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Blood, 15 October 2005, Vol. 106, No. 8, pp. 2605-2612.
Prepublished online as a Blood First Edition Paper on June 30, 2005; DOI 10.1182/blood-2005-04-1710.
 Previous Article | Table of Contents | Next Article 
REVIEW ARTICLES
Directing thrombin
David A. Lane,
Helen Philippou, and
James A. Huntington
From the Department of Haematology, Imperial College London, United Kingdom; the Academic Unit of Molecular Vascular Medicine, University of Leeds, Leeds, United Kingdom; and the Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
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Abstract
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Following initiation of coagulation as part of the hemostatic response to injury, thrombin is generated from its inactive precursor prothrombin by factor Xa as part of the prothrombinase complex. Thrombin then has multiple roles. The way in which thrombin interacts with its many substrates has been carefully scrutinized in the past decades, but until recently there has been little consideration of how its many functions are coordinated or directed. Any understanding of how it is directed requires knowledge of its structure, how it interacts with its substrates, and the role of any cofactors for its interaction with substrates. Recently, many of the interactions of thrombin have been clarified by crystal structure and site-directed mutagenesis analyses. These analyses have revealed common residues used for recognition of some substrates and overlapping surface exosites used for recognition by cofactors. As many of its downstream reactions are cofactor driven, competition between cofactors for exosites must be a dominant mechanism that determines the fate of thrombin. This review draws together much recent work that has helped clarify structure function relationships of thrombin. It then attempts to provide a cogent proposal to explain how thrombin activity is directed during the hemostatic response.
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Introduction: natural hemostatic substrates of thrombin
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Proteinases play crucial roles in biologic processes involved in organism homeostasis. Their actions require tight regulation and coordination. Failure of regulation and coordination leads to disease. The hemostasis system involves activation, regulation, and coordination of numerous proteinases. The complex reactions occur on activated or damaged cells, platelets, and endothelial cells. In normal physiologic conditions hemostasis is exquisitely initiated, controlled, and terminated. Genetic or physiologic perturbations, however, can lead to severe dysfunction, resulting in either hemorrhagic disorders or thromboembolic disease, illustrating the need for tight regulation of these proteolytic reactions.
Central to the functioning of hemostasis are the roles of thrombin. Numerous potential substrates have been identified for thrombin,1,2 suggesting diverse roles. Only those substrates related to hemostasis are considered here. A prime function of thrombin is the conversion of fibrinogen to fibrin. Two fibrinopeptides (FPs) are cleaved, FPA (the 16-residue N-terminal peptide from the A chain) and FPB (the 14-residue peptide from the B chain). Cleavage of FPA occurs first, and this forms a fibrin monomer (termed fibrin I) which spontaneously polymerizes to form protofibrils. Cleavage of FPB generates fibrin II protofibrils that undergo lateral aggregation. In the dynamic process of thrombin generation in blood, some of the early generated thrombin feeds back on the cascade system to activate factors V and VIII, enabling more sustained generation of thrombin.3,4 Thrombin activates factors V and VIII by excision of their central B domains. In factor V, R709, R1018, and R1545 are cleaved, leaving an A1-A2 domain fragment, noncovalently associated with an A3-C1-C2 domain fragment. In factor VIII, the corresponding cleavages are R372, R740, R1649, and R1689. This leaves an A1-A2 fragment associated with an A3-C1-C2 fragment. Thrombin cleaves the factor XIII A subunit at R37, releasing an aminoterminal activation peptide, resulting in exposure of its active site C314 and initiation of fibrin stabilization by covalent crosslinking. Thrombin generation occurring in the context of normal hemostasis is located on the surface of activated platelets that form the primary hemostatic plug. The platelet can be activated by a number of agonists. Thrombin specifically activates the platelet by interacting and cleaving protease-activated receptors (PARs).5 Cleavage of the PAR-1 receptor is at R41, revealing a tethered activating hexapeptide that self-associates with one of its extracellular loops, causing receptor-induced signaling. A second platelet activation mechanism involves proteolysis of glycoprotein V (GpV), part of the GpIb-IX-V complex on its surface, with the generation of hyperresponsive platelets.6 Thrombin can activate factor XI by proteolysis at R369 under rather defined conditions requiring anions such as dextran sulfate.7 However, in the presence of activated platelets, a more likely physiologic activation process involves GpIb.8 Thrombin may also indirectly enhance platelet adhesion. Recently, thrombin has been shown to proteolyze ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif) at multiple sites, leading to its inactivation.9 ADAMTS13 is the protease responsible for von Willebrand factor (VWF) processing. Von Willebrand factor is a key adhesive glycoprotein required for platelet adhesion to the site of injury. By inactivating ADAMTS13, thrombin can potentially aid platelet recruitment to the site of vessel injury. All of these reactions of thrombin with its substrates can be considered as procoagulant; that is, clot promoting.
Thrombin also participates in proteolytic interactions as part of endogenous anticoagulant pathways. First, thrombin generated in the vicinity of the endothelial cell surface is trapped in a high-affinity complex with the cell-surface proteoglycan thrombomodulin.10 Once bound as the thrombin-thrombomodulin complex, its substrate specificity is redirected from procoagulant to anticoagulant reactions. Procoagulant reactions are impeded and efficient activation of protein C occurs, by cleavage at R169 and release of an activation peptide. Second, in cleaving R393 of the reactive loop of the plasma SERPIN (serine protease inhibitor) antithrombin, it is trapped in an essentially irreversible complex and cleared from the circulation. This reaction is accelerated by the glycosaminoglycans heparin and heparan sulfate.11 Third, thrombin is similarly inhibited by cleavage of L444 of the SERPIN heparin cofactor II in the presence of the glycosaminoglycan, heparan sulfate, or the galactosaminoglycan, dermatan sulfate.12
The thrombin-thrombomodulin complex also activates a carboxypeptidase, thrombin-activated fibrinolysis inhibitor (TAFI) by proteolysis at R92,13,14 at a rate approximately 1000 times greater than thrombin alone. Activated TAFI is then able to remove lysine residues from fibrin that form favored binding sites for fibrinolytic proteins. Recent work has suggested that TAFI, when activated, may also have a broad antiinflammatory role. It modulates the proinflammatory properties of bradykinin, complement C5a, and thrombin-cleaved osteopontin with similar efficiency as its effect on fibrin.15
These multiple interactions in which thrombin participates raise the question of how it knows which substrate it should interact with and cleave during hemostasis. Does it, on the one hand, randomly cleave all of these substrates, or, on the other hand, is it directed in some way to cleave the substrates in a coordinated manner?
