The reaction with thrombomodulin is Ca2⫹ dependent.14 This mechanism is formally very similar to that described for protein C activation, but numerous differences exist. More of the structure of
TAFI Protein and TAFI Gene TAFI is a single-chain plasma protein of 401 amino acids.12 Its peptide molecular mass is 45,999 d. In gel electrophoresis in dodecyl sulfate, it migrates with an apparent molecular weight of 60,000 d, a feature that can be attributed to four glycosaminoglycan chains in the amino-terminal region of the molecule.13 It circulates in plasma at a concentration of approximately 100 nmol (6 g/mL), a value similar to that of protein C.14 The plasma level of TAFI appears to be strongly determined by polymorphisms in the promoter and 3⬘ untranslated region of the gene.15 It is cleaved after arginine 92 by thrombin, thrombin-thrombomodulin, and plasmin to yield a 92amino acid activation fragment from the amino terminus and the carboxypeptidase B-like enzyme, TAFIa, from the carboxyl terminus. The enzyme comprises 309 amino acids and migrates in gel electrophoresis in dodecyl sulfate with an apparent molecular weight of 35,000 d. The enzyme is highly homologous to the pancreatic carboxypeptidaseB.12 The enzyme, like that of the pancreas, is a Zn2⫹www.chestjournal.org
Figure 4. Mechanism of TAFI activation. TAFI can be generated from the binary thrombin-TAFI complex (T-TAFI) or the ternary thrombin-thrombomodulin-TAFI complex (T-TMTAFI). Kd and Km values are the dissociation constants for the corresponding interactions, and k1 and k2 are turnover numbers for TAFIa generation. The catalytic efficiency for the thrombomodulin-dependent reaction is 1,250-fold greater than that for the thrombomodulin-independent reaction. CHEST / 124 / 3 / SEPTEMBER, 2003 SUPPLEMENT
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thrombomodulin is required for TAFI activation.24,25 In addition, regions of thrombin that are sufficient for the two reactions differ.26,27 Furthermore, the thrombomodulin dependence of TAFI activation is not determined by the amino acids surrounding the cleavage site.28 One particularly notable difference involves methionine 388 of thrombomodulin. This residue is highly susceptible to oxidation, a reaction that can be accomplished by inflammatory cells.29 Oxidation of this residue reduces the effectiveness of thrombomodulin in protein C activation by approximately 90% but does not affect TAFI activation.24,29 This observation prompts the speculation that the balance between FN deposition and FN removal might be tipped strongly toward thrombosis in an inflammatory milieu due to the loss of the anticoagulant but not antifibrinolytic properties of thrombomodulin. The enzyme, TAFIa, is intrinsically unstable.30,31 The kinetics of decay are highly dependent on temperature. At body temperature the enzyme has a half-life of approximately 8 min. The antifibrinolytic potential is directly related to the half-life. Since no naturally occurring inhibitors of TAFIa have been found in the plasma, the intrinsic instability appears to reflect the physiologic mechanism for down-regulation. The region of the molecule associated with instability has been shown to encompass amino acids 302–330.30,32 A common polymorphism in the human population has been found at position 325. One form exhibits threonine at this position and the other isoleucine. The latter form is twice as stable and 60% more potent as an antifibrinolytic. Whether this fact has pathophysiologic consequences, however, has yet to be determined, although a recent report indicates that it is not associated with a tendency for myocardial infarction.33
deficiencies in the initiation phase. Rather, they seem to correlate more obviously with the failure to produce the propagation phase. A logical conundrum still exists, however, because clotting is associated with the initiation phase, which is nearly normal in hemophiliac blood. Studies36 –38 have shown, however, that plasmas deficient in components of the intrinsic pathway (factors VIII, IX, and XI, but not XII), when clotted via the extrinsic pathway, in the presence of tPA, exhibit subsequent fibrinolysis three times earlier than normal plasmas. The phenomenon has been designated “premature lysis.” The mechanism behind this phenomenon has been worked out. In normal plasma, the burst of thrombin following clotting is sufficient, even in the absence of thrombomodulin, to activate sufficient TAFI to suppress subsequent plasminogen activation and fibrinolysis. This does not happen in the hemophiliac plasma, however, because of the lack of the thrombin burst in the propagation phase. Consequently, the clots formed in the initiation phase dissolve more quickly. The concepts are presented in Figure 5. This premature lysis to date has only been demonstrated in vitro. Its existence, however, has led to the hypothesis that bleeding in patients with defects in the intrinsic pathway may be as much due to the failure to suppress fibrinolysis as it is to a failure to form a clot in the first place.
