SEARCH

SEARCH BY CITATION

Keywords:

  • cofactor;
  • exosite;
  • specificity;
  • structure;
  • substrate;
  • thrombin

Abstract

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

Summary.  Thrombin is the final protease generated in the blood coagulation cascade, and is the only factor capable of cleaving fibrinogen to create a fibrin clot. Unlike every other coagulation protease, thrombin is composed solely of its serine protease domain, so that once formed it can diffuse freely to encounter a large number of potential substrates. Thus thrombin serves many functions in hemostasis through the specific cleavage of at least a dozen substrates. The solution of the crystal structure of thrombin some 15 years ago revealed a deep active site cleft and two adjacent basic exosites, and it was clear that thrombin must utilize these unique features in recognizing its substrates. Just how this occurs is still being investigated, but recent data from thrombin mutant libraries and crystal structures combine to paint the clearest picture to date of the molecular determinants of substrate recognition by thrombin. In almost all cases, both thrombin exosites are involved, either through direct interaction with the substrate protein or through indirect interaction with a third cofactor molecule. The purpose of this article is to summarize recent biochemical and structural data in order to provide insight into the thrombin molecular recognition events at the heart of hemostasis.


Multiple functions of thrombin in hemostasis

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

Thrombin has been the focus of intense study since its discovery in the 19th century [1] and continues to command attention and research funds in the biomedical field. This is in part due to the position of thrombin at the end of the blood clotting cascade, and its unique ability to cleave fibrinogen to the polymerogenic fibrin. In the absence of efficient and timely thrombin generation, stable blood clots cannot form, resulting in hemorrhage. Conversely, unregulated thrombin activity results in dissemination of the clot beyond the site of tissue damage, causing thrombosis. Although critical, the role of thrombin in hemostasis is not limited to the cleavage of fibrinogen to fibrin. Over the years, many additional activities have been identified: it is now clear that thrombin is a major player in the early steps of blood coagulation; that it is a significant contributor to the so-called ‘thrombin burst’ through positive feedback mechanisms; that it functions to stabilize clots; and that it participates in attenuating its own procoagulant activity (for a recent review see Ref. [2]). The multiple functions of thrombin are summarized in Fig. 1. A fundamental question about thrombin is how a single protease domain possesses the determinants to specifically recognize such a large number of substrates; the answer requires an understanding of the unique structural features of thrombin.

image

Figure 1. The network of thrombin activities. Thrombin is generated from its zymogen prothrombin by cleavage at two sites by the prothrombinase complex, FVa and FXa and a negatively charged phospholipid. Initial thrombin formation is rapidly followed by the cleavage of fibrinogen and activation of platelets by cleavage of PAR1. Thrombin feeds back to stimulate its own formation by activating cofactors V and VIII, and by activating FXI, when bound to Gp1bα on the platelet surface. Stabilization of the fibrin clot is also effected by thrombin through activation of the transglutaminase FXIII, and by activating the metalloprotease TAFI when bound to TM. In addition to these procoagulant roles, thrombin is capable of undergoing a switch in specificity upon binding to TM to favor activation of the anticoagulant PC. Activated PC efficiently shuts off thrombin generation through cleavage inactivation of cofactors Va and VIIIa. Thrombin is finally inhibited and cleared from the circulation by circulating serpins AT and HCII, in a GAG-dependent fashion.

Download figure to PowerPoint

Structural features of thrombin

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

Thrombin is a trypsin-like member of the chymotrypsin family of serine proteases; meaning that its structure and catalytic mechanism are like the prototypic protease chymotrypsin, and that its preferred substrates, like trypsin, have a positively charged amino acid at the P1 position of the scissile bond (nomenclature of Schechter and Berger [3], where substrate residues are numbered from the scissile P1–P1′ bond toward the N- and C-termini, respectively). Balancing the multiple functions of thrombin in hemostasis, quite simply stated, is a matter of substrate specificity. As thrombin has lost the Gla and kringle domains, all determinants of substrate recognition are necessarily found on the catalytic domain. Thus, the solution of the first crystallographic structure of thrombin in 1989 [4] provided the framework for identifying the molecular interaction, which underlie the multiple functions of thrombin [5–7].