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Thrombin generation
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To address this question, it is necessary to consider how the activity of thrombin is controlled by its structure. During its activation by factor Xa, prothrombin is cleaved, first at R320 to generate meizothrombin and then at R271 to generate thrombin and fragment F1 + 2 (an alternative activation pathway is possible but may be less favored kinetically16 and is therefore ignored here). The F1 + 2 fragment contains the Gla and kringle domains, which function to localize prothrombin on membrane surfaces as part of the activation (or "prothrombinase") complex, that includes factor Va and factor Xa (Figure 1). Once prothrombin is activated, thrombin can escape the complex and is free to attack its substrates. Prothrombin is unique among the activated coagulation proteinases in that it completely loses the domains important for initial recognition interactions when it is activated to its serine proteinase derivative, thrombin. Loss of these domains allows thrombin to diffuse freely to encounter, recognize, cleave, and dissociate from its many substrates. Furthermore, their loss permits exposure of cryptic17 functional regions, the active site, and the charged anion binding regions, termed exosites,18 crucial for extending the range and specificity of thrombin's actions.
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Figure 1.. Prothrombin activation to thrombin. Prothrombin is first activated as part of the prothrombinase complex to meizothrombin by cleavage at R320. The Gla and kringle (K1 and K2) domains are cleaved at R271, allowing thrombin to diffuse away. Thrombin has 3 regions that are functionally inaccessible in prothrombin and become exposed on activation: the active site, exosite I, and exosite II.
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Thrombin structure and allostery
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The high-resolution structure of thrombin was considered in detail in a prescient and influential article by Bode et al.18 Thrombin is highly homologous to serine proteases such as chymotrypsin, with a serine residue (S195, chymotrypsin numbering) in an active site grove, along which its substrate must lie (Figure 2). Thrombin has critical features that control specificity, loops, and charged patches surrounding the active site pocket. The 60- and  -loops frame the canyonlike active site cleft containing S195. The 60-loop is hydrophobic with a structural rigidity provided by 2 adjacent Pro residues (P60b, P60c). It interacts with hydrophobic residues of the substrate, N-terminal to the cleavage site. Deletion mutant studies of the 60-loop have illustrated its role in controlling substrate activation.19,20 The -loop is more mobile, hydrophilic, and can make contact with residues C-terminal to the cleavage site.
Of importance for the discussion that follows are the exosites I and II (depicted ABEI and ABEII in Figure 2). Exosite I is centered on residues K36, H71, R73, R75, Y76, R77a, and K109/110, and exosite II includes residues R93, K236, K240, R101, and R233. The structural basis of the exosites and how they interact with cofactors has been reviewed comprehensively elsewhere21 and will not therefore be considered in detail here.
Another important loop contains a site for Na+ binding (Figure 2). Occupancy of its binding site controls some of the activities of thrombin and is important for the consideration of allostery. Allostery of thrombin is an intriguing and only partially resolved issue that can be considered from several (related) viewpoints. First, Na+ when bound to thrombin (denoted initially as the "fast" as opposed to the unoccupied "slow" form22) alters its function, increasing access of small substrates to the active site and favoring certain procoagulant (fibrinogen and PAR-1) rather than anticoagulant substrates. The residues energetically linked to Na+-induced allostery are D189, E217, D222, and Y225.23 Occupancy of its binding site affects the activity of thrombin, but, because Na+ concentration in blood is tightly controlled (140 mM), the site will be similarly occupied under all but the most extreme pathologic circumstances. Second, mutants such as E217K24 and W215A/E217A25 mimic, or adopt the conformation of, the slow form and exhibit greatly reduced procoagulant activities but retain or have inducible anticoagulant activities. Such mutants represent potential therapeutic anticoagulants.26 Four crystal structures of the slow form of thrombin have been proposed and show a collapsed active site pocket, but these differ appreciably in detail.24,25,27,28 Third, and of most importance to the present context, occupancy of exosites I and II may induce allosteric changes to the active site to promote catalysis. This is supported by functional analyses that show linkage between the exosites and the catalytic center,29,30 by mutational analysis of exosite residues of thrombin31 and by enhanced reactivity of inhibitors to thrombin, when it is complexed to thrombomodulin.32 Occupancy of exosite I of thrombin by thrombomodulin did not appear to alter the active site in a crystal structure of the complex,33 a finding that may be explained by use of an active site inhibitor to prevent proteolysis during crystallization. At its extreme, allostery might extend to reciprocal exosite inhibition,34 an idea that has, however, been firmly challenged.29
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Substrate recognition
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Of the very early actions of thrombin, the cleavage of FPA to form fibrin monomer is critical in determining the fate of the proteinase. Thrombin binds directly to the fibrinogen A chain, binding being mediated through part of exosite I (Figure 3A; Table 1) and also the active site cleft.36 Recognition of fibrinogen, as opposed to its isolated polypeptide chains or its proteolyzed N-terminal E domain, involves additional surface residues on the face containing the active site serine, identified by clotting experiments.37-39 Release of FPA is sufficient to induce fibrin polymerization, and, while the release of FPB follows closely that of FPA, it is determined by the relative specificity constants of thrombin for the A and B chains, and premature release of FPB does not affect polymerisation.40 Exosite I is also involved in the binding of thrombin to the B chain to cleave FPB (Table 1). Another early action of thrombin is activation of factor V, needed to promote further generation of thrombin. Numerous surface residues on thrombin are involved with residues of exosites I and II and the Na+ loop required for cleavage of R70941 (Figure 3B; Table 1). Factor VIII cleavage activation by thrombin involves a similar wide distribution of residues (Table 1). Cleavage at R372 requires residues in both exosites, while cleavage at R1689 does not require exosite II.42 It should be noted that each of these early reactions of thrombin are direct between it and the substrates, and no cofactors are involved in acceleration of the cleavage reactions. This contrasts with the substrate reactions below, which are all assisted by cofactors.