Fibrinolysis and the Intrinsic Pathway of Coagulation A comprehensive analysis has been made of the temporal course of events associated with the clotting of whole blood when the process is initiated through the tissue factor-dependent (extrinsic) pathway.34,35 The process begins with an initiation phase, which lasts approximately 4.5 min. In this phase, thrombin at low levels catalyzes in sequence platelet activation, factor V activation, factor XIII activation, and FPA release. Over this interval, the thrombin level reaches approximately 25 nmol (2.5 U/mL). At the end of this phase, clotting occurs (4.7 min). In blood from normal individuals, the initiation phase is immediately followed by a propagation phase in which the intrinsic pathway is triggered and a relatively enormous burst of thrombin occurs within the clot, peaking at a concentration of approximately 850 nmol. This is then consumed fairly rapidly over the next few minutes by antithrombin and other protease inhibitors. In blood from people with deficiencies in the intrinsic pathway (eg, hemophilia A or B), the initiation phase is similar to that of blood from normal individuals, although clotting is delayed modestly. Strikingly, however, the large burst of thrombin following clotting is extremely diminished or absent. The dire bleeding tendencies in hemophilia A and B do not seem to be rationalized very well by modest 36S
Figure 5. Suppression of fibrinolysis through the intrinsic pathway. Initiation of coagulation via the extrinsic pathway generates small amounts of thrombin (IIa) from prothrombin (II) in the initiation phase. This is sufficient to form a clot by converting fibrinogen (FGN) to FN. It is also sufficient to initiate the intrinsic pathway through the thrombin-catalyzed activation of factor XI. This leads to a large burst of thrombin formation within the clot. The thrombin level is sufficiently high in this phase to activate enough TAFI to suppress plasminogen (Pg) activation to plasmin (Pn) and thereby attenuate digestion of the clot to soluble FDPs. Thrombin: Physiology and Pathophysiology
Some Potential Clinical Implications of the TAFI System Insufficient time has passed since the discovery of the TAFI system to have acquired enough information to determine definitively whether it will be relevant with respect to pathophysiology, diagnosis, or treatment. Epidemiologic studies and work in animals, however, suggest that it might. For example, elevated levels of plasma TAFI have been associated with a mildly elevated risk for venous thrombosis,39 and it is significantly elevated (50%) in patients with type 2 diabetes.40 In a large cohort studied in France, an increase in the TAFI antigen level was found to be a risk factor for angina pectoris; interestingly, the same result was not seen in a study in Northern Ireland.41 TAFI levels measured both by antigen detection and functional assays were found to be significantly lower than normal in disseminated intravascular coagulation.42 Similar measurements in acute promyelocytic leukemia showed that antigen levels were normal, but levels measured on the basis of function were reduced approximately 60%.43 The suggestion made in the report of this study was that the acquired TAFI deficiency in acute promyelocytic leukemia may contribute to the severity of the hemorrhagic diathesis in this disease because of an impairment in the capacity of the coagulation system to protect the clot from the fibrinolytic system. Levels of TAFI were found to be very low in patients with liver cirrhosis and even undetectable in patients with advanced hepatocellular disease.44,45 Recombinant factor VIIa, used for therapy of hemophiliacs with inhibitors to factor VIII, was shown to prolong clot lysis in vitro.46 The effect was shown to be TAFI dependent. The authors concluded that recombinant factor VIIa both accelerates clot formation and inhibits fibrinolysis in factor VIII-deficient plasma. They also reported a large individual variability in antifibrinolytic potential of recombinant factor VIIa in the population