Thrombin is traditionally viewed in the ‘standard’ orientation with the active site facing and the peptide substrate running from left to right, from its N[RIGHTWARDS ARROW]C-termini (Fig. 2). A stereo representation is necessary to appreciate the depth of the active site cleft, which for thrombin is often referred to as a ‘canyon’. The walls of the canyon are formed by the insertion loops above and below the active site. These loops are longer than those found in the parent molecule chymotrypsin, and are known as the 60 and γ-insertion loops. The 60-loop is composed of Leu60, Tyr60a, Pro60b, Pro60c, Trp60d, Asp60e, Lys60f, Asn60g, Phe60h, and Thr60i, with Asn60g glycosylated. It is evident from the sequence that the 60-loop is hydrophobic in nature, with the two consecutive prolines serving to rigidify. The 60-loop, thus provides a rigid, hydrophobic cap over the active site and mediates contacts with the hydrophobic substrate residues N-terminal to the scissile bond. In contrast, the γ-loop is more hydrophilic and flexible in nature; composed of Thr147, Trp147a, Thr147b, Ala147c, Asn147d, and Val147f. It is often incompletely modeled in crystal structures due to its inherent mobility, but, as it is adjacent to the active site cleft of thrombin, it can contact substrate residues C-terminal to the scissile bond, and can make contacts with the body of substrate proteins. In forming the canyon, the loops also serve to restrict access to the catalytic site of thrombin to proteins with long, flexible substrate loops, or to proteins with complementary surfaces which mediate contact with the loops.

image

Figure 2. Thrombin geography. Stereo pairs of surface representations of thrombin are shown in the standard orientation, colored according to electrostatic potential, A, (red for negative and blue for positive), or hydrophobicity (green), B. The active site is occupied in this figure by the reactive center loop of AT from P4 to P2′ (yellow rods). Substrate recognition within the active site depends on favorable interactions between the P1 residue and the deep acidic S1 pocket, and between hydrophobic residues N-terminal to P1 (often P2 proline and an aromatic residue at P4) in the hydrophobic groove known as the aryl-binding pocket. The active site cleft of thrombin is unusually deep due to the presence of the 60- and γ-insertion loops above and below the active site. Two basic exosites on the surface of thrombin have been identified as critical for substrate and cofactor recognition: the so-called anion-binding exosites I and II (ABEI and ABEII). Although the figure depicts a wide open thrombin active site, thrombin can exist in a less active, closed conformation in the absence cofactor, substrate or Na+, which co-ordinates near the site indicated.

Download figure to PowerPoint

The original structure of thrombin contained the covalent inhibitor D-Phe-Pro-Arg-chloromethylketone (PPACK). This inhibitor mimicked natural substrate interactions and helped explain the preference for an arginine at the P1 position, a proline at the P2 position, and a hydrophobic (preferably aromatic) residue at P4 (the D-Phe side chain occupies the position normally occupied by the P4 of the natural l-stereoisomer). This and subsequent structures of thrombin with peptides elucidated the substrate interactions N-terminal of the scissile bond, but no information was obtained about the C-terminal interactions until much later. One thing that is clear from an analysis of thrombin substrate sequences is that only a part of the information which determines specificity is to be found there (Table 1). Exosite interactions, outside the active site, either direct or cofactor mediated, are required to accelerate the formation of, or to stabilize the initial thrombin-substrate complexes (Michaelis complexes) sufficiently so that cleavage of the peptide bond can proceed.

Table 1.  Natural substrate sequences of thrombin
 P4P3P2P1P1P2P3Cofactor
Fibrinogen (A)GlyGlyValArgGlyProArgNone
Fibrinogen (B)PheSerAlaArgGlyHisArgNone
FV (709)LeuGlyIleArgSerPheArgNone
FV (1018)LeuSerProArgThrPheHisNone
FV (1545)TrpTyrLeuArgSerAsnAsnNone
FVIII (372)IleGlnIleArgSerValAlaNone
FVIII (740)IleGluProArgSerPheSerNone
FVIII (1689)GlnSerProArgSerPheGlnNone
FXIIIGlyValProArgGlyValAsnFibrin
PAR1LeuAspProArgSerPheLeuGpIbα
PAR4ProAlaProArgGlyTyrProGpIbα
FXIIleLysProArgIleValGlyGpIbα
PCValAspProArgLeuIleAspTM
TAFIValSerProArgAlaSerAlaTM
ATIleAlaGlyArgSerLeuAsnHeparin
HCIIPheMetProLeuSerThrGlnHeparin