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Table 1.. Interaction of the exosites (I and II) of thrombin in substrate recognition (depicted as yes or no) and with cofactors that accelerate the substrate reactions
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Figure 2.. The structural features of thrombin. A stereo view of the surface thrombin in the standard orientation reveals the main features of the protease. The surface is colored according to electrostatic potential, with red and blue representing negatively and positively charged patches, respectively. In this orientation, the active site is centered so that a peptide substrate will run from the left to the right, from its N- to C-termini. Here, the P4 to P2' residues of antithrombin are depicted as found in the structure of the Michaelis complex, with the reactive center bond (P1-P1') indicated by the yellow arrow. The active site cleft is flanked above by the 60-loop, and below by the -loop making the cleft unusually deep. The 2 major anion binding exosites (ABEI and ABEII) are indicated by the ovals. The adjacent Na+ binding site is also indicated.
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Proteolytic activation of platelets requires cleavage of PARs. In crystal structures of binding of receptor-based peptides to thrombin, the active site and exosite I were shown to be involved43 (Table 1). The residues on thrombin required for activation of PAR-1 and PAR-4 have been quantitatively assessed using functional analysis and are very similar to those used for recognition of fibrinogen.44,45 There are some differences between PAR-1 and PAR-4, with the former being more dependent on exosite I interaction than the latter.44,46
A restricted common set of surface residues on thrombin around the Na+ loop and active site cleft is important for activity of a number of substrates (in the absence of any cofactor), including antithrombin,37 protein C,39 and factor XIII.47 Use of the same, or a similar, subset of surface residues might suggest competition between these substrates for thrombin; however, it is unlikely that such direct substrate competition will be an important determinant of the direction or fate of thrombin. This is because of the large increases in efficiencies of substrate cleavage obtained in the presence of the cofactors (see next section). In the absence of these cofactors, the substrate reactions are inefficient, and it is unlikely that the unassisted reactions will take place in vivo.
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The role of cofactors; variation on a theme
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The way in which exosites and cofactors function became evident from their roles in anticoagulant pathways localized on the surface of the endothelium (a recent more general review of the importance of exosites in substrate recognition can be found elsewhere48). The protein C anticoagulant pathway is important for the down-regulation of thrombin generation. Thrombin activates protein C, which then proteolytically inactivates factors Va and VIIIa. However, the catalytic efficiency of this activation reaction is very low (kcat/Km, 5.6 x 102 M–1s–1).10 This is increased approximately 1500-fold by thrombomodulin alone and approximately 10 000-fold by thrombomodulin in the presence of a phospholipid membrane (kcat/Km, 5.9 x 106 M–1s–1). Thrombomodulin achieves this increase in efficiency by binding to thrombin at exosite I with very high affinity, binding being mediated by its 5th and 6th epidermal growth factor (EGF)–like domains.33,49 Protein C approaches this complex and associates weakly to thrombomodulin, by an interaction between a positively charged exosite on protein C and the 4th EGF-like domain of thrombomodulin,50 in an orientation that favors its activation. Thrombomodulin thereby functions as a cofactor in the activation of protein C by thrombin, approximating the 2 proteins (Figure 4A) and enabling allosteric changes in thrombin.31,53 Two separate regions of thrombin are required for this reaction: exosite I for thrombomodulin binding, and the active site for protein C activation (Table 1). However, exosite II can also be involved by binding to the chondroitin sulfate moiety present on a fraction of thrombomodulin molecules54 (Figure 4A; Table 1).
The thrombin-thrombomodulin complex is also able to activate TAFI. As described for protein C activation, exosite I is used to bind to the thrombomodulin,39 but exosite II can also again be involved when chondroitin sulfate is attached to the receptor (Table 1).
Antithrombin inhibits thrombin directly in a second anticoagulant pathway. Thrombin recognizes the reactive site loop of antithrombin as a substrate but is trapped during the proteolysis reaction. Antithrombin inhibition of thrombin is relatively inefficient. Catalytic efficiency has the modest value of 6.8 x 103 M–1s–1.55 A fraction of heparin and heparan sulfate chains contain high-affinity sites for antithrombin binding and also provide a template for the efficient diffusion of thrombin. Thrombin is then "approximated" to antithrombin by its binding to an adjacent site on the glycosaminoglycan56,57 (Figure 4B). For this approximation, thrombin uses exosite II to bind to the sulfate groups of the glycosaminoglycan (Table 1). Two sites on thrombin are therefore involved in this cofactor reaction, exosite II and the active site. The outcome of this cofactor participation in the antithrombin/thrombin reaction is an approximate 20 000-fold increase in efficiency, to a kcat/Km of 1.2 x 108 M–1s–1.55
Thrombin is inhibited by another SERPIN, heparin cofactor II, in a reaction accelerated up to 70 000-fold by heparin, heparan sulfate, and dermatan sulfate.58,59 The mechanism is more complex than the heparin-accelerated inhibition of thrombin by antithrombin.12 In addition to a template acceleration reaction mediated by exosite II, the polysaccharide releases a sequestered highly acidic N-terminal tail of heparin cofactor II, which is then able to bind to exosite I and position the thrombin for attempted cleavage of the Leu-Ser reactive center bond (Table 1). This mechanism is supported by crystallographic and mutagenesis studies.60,61
Exosites and cofactors are also involved in procoagulant reactions on the surface of the platelet. GpIb plays a critical role in initial adhesion reactions between platelets and the subendothelium, mediated through VWF. It also functions as a cofactor in the activation of PARs62,63 and GpV6 (Figure 4C-D). Its cofactor function is similar to the examples already given in the sense that an approximation is achieved. It differs, however, in that the enhanced cleavage arises from substrate receptors merely being in proximity to GpIb. Crystal structure analyses have demonstrated both exosite I and II binding interactions with GpIb.51,64 The weight of evidence from functional analyses indicate that exosite II interaction with GpIb is critical in these cofactor reactions65 (Table 1), but the additional exosite I interactions may play a crosslinking role in platelet activation and signaling.51 Thrombin bound to GpIb through exosite II is also positioned to activate factor XI on stimulated platelets. Factor XI bound via its Apple 3 domain66 to leucine-rich repeats67 of adjacent GpIb is recognized by thrombin in part through exosite I interaction68 (Figure 4E; Table 1).