studied. These studies and others like them all suggest that TAFI may be linked to certain pathologic conditions, but to date no pathologic condition has been identified that can be linked directly to a defect in the TAFI system. Studies in animal models show that TAFIa functions as an antifibrinolytic agent in vivo. Early work by Redlitz et al47 showed that a carboxypeptidase B-like activity is induced when thrombolysis is initiated in a canine thrombolysis model. The activation of TAFI in another dog model of coronary artery thrombosis was shown to be inhibited by the thrombin inhibitor melagatran.48 Klement et al49 investigated the impact of a TAFIa inhibitor on tPA-induced clot lysis in a rabbit model of arterial thrombolysis. The inhibitor was a peptide isolated from the potato called potato tuber carboxypeptidase inhibitor (PTCI). They found that when the inhibitor was administered along with tPA, numerous parameters associated with thrombolysis were enhanced. The time to reperfusion was reduced by an approximate factor of three. Vessel patency was similarly improved. In their experiments, they measured both clot accretion and lysis. The TAFIa inhibitor did not influence accretion but it substantially promoted clot lysis. Similar studies were carried out by Nagashima et al50 in a rabbit model of venous thrombolwww.chestjournal.org
ysis, where the impact of PTCI on tPA-mediated clot lysis was determined. Indwelling clots were removed and weighted 90 min after initiation of the thrombolytic treatment, which consisted of a bolus of tPA, with or without PTCI, followed by continuous infusion of tPA, with or without PTCI. They found that the weight of the thrombus was not affected by PTCI when it was administered along with a saline solution control. When it was administered along with tPA, however, the clot weight was reduced to approximately one half of the control weight. tPA alone at the same dose reduced clot weight only 26%. This combined effect of tPA and PTCI could be obtained with three times the dose of tPA in the absence of PTCI. Thus, the TAFIa inhibitor effectively tripled the effect of a given dose of tPA. In addition, when PTCI was administered along with the higher dose of tPA, the clots were completely digested. A similar apparent tripling of the effectiveness of tPA was reported by Refino et al51 in another animal model of thrombolysis. These studies suggest that TAFIa inhibitors could be fruitfully employed as adjuvants in thrombolytic therapy. Whether drugs will inhibit TAFIa or suppress TAFI activation in order to promote endogenous fibrinolysis and thereby offer long-term protection from undesirable thrombotic events is not yet known. To date, no work in animals along these lines has been reported. Most work in vitro, however, indicates that TAFIa modulation affects the balance between FN deposition and removal, and thereby provides solid rationale for pursuing the appropriate investigations. The TAFI knockout mouse has been obtained.52 The mice are viable, and the absence of TAFI in this species does not create a phenotype, which suggests that TAFIa inhibition would not be unacceptably dangerous. In the case of hemophilia, a reasonable argument can be made that an agent that would enhance TAFI activation might help alleviate bleeding. An obvious choice for such an agent would be a soluble recombinant thrombomodulin engineered to promote TAFI activation but not protein C activation. In vitro, soluble thrombomodulin has been shown to correct the premature lysis of clots in hemophiliac plasma,36 because in the presence of thrombomodulin the thrombin generated in the initiation phase is adequate to activate TAFI, even in the absence of the thrombin burst, which occurs in normal plasma during the propagation phase. Since the elements of thrombomodulin structure needed to activate TAFI and protein C differ somewhat, the acquisition of a form of thrombomodulin selective for TAFI activation is feasible. In fact, two potential variants are currently known. One with oxidized methionine 388 is effective with TAFIa but not with protein C.29 In addition, a construct with tyrosine 348 replaced with alanine functions normally in TAFI activation but not protein C activation.24 Whether measurements of TAFI, TAFIa, the inactivated form of TAFIa, or other proteolytic fragments will prove useful for diagnosis, prognosis, or risk assessment is not yet known. This is also true for analyzing numerous polymorphisms in both the translated and untranslated portions of the TAFI gene. Several enzyme-linked immunosorbent assays are available commercially, however. In CHEST / 124 / 3 / SEPTEMBER, 2003 SUPPLEMENT