Two exosites of significance are the basic anion-binding exosites I and II (ABEI and II, or alternatively referred to as exosites I and II). Exosite I is adjacent to the P′ side of the active site cleft, and is also known as the fibrinogen recognition exosite. Exosite II is the more basic of the two exosites, and was originally identified as the putative heparin-binding site of thrombin. With an increasing number of studies characterizing thrombin mutants, it is becoming clear that all of the natural substrates and cofactors of thrombin utilize one or both anion-binding exosites. Thrombin specificity can thus be understood as a competition for three sites: the active site, and exosites I and II. The purpose of this article is to concisely summarize the relevant biochemical and structural data that, over the last 15 years, have combined to elucidate the complex network of thrombin molecular recognition events which lie at the heart of hemostasis.

Method

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

In dealing with thrombin mutagenesis data, point mutations known to affect thrombin's catalytic activity (i.e. two-fold or greater reduction in rate of S2238 substrate cleavage) and those seen to participate in critical structural contacts (thus not available for direct intermolecular interactions) were excluded from the list of residues potentially mediating direct substrate or cofactor interaction. It was also necessary to exclude some of the structurally defined thrombin interactions on the basis of biochemical data to the contrary. In this article, thrombin is numbered according to the chymotrypsin template scheme [4], and combined mutations are indicated by a forward slash between residues. Figures were made using MolScript [8], BobScript [9], Spock and Raster3D [10].

Fibrinogen

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

Thrombin cleavage of fibrinogen at the N-termini of the A and B chains leads to the formation of a fibrin monomer capable of linear and lateral self-association, resulting in a fibrin clot [11]. Fibrinogen is a substrate whose recognition by thrombin is directly mediated by active site and exosite I interactions to form a Michaelis complex defined by a Km in the micromolar range [12]. Two mutagenesis studies [13,14] identified 15 thrombin residues which potentially mediate direct contact with fibrinogen: K36, R67, H71, R73, Y76, R77a, K81, and K109/110 in exosite I; D186a/K186d near the Na+-binding region; and, K60f and R173/R175/D178 which border the active site cleft. Several structural studies have cocrystallized thrombin with fibrinopeptide A, but with mixed results [15–19]. The most complete peptide structure is of bovine thrombin with an uncleavable analog of residues 7–19 of human fibrinopeptide A [16]. The three monomers in the asymmetric unit revealed conserved contacts from P10 to P2′, with P3′ in two different orientations. The P10–P1 interactions were consistent with those seen in other structures [15,17–19], and involve the burying the hydrophobic face of a one-turn helix into the aryl-binding pocket of thrombin (of primary importance is P9 Phe), and normal interactions of the P1 Arg in the acidic S1 pocket. In 2004, the structure of human thrombin bound to the central E domain of human fibrin was solved, revealing the predicted exosite I interaction interface [20]. Thrombin residues seen in direct contact with fibrin were F34, S36a, L65, Y76, R77a, I82, and K110. Although the interface interposes the basic exosite I of thrombin with an acidic surface of fibrin, the majority of the contacts, and presumably the major energetic contribution, derive from the apposition of hydrophobic surfaces on the two molecules (F34, L65, Y76 and I82 on thrombin and F35 and A68 on the Aα and Bβ chains of fibrin, respectively). Because the structural data agree with the mutagenesis data, a figure of the active site and exosite interactions between thrombin and fibrinogen was constructed using the structural data alone (Fig. 3C, active site contacts from 1UCY in white, and fragments of Aα, Bβ and γ chains colored magenta, cyan and yellow, respectively). It is possible that other contacts exist in the 17 residue stretch which links the C-terminus of the substrate peptide to the Aα chain of the E domain, but these are not expected to be important. The exosite interactions are predicted to be preserved in thrombin cleavage of the Bβ chain of fibrinogen [20], but no structural information is available on the active site interactions involved.