The principle of cofactor-enhanced proteolysis is subject to an interesting variation during factor XIII activation by thrombin. Thrombin activates factor XIII by cleaving a 37-residue activation peptide from the factor XIII A chain. On its own this is not a highly efficient reaction (kcat/Km, 1.4 x 105 M–1s–1).69,70 It has been known for many years that polymerizing fibrin accelerates the activation of factor XIII, but there was no understanding of how this was achieved. Thrombin uses fibrin as a cofactor, but there are unique aspects of this. The cofactor has to be first generated at the site of vascular injury by cleavage of FPA from fibrinogen. It is the subsequent process of polymerization that generates the cofactor. Once thrombin has bound to fibrinogen and cleaved the fibrinopeptides, it remains bound to the fibrin that is formed, in the N-terminal region termed the E domain (Figure 5). It has now been shown that part of exosite I is important for this binding and enhanced activation of factor XIII.47 But how does the needed approximation of factor XIII occur? Factor XIII is known to bind with moderate affinity to the C-terminal D domain of A/' fibrinogen. This domain engages with complementary sites of the N-terminal E domain to induce polymerization. It is plausible that thrombin is brought to its substrate, factor XIII, by the polymerization process47 (Figure 5), and the catalytic efficiency is thereby increased approximately 80-fold to 1.2 x 107 M–1s–1.69,71
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Figure 5.. Self-generated cofactor-assisted substrate interactions of thrombin. Once thrombin has cleaved FPA from fibrinogen, fibrin monomer is formed, to which the thrombin remains attached via exosite I interactions at the N-terminal E domain (A). Factor XIII (FXIII) is also loosely bound to fibrin, but at the C-terminal D domain (B). The fibrin monomers will spontaneously polymerize. It has been proposed that this approximates the thrombin and factor XIII (C), resulting in efficient cleavage of the activation peptide (AP).47 Fibrin(ogen) polypeptide chains are colored as in figure 3A.
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Cofactor competition for exosites
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The finding that residues on exosite I involved in the fibrin-enhanced activation of factor XIII comprise a discreet subset of those required for the thrombomodulin-enhanced activation of protein C by thrombin, raised the interesting issue of competition between cofactors for exosites.47 If, as suggested in the preceding section, cofactors drive many of the reactions of thrombin, competition between them for the exosites might be expected to determine the fate of thrombin.
A cofactor has a potential choice of either of the exosites with which to interact. How is this choice made? It is known that exosite II interactions are more dependent on salt concentration than are exosite I interactions, suggesting strong ionic interactions. A first consideration of this has been made elsewhere21 on the basis of peptide sequences from cofactors known to bind to the exosites. A number of physiologic and nonphysiologic peptide ligands were considered in terms of the overall properties, acidic, basic, or hydrophobic. Predictably, both exosites required peptides with a high percentage of acidic residues, 50% for exosite I and 72% for exosite II, but the hydrophobic content was somewhat lower for exosite II. The average fractional net negative charge to the hydrophobic fraction was consequently higher for exosite II binding peptides, 2.5 compared with 1.2.
Competition for thrombin exosites is nicely illustrated by recent crystal structures of thrombin complexes with fibrin, thrombomodulin, GpIb, and heparin.33,36,51,72 In Figure 6A, the structure of fibrin (ribbon, with yellow, green, and blue polypeptide chains) bound to thrombin is shown. The contact region on exosite I is depicted in red. The contact of thrombomodulin (green and blue ribbon EGF domains) with thrombin is shown in Figure 6B, and the red contact region on exosite I can be seen to be overlapping that of fibrin. These structures predict competition between thrombomodulin and fibrin but not its outcome. Because the affinity of thrombin for thrombomodulin is high ( 1 nM) and that of thrombin for fibrin is low (1 µM), the former will always be preferred. As shown in Figure 6C, GpIb (purple ribbon) binds to exosite II (red), using many of the same residues as does heparin (ball and stick) (Figure 6D). Once again, competition will depend in part on relative affinities. Heparin (and presumably heparan sulfate) binds to thrombin with an approximately 1 µM dissociation constant,73 similar to the affinity of thrombin for GpIb as an isolated peptide65 (it may be greater on the intact platelet74,75). The local concentration of heparan sulfate will be much higher over the intact endothelium where the competition is staged, giving it a clear advantage.
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How thrombin is directed
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Thus, the many reactions of thrombin therefore involve either direct or cofactor-assisted recognition events involving common exosites. How are these events orchestrated in vivo when the hemostatic plug is being generated? A very early if not initiating event in hemostasis will be the interaction of the platelet with the damaged endothelium, mediated through von Willebrand factor-GpIb interactions (Figure 7A) and collagen-receptor interactions (not shown). Further, platelets will be recruited to this plug in interactions mediated by adhesive proteins such as fibrinogen with other glycoprotein receptors. Engagement of the glycoproteins results in intracellular signaling and cellular activation. The activated platelet plug is induced to provide a surface of negatively charged phospholipid on which coagulation factors can assemble. Tissue factor (TF) is exposed by vessel injury, and this leads to factor V and factor VIII activation and the generation of the first traces of thrombin on this surface. Thrombin activates more factor V and VIII (Figure 7Bi), aided by the large spread of surface residues used for recognition. The affinity of thrombin for the N terminus of fibrinogen is modest, approximately 7 µM, but the concentration of fibrinogen is high ( 15 µM as a monomer), and much (but, crucially, not all) of the thrombin generated will associate with fibrinogen (Figure 7Bii). Recognition of fibrinogen may again be favored by the large number of residues spread over the surface of thrombin that participate in binding. Productive binding results in cleavage of FPA with formation of fibrin monomers. As the fibrin begins to polymerize, it will retain thrombin bound to a subset of those residues in exosite I used for initial recognition. The newly generated polymer will provide the cofactor required for accelerated factor XIII activation. This ensures that factor XIIIa is generated where it is needed, on the fibrin clot surface, where it acts to crosslink the fibrin (Figure 7Bv).
