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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.
REVIEW ARTICLES Directing thrombinFrom 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.
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.
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 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?
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.
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
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.
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.
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
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
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).
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
We thank Dr Jim Crawley for his help in the preparation of the figures.
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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Abstract
Introduction: natural hemostatic...