imageimage

Figure 3. Crystallographic and mutagenic identification of thrombin exosite and active site interactions. Thrombin is shown in three orientations: in the standard orientation with the active site facing, center; rotated −90° with exosite I facing, left; and, rotated +90° with exosite II facing, right. The first two panels are colored according to electrostatic and hydrophobic potential, as before, to illustrate the nature of the active site and two exosites. All other panels are colored to indicate interaction surfaces on thrombin: blue indicates residues whose mutations specifically affect the rate of substrate cleavage, presumably through direct interaction; and, red indicates thrombin surfaces less than 4 Å distant from substrate or cofactor residues which have been observed crystallographically. This figure emphasizes thrombin's exosite interactions, and although it is understood that the active site is necessarily involved in all substrate recognition events, only active site interactions which have been crystallographically defined are shown. Details of individual panels are given in the appropriate section in the text.

Factors V and VIII

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

Factors V and VIII are required cofactors for the activities of the prothrombinase and Xase complexes, respectively, and their activation by thrombin thereby upregulates thrombin generation through positive feedback [2]. Both factors are cleaved in multiple places by thrombin to release the central B domains from the active components, factors Va (FVa) and VIIIa (FVIIIa) (Table 1, and for current reviews see Refs. [21,22]). Consistent with earlier work [23], two recent studies using the same library of thrombin variants have identified several residues which potentially mediate direct thrombin contact with FV [24] and FVIII [25]: K36, H71, R73, R75, Y76, R77a, K109/110, R93/R97/E97a, R101, R233/K236/Q239, and K60f for FV; and, K36, H71, R73, R75, Y76, R101 and D186a/K186d for FVIII. Of the identified residues, K36, H71, R73, R75, Y76, R77a and K109/110 are in exosite I, and R93/R97/E97a, R101, and R233/K236/Q239 are in exosite II. All studies support the absolute involvement of exosite I in recognition FV and FVIII, but the role of exosite II in recognition of individual cleavage sites is less clear. Although no structural data exist for either active site or exosite interactions, it can be concluded from the biochemical studies that in addition to the active site, both exosites I and II are involved in thrombin recognition of FV and FVIII (Fig. 3D,E).

Factor XIII

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

Factor XIII and all subsequent thrombin substrates discussed in this article require a cofactor for effective recognition and cleavage by thrombin (Table 1). FXIII is a heterotetrameric zymogen composed of two catalytic A subunits and two carrier B subunits (for review see Ref. [26]). Thrombin cleavage at a single position in the A chains (R37) results in the release of the B-chains and exposure of the catalytic site. The activated transglutaminase, FXIIIa, covalently cross-links glutamine residues with lysines on adjacent fibrin monomers to stabilize the nascent fibrin clot. Thrombin thus plays a critical role in both activating FXIII and in creating its substrate fibrin. Not surprisingly, the cofactor which accelerates the activation of FXIII by thrombin is fibrin (80-fold acceleration) [27]. Exosite I of thrombin is thus indirectly involved in FXIII recognition through its interaction with fibrin (described above). A mutagenesis study has identified residues adjacent to the active site (R173/R175/D178) as affecting FXIII cleavage [28], but no direct exosite interactions appear to be involved in the recognition of the FXIII by thrombin. For FXIII, as with all thrombin substrates, an interaction with the active site of thrombin is a critical part of the molecular recognition event; the details of which have been revealed by a crystal structure of the FXIII activation peptide with thrombin [29]. The crystal structure showed residues P10 to P1 of the activation peptide (white rods in Fig. 3F) in a similar conformation as fibrinopeptide Aα (see above), and conserved S1 and aryl-binding pocket (P9 Val) contacts.