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Figure 7.. How thrombin is directed. Schematic diagram showing many of the interactions of thrombin over the damaged endothelium as part of the hemostatic response. The direction of thrombin is indicated by use of A, B (i, ii, iii, iv, and v simultaneously), C, D, E (i and ii simultaneously), F. Fibrinogen is shown as a simple domain scheme for simplicity. TF indicates tissue factor.
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Thrombin located on the fibrin clot can migrate, because it is bound through a limited subset of exosite I residues, and its affinity for fibrin is weak. Proximity to GpIb will enable its binding primarily through exosite II (Figure 7Biii). This glycoprotein will be engaged with VWF, linking the plug to the exposed subendothelium. Thrombin in the vicinity of unattached GpIb will be able to protect VWF from ADAMTS13 action by proteolysis (Figure 7Biv). This protection, mediated by recognition of ADAMTS13 through exosite I of thrombin,9 prevents destabilization of the plug by ADAMTS13 cleavage of VWF. Thrombin bound to GpIb through exosite 2 interaction will also be positioned for activation of PARs (Figure 7Biii) (the recognition of which is largely through exosite I) and for GpV cleavage. Complex cell-signaling pathways will be triggered at this stage by proteolysis, nonproteolytic binding of thrombin, and possibly by bivalent thrombin interactions using both exosites.6,64 In situations of extreme hemostatic challenge, factor XI will be activated by thrombin on the platelet surface (Figure 7C) by a mechanism involving both exosites and GpIb.68 This will enhance downstream thrombin generation. Essentially unregulated and therefore uncontrollable thrombin generation can then occur locally over the damaged endothelium (Figure 7D). Thrombin will continue to be active until the whole platelet plug is surrounded and stabilized with crosslinked fibrin. However, as the clot continues to expand, it will reach areas of intact endothelium. Here it will encounter thrombomodulin (Figure 7Ei). Thrombomodulin has very high affinity for thrombin, much higher than the affinity of thrombin for fibrin. Crucially, both cofactors (fibrin and thrombomodulin) use exosite I and will therefore compete for binding. This competition will only have one outcome, thrombin bound to thrombomodulin. Thrombomodulin will function as a sink, draining and redirecting thrombin activity (from procoagulant to anticoagulant), thereby protecting the intact endothelium from multiple thrombin actions and obstructive thrombus formation.
Heparan sulfate and GpIb both use exosite II. In areas of damaged vessel with abundant platelet adhesion, binding to GpIb will be favored. However, over the intact endothelium, the more abundant heparan sulfate will tend to dominate in competition for exosite II and thrombin (bound by exosite II) will be neutralized by approximated antithrombin (Figure 7Eii). A potentially more complicated competitive interaction for GpIb can be invoked for the second serpin thrombin inhibitor, heparin cofactor II. This inhibitor when bound to its glycosaminoglycan/galactosaminoglycan cofactor will be in a partially activated state with its acidic N-terminal tail displaced and ready to receive thrombin. The latter will be approximated by exosite II contact with sulfate residues in the polysaccharide and by interaction of the N-terminal tail with exosite I. However, in the physiologic context, heparin cofactor II has at best a secondary role in the control of the normal hemostatic clot.76 Should any thrombin break past the functional barrier presented by thrombomodulin, endothelial cell heparan sulfate with bound antithrombin will act as a backup anticoagulant mechanism, with thrombin binding to the glycosaminoglycan side chains through exosite II (Figure 7F). Antithrombin scavenging action may not be restricted to the endothelium. As the hemostatic plug grows into the lumen of the vessel, the thrombin will be associating and dissociating with fibrin, and, because the equilibrium binding affinity is weak, the dissociated thrombin will be a target for free antithrombin. Although efficiency of the thrombin inhibition by direct antithrombin interaction is low, it may be sufficient to limit luminal plug growth. All generated thrombin is eventually cleared from the circulation in complex with either antithrombin, heparan cofactor II, or less specific inhibitors, by receptor-mediated processes.77
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Acknowledgements
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We thank Dr Jim Crawley for his help in the preparation of the figures.
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Footnotes
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Submitted April 27, 2005;
accepted June 5, 2005.
Prepublished online as Blood First Edition Paper, June 30, 2005; DOI 10.1182/blood-2005-04-1710.
Supported by grants from the British Heart Foundation and the Medical Research Council.
Reprints: David A. Lane, Department of Haematology, Imperial College London, Hammersmith Hospital Campus, Du Cane Rd, London W12 ONN, United Kingdom; e-mail: d.lane{at}imperial.ac.uk.