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addition, commercial assays exist that are based on the measurement of carboxypeptidase B-like activity after intentional activation of TAFI in the sample. A specific assay for endogenous TAFIa, however, has proven difficult to obtain. This is due, in large part, to the existence of a constitutively active carboxypeptidase B-like enzyme known as carboxypeptidase N.53 It is present at a relatively high concentration in plasma and provides an unacceptably high background activity for measuring TAFIa with small and not sufficiently selective substrates. Our laboratory has devised an assay based on attenuation by TAFIa of the plasminogen activator cofactor activity of high-molecular-soluble FN degradation products. Preliminary work has shown that endogenous TAFIa levels in normal plasma are remarkably low, on the order of 5 to 10 pmol (the TAFI zymogen level is approximately 100 nmol or 100,000 pmol). In collaboration with Dr. Fletcher Taylor, we have found that continuous infusion of the baboon with thrombin at a low level over a period of 1 h leads to a progressive increase in the plasma TAFIa concentration up to a level of 2,000 pmol. On termination of the thrombin infusion, the TAFIa level decreases approximately exponentially, with a half-life of approximately 10 min. Thus, TAFI is activated in vivo in response to thrombin, and the concentration of TAFIa achieved is sufficient to seriously attenuate fibrinolysis.14,31 Its relatively short half-life would suggest that elevated TAFIa would reflect events that are currently happening, rather than those that happened in the past.
Summary The coagulation and fibrinolytic cascades are generally thought of as separate and independent entities. Recent observations, however, suggest that they are directly linked to one another. This occurs not only through the TAFI pathway, but also through reactions whereby plasmin, for example, can both activate and/or inactivate coagulation factors V and IX.54,55 Consequently, methods for more efficacious control of bleeding or thrombosis will likely be obtained from insights gained through studies aimed at acquiring a more global appreciation of the mechanisms involved in regulating not just coagulation or fibrinolysis, but rather in regulating the balance between the deposition and removal of FN.
References 1 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991; 30:10363–10370 2 Mann KG, Bovill EG, Krishnaswamy S. Surface-dependent reactions in the propagation phase of blood coagulation. Ann N Y Acad Sci 1991; 614:63–75 3 Collen D, Lijnen HR. Basic and clinical aspects of fibrinolysis and thrombolysis. Blood 1991; 78:3114 –3124 4 Irigoyen JP, Munoz-Canoves P, Montero L, et al. The plasminogen activator system: biology and regulation. Cell Mol Life Sci 1999; 56:104 –132 5 Giles AR, Nesheim ME, Herring SW, et al. The fibrinolytic potential of the normal primate following the generation of thrombin in vivo. Thromb Haemost 1990; 63:476 – 481 6 Esmon CT. Regulation of blood coagulation. Biochim Biophys Acta 2000; 1477:349 –360 38S