GpIbα

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

Glycoprotein Ibα is one of four integral membrane proteins comprising the platelet receptor complex Gp Ib-IX-V (for reviews see Refs. [30,31]). GpIbα interacts with von Willebrand factor in a molecular event which adheres platelets to the site of tissue damage, but it also serves as a thrombin cofactor for at least three substrate cleavage events (PARs 1 and 4, and FXI, see below). Two very different crystal structures of GpIbα complexed to thrombin were reported in back-to-back articles in 2003 [32,33]. The structures show different GpIbα interactions with both thrombin exosites I and II, and some considerable effort was made to reconcile the structures with one another [34,35] and with the fact that solution studies conclusively limit the interaction to exosite II. This has led to some confusion in the field. However, it is clear from a careful examination of the biochemical and structural data that only the thrombin interactions involving exosite II are relevant to the cofactor activity of GpIbα. Two mutagenesis studies published in 2001 are of crucial importance in arriving at this conclusion. Wardell et al. [36] showed that exosite I variants R73E and R70E had no effect on thrombin binding to GpIbα, but that exosite II variants R93E and K236E decrease the affinity of the interaction by 10 and 25-fold, respectively. In addition, the weak ligand heparin, which binds to exosite II, decreased the affinity of the thrombin-GpIbα interaction, but the very strong exosite I ligand hirudin had no effect. The second study by De Cristofaro et al. [37] similarly found that alanine mutations in exosite I had no effect on GpIbα binding, but that those in exosite II decreased affinity appreciably. In addition, the loss of thrombin affinity for GpIbα correlated with the magnitude loss in platelet activation activity. When the two GpIbα-thrombin structures are compared with one another, that determined by Dumas et al. stands out as the most consistent with the biochemical data. The thrombin-binding region of GpIbα is composed of 10 negatively charged side chains: D269, E270, D272, D274, D277, E281, E282, D283, and two sulfated tyrosines at 276 and 279 [38,39]. In the structure by Celikel et al., only three salt bridges between exosite II and the acidic region are found; whereas a much more extensive interaction is observed in the structure reported by Dumas et al., involving five ionic interactions. In the Dumas structure residues R126, K236, K233, R101 and K240 make salt bridges, and K235 and R93 are in proximity to contribute. The dependence of the GpIbα-thrombin interaction on ionic strength is also consistent with the Dumas structure, revealing an electrostatic contact of four-to-five ionic interactions [36] (shown as exosite II-docked white rods in Fig. 3G,H).

PARs

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

In one of the most important procoagulant actions of thrombin, platelets are activated by the cleavage of G-protein coupled, protease-activated receptors (PARs) 1 and 4 (for review see Ref. [40]). Thrombin cleaves PARs at a single site resulting in the release of the N-terminal activation peptide. PAR1 is the primary thrombin receptor on platelets, requiring picomolar thrombin concentrations for effective activation, while PAR4 cleavage is only relevant at high thrombin concentrations (>10 nm). The rapidity of PAR1 cleavage is conferred by exosite interactions. The extended substrate sequence of PAR1 contains a hirudin-like domain C-terminal to the scissile bond (DKYEPFWEDEE), absent in PAR4, which has been demonstrated by mutagenesis and competition studies to interact with exosite I [41–44]. A cocrystallization study of thrombin with the PAR1 recognition sequence has also been undertaken [45], but with mixed results. Several structures were solved, but in none was the peptide fully bound in a productive fashion. However, one structure (PDB ID code 1NRS) revealed the active site interactions from P4 to P1 (38LDPR41) and another revealed the exosite I interactions (1NRN) with the hydrophobic sequence (51KYEPFW56). At the time of the publication of this study, little was known about the P′ side interactions, but the orientation of the two fragments from the two separate structures was consistent with productive peptide–thrombin interactions. I have made a model of the entire PAR1 peptide–thrombin interaction based on the fixed position of substrate sequence LDPR from 1NRS and the exosite I-interacting residues KYEPFW from 1NRN. The conformation of the intervening sequence was taken from the structure of the antithrombin (AT) reactive center loop in the active site cleft of thrombin [46]. The conserved P1′ Ser was fixed, and the hydrophobic P2′ Leu of AT was replaced by Phe of PAR1 and then fixed. The intervening residues were fit loosely according to the AT structure and energy minimized for 200 steps using the program CNS [47]. The result is shown as white active site and exosite-bound rods in Fig. 3G, and the model is available upon request. In addition to the exosite I and active site interactions, thrombin cleavage of PAR1 (and presumably PAR4) is also exosite II dependent due to the cofactor effect of GpIbα, which accelerates the rate of reaction by six-to-seven-fold [48].