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References
|
|---|
- Stubbs MT, Bode W. The clot thickens: clues provided by thrombin structure. Trends Biochem Sci. 1995;20: 23-28.[CrossRef][Medline]
[Order article via Infotrieve]
- Di Cera E. Thrombin interactions. Chest. 2003;124: 11S-17S.[Abstract/Free Full Text]
- Hemker HC, Beguin S. Thrombin generation in plasma: its assessment via the endogenous thrombin potential. Thromb Haemost. 1995;74: 134-138.[Medline]
[Order article via Infotrieve]
- Brummel KE, Paradis SG, Butenas S, Mann KG. Thrombin functions during tissue factor-induced blood coagulation. Blood. 2002;100: 148-152.[Abstract/Free Full Text]
- Coughlin SR. Thrombin signalling and protease-activated receptors. Nature. 2000;407: 258-264.[CrossRef][Medline]
[Order article via Infotrieve]
- Ramakrishnan V, DeGuzman F, Bao M, Hall SW, Leung LL, Phillips DR. A thrombin receptor function for platelet glycoprotein Ib-IX unmasked by cleavage of glycoprotein V. Proc Natl Acad Sci U S A. 2001;98: 1823-1828.[Abstract/Free Full Text]
- Gailani D, Broze GJ Jr. Factor XI activation in a revised model of blood coagulation. Science. 1991;253: 909-912.[Abstract/Free Full Text]
- Baglia FA, Badellino KO, Li CQ, Lopez JA, Walsh PN. Factor XI binding to the platelet glycoprotein Ib-IX-V complex promotes factor XI activation by thrombin. J Biol Chem. 2002;277: 1662-1668.[Abstract/Free Full Text]
- Crawley JT, Lam JK, Rance JB, Mollica LR, O'Donnell JS, Lane DA. Proteolytic inactivation of ADAMTS13 by thrombin and plasmin. Blood. 2005;105: 1085-1093.[Abstract/Free Full Text]
- Esmon CT. Molecular events that control the protein C anticoagulant pathway. Thromb Haemost. 1993;70: 29-35.[Medline]
[Order article via Infotrieve]
- Olson ST, Bjork I, Bock SC. Identification of critical molecular interactions mediating heparin activation of antithrombin: implications for the design of improved heparin anticoagulants. Trends Cardiovasc Med. 2002;12: 198-205.[CrossRef][Medline]
[Order article via Infotrieve]
- Tollefsen DM. Heparin cofactor II. Adv Exp Med Biol. 1997;425: 35-44.[Medline]
[Order article via Infotrieve]
- Nesheim M. Thrombin and fibrinolysis. Chest. 2003;124: 33S-39S.[Abstract/Free Full Text]
- Bouma BN, Meijers JC. Thrombin-activatable fibrinolysis inhibitor (TAFI, plasma procarboxypeptidase B, procarboxypeptidase R, procarboxypeptidase U). J Thromb Haemost. 2003;1: 1566-1574.[CrossRef][Medline]
[Order article via Infotrieve]
- Myles T, Nishimura T, Yun TH, et al. Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation. J Biol Chem. 2003;278: 51059-51067.[Abstract/Free Full Text]
- Orcutt SJ, Krishnaswamy S. Binding of substrate in two conformations to human prothrombinase drives consecutive cleavage at two sites in prothrombin. J Biol Chem. 2004;279: 54927-54936.[Abstract/Free Full Text]
- Wu Q, Picard V, Aiach M, Sadler JE. Activation-induced exposure of the thrombin anion-binding exosite. Interactions of recombinant mutant prothrombins with thrombomodulin and a thrombin exosite-specific antibody. J Biol Chem. 1994;269: 3725-3730.[Abstract/Free Full Text]
- Bode W, Turk D, Karshikov A. The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. Protein Sci. 1992;1: 426-471.[Medline]
[Order article via Infotrieve]
- Le Bonniec BF, Guinto ER, MacGillivray RT, Stone SR, Esmon CT. The role of thrombin's Tyr-Pro-Pro-Trp motif in the interaction with fibrinogen, thrombomodulin, protein C, antithrombin III, and the Kunitz inhibitors. J Biol Chem. 1993;268: 19055-19061.[Abstract/Free Full Text]
- Rezaie AR. Reactivities of the S2 and S3 subsite residues of thrombin with the native and heparin-induced conformers of antithrombin. Protein Sci. 1998;7: 349-357.[Medline]
[Order article via Infotrieve]
- Huntington J. Molecular recognition mechanisms of thrombin. J Thromb Haemost. 2005;3: 1861-1872.[CrossRef][Medline]
[Order article via Infotrieve]
- Wells CM, Di Cera E. Thrombin is a Na(+)-activated enzyme. Biochemistry. 1992;31: 11721-11730.[CrossRef][Medline]
[Order article via Infotrieve]
- Pineda AO, Carrell CJ, Bush LA, et al. Molecular dissection of Na+ binding to thrombin. J Biol Chem. 2004;279: 31842-31853.[Abstract/Free Full Text]
- Carter WJ, Myles T, Gibbs CS, Leung LL, Huntington JA. Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery. J Biol Chem. 2004;279: 26387-26394.[Abstract/Free Full Text]
- Pineda AO, Savvides SN, Waksman G, Di Cera E. Crystal structure of the anticoagulant slow form of thrombin. J Biol Chem. 2002;277: 40177-40180.[Abstract/Free Full Text]
- Gruber A, Cantwell AM, Di Cera E, Hanson SR. The thrombin mutant W215A/E217A shows safe and potent anticoagulant and antithrombotic effects in vivo. J Biol Chem. 2002;277: 27581-27584.[Abstract/Free Full Text]
- Huntington JA, Esmon CT. The molecular basis of thrombin allostery revealed by a 1.8 A structure of the "slow" form. Structure (Camb). 2003;11: 469-479.