7 Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. J Biol Chem 1996; 271:16603–16608 8 Bajzar L. Thrombin activatable fibrinolysis inhibitor and an antifibrinolytic pathway. Arterioscler Thromb Vasc Biol 2000; 20:2511–2518 9 Wang W, Boffa MB, Bajzar L, et al. A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor. J Biol Chem 1998; 273:27176 –27181 10 Nesheim M, Wang W, Boffa M, et al. Thrombin, thrombomodulin and TAFI in the molecular link between coagulation and fibrinolysis. Thromb Haemost 1997; 78:386 –391 11 Collen D, Lijnen HR. Molecular and cellular basis of fibrinolysis. In: Hoffman R, Benz E, Shattil SJ, et al, eds. Hematology: basic principles and practice. New York, NY: Churchill Livingstone, 1991; 1232–1242 12 Eaton DL, Malloy BE, Tsai SP, et al. Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma. J Biol Chem 1991; 266:21833–21838 13 Boffa MB, Wang W, Bajzar L, et al. Plasma and recombinant thrombin-activable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/ thrombomodulin-dependent activation, thermal stability, and enzymatic properties. J Biol Chem 1998; 273:2127–2135 14 Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. J Biol Chem 1995; 270:14477–14484 15 Henry M, Aubert H, Morange PE, et al. Identification of polymorphisms in the promoter and the 3⬘ region of the TAFI gene: evidence that plasma TAFI antigen levels are strongly genetically controlled. Blood 2001; 97:2053–2058 16 Marx PF, Bouma BN, Meijers JC. Role of zinc ions in activation and inactivation of thrombin-activatable fibrinolysis inhibitor. Biochemistry 2002; 41:1211–1216 17 Tan AK, Eaton DL. Activation and characterization of procarboxypeptidase B from human plasma. Biochemistry 1995; 34:5811–5816 18 Wang W, Hendriks DF, Scharpe SS. Carboxypeptidase U, a plasma carboxypeptidase with high affinity for plasminogen. J Biol Chem 1994; 269:15937–15944 19 Campbell W, Okada H. An arginine specific carboxypeptidase generated in blood during coagulation or inflammation which is unrelated to carboxypeptidase N or its subunits. Biochem Biophys Res Commun 1989; 162:933–939 20 Vanhoof G, Wauters J, Schatteman K, et al. The gene for human carboxypeptidase U (CPU): a proposed novel regulator of plasminogen activation; maps to 13q14.11. Genomics 1996; 38:454 – 455 21 Boffa MB, Reid TS, Joo E, et al. Characterization of the gene encoding human TAFI (thrombin-activable fibrinolysis inhibitor; plasma procarboxypeptidase B). Biochemistry 1999; 38:6547– 6558 22 Mosnier LO, Buijtenhuijs P, Marx PF, et al. Identification of thrombin activatable fibrinolysis inhibitor (TAFI) in human platelets. Blood 2003; 101:4844 – 4846 23 Anderson GM, Shaw AR, Shafer JA. Functional characterization of promoter elements involved in regulation of human B beta-fibrinogen expression: evidence for binding of novel activator and repressor proteins. J Biol Chem 1993; 268: 22650 –22655 24 Wang W, Nagashima M, Schneider M, et al. Elements of the primary structure of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation. J Biol Chem 2000; 275:22942–22947 25 Kokame K, Zheng X, Sadler JE. Activation of thrombin-activable fibrinolysis inhibitor requires epidermal growth factor-like doThrombin: Physiology and Pathophysiology
26
27 28
29
30
31
32
33 34 35 36
37
38
39
main 3 of thrombomodulin and is inhibited competitively by protein C. J Biol Chem 1998; 273:12135–12139 Hall SW, Nagashima M, Zhao L, et al. Thrombin interacts with thrombomodulin, protein C, and thrombin-activatable fibrinolysis inhibitor via specific and distinct domains. J Biol Chem 1999; 274:25510 –25516 Bell R, Stevens WK, Jia Z, et al. Fluorescence properties and functional roles of tryptophan residues 60d, 96, 148, 207, and 215 of thrombin. J Biol Chem 2000; 275:29513–29520 Schneider M, Nagashima M, Knappe S, et al. Amino acid residues in the P6-P’3 region of thrombin-activable fibrinolysis inhibitor (TAFI) do not determine the thrombomodulin dependence of TAFI activation. J Biol Chem 2002; 277: 9944 –9951 Glaser CB, Morser J, Clarke JH, et al. Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity: a potential rapid mechanism for modulation of coagulation. J Clin Invest 1992; 90:2565–2573 Boffa MB, Bell R, Stevens WK, et al. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor. J Biol Chem 2000; 275:12868 –12878 Schneider M, Boffa M, Stewart R, et al. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme. J Biol Chem 2002; 277:1021–1030 Marx PF, Hackeng TM, Dawson PE, et al. Inactivation of active thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage. J Biol Chem 2000; 275:12410 –12415 Morange PE, Henry M, Frere C, et al. Thr325IIe polymorphism of the TAFI gene does not influence the risk of myocardial infection. Blood 2002; 99:1878 –1879 Brummel KE, Paradis SG, Butenas S, et al. Thrombin functions during tissue factor-induced blood coagulation. Blood 2002; 100:148 –152 Butenas S, van ’t Veer C, Cawthern K, et al. Models of blood coagulation. Blood Coagul Fibrinolysis 2000; 11(suppl 1):S9– S13 Broze GJ Jr, Higuchi DA. Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma. Blood 1996; 88:3815– 3823 Mosnier LO, Lisman T, van den Berg HM, et al. The defective down regulation of fibrinolysis in haemophilia A can be restored by increasing the TAFI plasma concentration. Thromb Haemost 2001; 86:1035–1039 Von dem Borne PA, Bajzar L, Meijers JC, et al. Thrombinmediated activation of factor XI results in a thrombinactivatable fibrinolysis inhibitor-dependent inhibition of fibrinolysis. J Clin Invest 1997; 99:2323–2327 van Tilburg NH, Rosendaal FR, Bertina RM. Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis. Blood 2000; 95:2855–2859
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40 Yano Y, Kitagawa N, Gabazza EC, et al. Increased plasma thrombin-activatable fibrinolysis inhibitor levels in normotensive type 2 diabetic patients with microalbuminuria. J Clin Endocrinol Metab 2003; 88:736 –741 41 Morange PE, Juhan-Vague I, Scarabin PY, et al. Association between TAFI antigen and Ala147Thr polymorphism of the TAFI gene and the angina pectoris incidence. Thromb Haemost 2003; 89:554 –560 42 Watanabe R, Wada H, Watanabe Y, et al. Activity and antigen levels of thrombin-activatable fibrinolysis inhibitor in plasma of patients with disseminated intravascular coagulation. Thromb Res 2001; 104:1– 6 43 Meijers JC, Oudijk EJ, Mosnier LO, et al. Reduced activity of TAFI (thrombin-activatable fibrinolysis inhibitor) in acute promyelocytic leukaemia. Br J Haematol 2000; 108:518 –523 44 Van Thiel DH, George M, Fareed J. Low levels of thrombin activatable fibrinolysis inhibitor (TAFI) in patients with chronic liver disease. Thromb Haemost 2001; 85:667– 670 45 Lisman T, Leebeek FW, Mosnier LO, et al. Thrombinactivatable fibrinolysis inhibitor deficiency in cirrhosis is not associated with increased plasma fibrinolysis. Gastroenterology 2001; 121:131–139 46 Lisman T, Mosnier LO, Lambert T, et al. Inhibition of fibrinolysis by recombinant factor VIIa in plasma from patients with severe hemophilia A. Blood 2002; 99:175–179 47 Redlitz A, Nicolini FA, Malycky JL, et al. Inducible carboxypeptidase activity: a role in clot lysis in vivo. Circulation 1996; 93:1328 –1330 48 Mattsson C, Bjorkman JA, Abrahamsson T, et al. Local proCPU (TAFI) activation during thrombolytic treatment in a dog model of coronary artery thrombosis can be inhibited with a direct, small molecule thrombin inhibitor (melagatran). Thromb Haemost 2002; 87:557–562 49 Klement P, Liao P, Bajzar L. An inhibitor of activated TAFI enhances thrombolysis. Blood 1998; 92:1967–1975 50 Nagashima M, Werner M, Wang M, et al. An inhibitor of activated thrombin-activatable fibrinolysis inhibitor potentiates tissue-type plasminogen activator-induced thrombolysis in a rabbit jugular vein thrombolysis model. Thromb Res 2000; 98:333–342 51 Refino CJ, Schmitt D, Pater C, et al. Carboxypeptidase inhibitor markedly improves the potency of tPA in vivo. Fibrinolysis Proteolysis 1998; 12:29 52 Nagashima M, Yin ZF, Zhao L, et al. Thrombin-activatable fibrinolysis inhibitor (TAFI) deficiency is compatible with murine life. J Clin Invest 2002; 109:101–110 53 Skidgel RA. Human carboxypeptidase N: lysine carboxypeptidase. Methods Enzymol 1995; 248:653– 663 54 Lee CD, Mann KG. Activation/inactivation of human factor V by plasmin. Blood 1989; 73:185–190 55 Samis JA, Ramsey GD, Walker JB, et al. Proteolytic processing of human coagulation factor IX by plasmin. Blood 2000; 95:943–951
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