- Pineda AO, Zhang E, Guinto ER, Savvides SN, Tulinsky A, Di Cera E. Crystal structure of the thrombin mutant D221A/D222K: the Asp222: Arg187 ion-pair stabilizes the fast form. Biophys Chem. 2004;112: 253-256.[CrossRef][Medline]
[Order article via Infotrieve]
- Verhamme IM, Olson ST, Tollefsen DM, Bock PE. Binding of exosite ligands to human thrombin. Re-evaluation of allosteric linkage between thrombin exosites I and II. J Biol Chem. 2002;277: 6788-6798.[Abstract/Free Full Text]
- Croy CH, Koeppe JR, Bergqvist S, Komives EA. Allosteric changes in solvent accessibility observed in thrombin upon active site occupation. Biochemistry. 2004;43: 5246-5255.[CrossRef][Medline]
[Order article via Infotrieve]
- Rezaie AR, Yang L. Thrombomodulin allosterically modulates the activity of the anticoagulant thrombin. Proc Natl Acad Sci U S A. 2003;100: 12051-12056.[Abstract/Free Full Text]
- Yang L, Manithody C, Walston TD, Cooper ST, Rezaie AR. Thrombomodulin enhances the reactivity of thrombin with protein C inhibitor by providing both a binding site for the serpin and allosterically modulating the activity of thrombin. J Biol Chem. 2003;278: 37465-37470.[Abstract/Free Full Text]
- Fuentes-Prior P, Iwanaga Y, Huber R, et al. Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex. Nature. 2000;404: 518-525.[CrossRef][Medline]
[Order article via Infotrieve]
- Fredenburgh JC, Stafford AR, Weitz JI. Evidence for allosteric linkage between exosites 1 and 2 of thrombin. J Biol Chem. 1997;272: 25493-25499.[Abstract/Free Full Text]
- Cote HC, Lord ST, Pratt KP. -Chain dysfibrinogenemias: molecular structure-function relationships of naturally occurring mutations in the chain of human fibrinogen. Blood. 1998;92: 2195-2212.[Free Full Text]
- Pechik I, Madrazo J, Mosesson MW, Hernandez I, Gilliland GL, Medved L. Crystal structure of the complex between thrombin and the central "E" region of fibrin. Proc Natl Acad Sci U S A. 2004;101: 2718-2723.[Abstract/Free Full Text]
- Tsiang M, Jain AK, Dunn KE, Rojas ME, Leung LL, Gibbs CS. Functional mapping of the surface residues of human thrombin. J Biol Chem. 1995;270: 16854-16863.[Abstract/Free Full Text]
- Hall SW, Gibbs CS, Leung LL. Identification of critical residues on thrombin mediating its interaction with fibrin. Thromb Haemost. 2001;86: 1466-1474.[Medline]
[Order article via Infotrieve]
- Hall SW, Nagashima M, Zhao L, Morser J, Leung LL. Thrombin interacts with thrombomodulin, protein C, and thrombin-activatable fibrinolysis inhibitor via specific and distinct domains. J Biol Chem. 1999;274: 25510-25516.[Abstract/Free Full Text]
- Mullin JL, Gorkun OV, Binnie CG, Lord ST. Recombinant fibrinogen studies reveal that thrombin specificity dictates order of fibrinopeptide release. J Biol Chem. 2000;275: 25239-25246.[Abstract/Free Full Text]
- Myles T, Yun TH, Hall SW, Leung LL. An extensive interaction interface between thrombin and factor V is required for factor V activation. J Biol Chem. 2001;276: 25143-25149.[Abstract/Free Full Text]
- Myles T, Yun TH, Leung LL. Structural requirements for the activation of human factor VIII by thrombin. Blood. 2002;100: 2820-2826.[Abstract/Free Full Text]
- Mathews II, Padmanabhan KP, Ganesh V, et al. Crystallographic structures of thrombin complexed with thrombin receptor peptides: existence of expected and novel binding modes. Biochemistry. 1994;33: 3266-3279.[CrossRef][Medline]
[Order article via Infotrieve]
- Ayala YM, Cantwell AM, Rose T, Bush LA, Arosio D, Di Cera E. Molecular mapping of thrombin-receptor interactions. Proteins. 2001;45: 107-116.[CrossRef][Medline]
[Order article via Infotrieve]
- Myles T, Le Bonniec BF, Stone SR. The dual role of thrombin's anion-binding exosite-I in the recognition and cleavage of the protease-activated receptor 1. Eur J Biochem. 2001;268: 70-77.[Medline]
[Order article via Infotrieve]
- Jacques SL, Kuliopulos A. Protease-activated receptor-4 uses dual prolines and an anionic retention motif for thrombin recognition and cleavage. Biochem J. 2003;376: 733-740.[CrossRef][Medline]
[Order article via Infotrieve]
- Philippou H, Rance J, Myles T, et al. Roles of low specificity and cofactor interaction sites on thrombin during factor XIII activation. Competition for cofactor sites on thrombin determines its fate. J Biol Chem. 2003;278: 32020-32026.[Abstract/Free Full Text]
- Krishnaswamy S. Exosite-driven substrate specificity and function in coagulation. J Thromb Haemost. 2005;3: 54-67.[CrossRef][Medline]
[Order article via Infotrieve]
- Ye J, Liu LW, Esmon CT, Johnson AE. The fifth and sixth growth factor-like domains of thrombomodulin bind to the anion-binding exosite of thrombin and alter its specificity. J Biol Chem. 1992;267: 11023-11028.[Abstract/Free Full Text]
- Yang L, Rezaie AR. The fourth epidermal growth factor-like domain of thrombomodulin interacts with the basic exosite of protein C. J Biol Chem. 2003;278: 10484-10490.[Abstract/Free Full Text]
- Dumas JJ, Kumar R, Seehra J, Somers WS, Mosyak L. Crystal structure of the GpIbalpha-thrombin complex essential for platelet aggregation. Science. 2003;301: 222-226.[Abstract/Free Full Text]
- Sadler JE. A menage a trois in two configurations. Science. 2003;301: 177-179.[Abstract/Free Full Text]
- Lu G, Chhum S, Krishnaswamy S. The affinity of protein c for the thrombin.thrombomodulin complex is determined in a primary way by active site-dependent interactions. J Biol Chem. 2005;280: 15471-15478.[Abstract/Free Full Text]
- Bourin MC, Ohlin AK, Lane DA, Stenflo J, Lindahl U. Relationship between anticoagulant activities and polyanionic properties of rabbit thrombomodulin. J Biol Chem. 1988;263: 8044-8052.[Abstract/Free Full Text]
- Rezaie AR, Olson ST. Calcium enhances heparin catalysis of the antithrombin-factor Xa reaction by promoting the assembly of an intermediate heparin-antithrombin-factor Xa bridging complex. Demonstration by rapid kinetics studies. Biochemistry. 2000;39: 12083-12090.[CrossRef][Medline]
[Order article via Infotrieve]
- Dementiev A, Petitou M, Herbert JM, Gettins PG. The ternary complex of antithrombin-anhydrothrombinheparin reveals the basis of inhibitor specificity. Nat Struct Mol Biol. 2004;11: 863-867.[CrossRef][Medline]
[Order article via Infotrieve]
- Li W, Johnson DJ, Esmon CT, Huntington JA. Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin. Nat Struct Mol Biol. 2004;11: 857-862.[CrossRef][Medline]
[Order article via Infotrieve]
- Derechin VM, Blinder MA, Tollefsen DM. Substitution of arginine for Leu444 in the reactive site of heparin cofactor II enhances the rate of thrombin inhibition. J Biol Chem. 1990;265: 5623-5628.[Abstract/Free Full Text]
- Verhamme IM, Bock PE, Jackson CM. The preferred pathway of glycosaminoglycan-accelerated inactivation of thrombin by heparin cofactor II. J Biol Chem. 2004;279: 9785-9795.[Abstract/Free Full Text]
- Baglin TP, Carrell RW, Church FC, Esmon CT, Huntington JA. Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism. Proc Natl Acad Sci U S A. 2002;99: 11079-11084.[Abstract/Free Full Text]
- Fortenberry YM, Whinna HC, Gentry HR, Myles T, Leung LL, Church FC. Molecular mapping of the thrombin-heparin cofactor II complex. J Biol Chem. 2004;279: 43237-43244.[Abstract/Free Full Text]
- De Candia E, Hall SW, Rutella S, Landolfi R, Andrews RK, De Cristofaro R. Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets. J Biol Chem. 2001;276: 4692-4698.[Abstract/Free Full Text]
- De Cristofaro R, De Candia E, Landolfi R, Rutella S, Hall SW. Structural and functional mapping of the thrombin domain involved in the binding to the platelet glycoprotein Ib. Biochemistry. 2001;40: 13268-13273.[CrossRef][Medline]
[Order article via Infotrieve]
- Celikel R, McClintock RA, Roberts JR, et al. Modulation of alpha-thrombin function by distinct interactions with platelet glycoprotein Ibalpha. Science. 2003;301: 218-221.[Abstract/Free Full Text]
- Li CQ, Vindigni A, Sadler JE, Wardell MR. Platelet glycoprotein Ib alpha binds to thrombin anion-binding exosite II inducing allosteric changes in the activity of thrombin. J Biol Chem. 2001;276: 6161-6168.[Abstract/Free Full Text]
- Baglia FA, Gailani D, Lopez JA, Walsh PN. Identification of a binding site for glycoprotein Ibalpha in the Apple 3 domain of factor XI. J Biol Chem. 2004;279: 45470-45476.[Abstract/Free Full Text]
- Baglia FA, Shrimpton CN, Emsley J, et al. Factor XI interacts with the leucine-rich repeats of glycoprotein Ibalpha on the activated platelet. J Biol Chem. 2004;279: 49323-49329.[Abstract/Free Full Text]
- Yun TH, Baglia FA, Myles T, et al. Thrombin activation of factor XI on activated platelets requires the interaction of factor XI and platelet glycoprotein Ib alpha with thrombin anion-binding exosites I and II, respectively. J Biol Chem. 2003;278: 48112-48119.[Abstract/Free Full Text]
- Janus TJ, Lewis SD, Lorand L, Shafer JA. Promotion of thrombin-catalyzed activation of factor XIII by fibrinogen. Biochemistry. 1983;22: 6269-6272.[CrossRef][Medline]
[Order article via Infotrieve]
- Ariens RA, Philippou H, Nagaswami C, Weisel JW, Lane DA, Grant PJ. The factor XIII V34L polymorphism accelerates thrombin activation of factor XIII and affects cross-linked fibrin structure. Blood. 2000;96: 988-995.[Abstract/Free Full Text]
- Naski MC, Lorand L, Shafer JA. Characterization of the kinetic pathway for fibrin promotion of alpha-thrombin-catalyzed activation of plasma factor XIII. Biochemistry. 1991;30: 934-941.[CrossRef][Medline]
[Order article via Infotrieve]
- Carter WJ, Cama E, Huntington JA. Crystal structure of thrombin bound to heparin. J Biol Chem. 2005;280: 2745-2749.[Abstract/Free Full Text]
- Streusand VJ, Bjork I, Gettins PG, Petitou M, Olson ST. Mechanism of acceleration of antithrombin-proteinase reactions by low affinity heparin. Role of the antithrombin binding pentasaccharide in heparin rate enhancement. J Biol Chem. 1995;270: 9043-9051.[Abstract/Free Full Text]
- Harmon JT, Jamieson GA. Activation of platelets by alpha-thrombin is a receptor-mediated event. D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin, but not N alpha-tosyl-L-lysine chloromethyl ketone-thrombin, binds to the high affinity thrombin receptor. J Biol Chem. 1986;261: 15928-15933.[Abstract/Free Full Text]
- Mazzucato M, Marco LD, Masotti A, Pradella P, Bahou WF, Ruggeri ZM. Characterization of the initial alpha-thrombin interaction with glycoprotein Ib alpha in relation to platelet activation. J Biol Chem. 1998;273: 1880-1887.[Abstract/Free Full Text]
- He L, Vicente CP, Westrick RJ, Eitzman DT, Tollefsen DM. Heparin cofactor II inhibits arterial thrombosis after endothelial injury. J Clin Invest. 2002;109: 213-219.[CrossRef][Medline]
[Order article via Infotrieve]
- Wells MJ, Sheffield WP, Blajchman MA. The clearance of thrombin-antithrombin and related serpin-enzyme complexes from the circulation: role of various hepatocyte receptors. Thromb Haemost. 1999;81: 325-337.[Medline]
[Order article via Infotrieve]
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Abstract
Introduction: natural hemostatic...