Factor XI

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

It has only recently become clear that thrombin is the preferred physiological activator of FXI [49–52] (and reviewed in Ref. [53]), with efficient activation occuring exclusively on the surface of platelets where thrombin and FXI are colocalized through separate GpIbα interactions. One study has investigated the thrombin residues involved [54] and found direct exosite I interactions between thrombin and FXI, and a dependence of exosite II for GpIbα binding (discussed above). Exosite I residues H71, R73, R75, Y76, R77a, K110, and K109/110 are involved in direct contacts, possibly to the A1 domain of FXI [55] (Fig. 3H).

Thrombomodulin

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

Thrombomodulin (TM) [56] is a single chain glycoprotein of 557 amino acids in length which is found in high density on the surface of the endothelial cells which line blood vessels (for recent reviews see Refs. [57–59]). It is composed of five regions: (1) the N-terminal lectin-like domain; (2) six consecutive EGF-like repeats; (3) a proteoglycan-like Ser-Thr-rich region which is highly O-glycosylated; (4) a single transmembrane helix; and, (5) the C-terminal cytoplasmic tail (Fig. 4). The molecular details of the thrombin–TM interaction were revealed in 2000 by the structure of thrombin bound to a minimal TM fragment [60] containing the final three EGF-like repeats, TME456 [61]. As expected from biochemical studies [62–66], EGF repeats five and six interact directly with exosite I of thrombin, with a buried interface of ∼900 Å2 (yellow ribbon in Fig. 3I,J). While the interface interposes surfaces with complementary electrostatic properties, only one salt bridge was observed between thrombin and TM (Lys110 of thrombin and Asp461 of TM), and the majority of the energy of the interaction is provided by hydrophobic contacts. The more basic exosite II of thrombin can also be involved in the TM interaction [67]. A chondroitin sulfate (CS) moiety found on about 20% of TM molecules in the vascular endothelium and about 30%–35% of those lining the arteries [68] improves the affinity of TM for thrombin by an order-of-magnitude [69,70]. Chondroitin 4-sulfate [71] is similar to heparin in its composition and sulfation pattern and would be predicted to bind in a similar fashion (rods in Fig. 3I,J). The avidity of the thrombin–TM interaction is great, with an overall dissociation constant in the nanomolar range [66,72]. Such a tight interaction ensures that TM will effectively compete for any thrombin that has diffused away from the clot onto the surface of the adjacent intact endothelium. Once bound to TM, thrombin is no longer capable of cleaving any of its many procoagulant substrates [73,74] (with the exception of the fibrinolysis inhibitor TAFI), but its ability to activate the anticoagulant protein C (PC) is enhanced by three orders-of-magnitude.

image

Figure 4. The mechanism by which TM confers thrombin specificity for PC. TM (yellow) is an integral membrane protein, which is expressed on the surface of endothelial cells which line the vasculature. It is a multidomain protein composed of an N-terminal lectin-like domain, six consecutive EGF-like domains, a Ser/Thr-rich proteoglycan domain, a single transmembrane helix, and a small C-terminal cytoplasmic domain. The proteoglycan domain is heterogeneous in its sulfation, so that about one third of TM molecules possess a single highly sulfated CS moiety. Thrombin (red, IIa) which diffuses away from the clot can encounter TM on the surface of the intact endothelium, where it engages the final two EGF domains of TM in a tight interaction on exosite I. Exosite II will also be occupied by the CS moiety of TM, when present, significantly strengthening the interaction between thrombin and TM. In this manner, thrombin is incapable of interacting with any of its procoagulant substrates which rely on exosite I, and to some degree, exosite II binding. Thrombin specificity for PC (green) is partially conferred by their colocalization on the endothelial cell surface through the docking of the Gla domain of PC onto its receptor EPCR; this also ensures correct positioning of the activation loop of PC relative to the active site of TM-docked thrombin. The encounter complex is further stabilized by direct contact between TM EGF domain 4 and the body of PC. The result of this quarternary complex is the activation of protein C (APC), which then shuts down thrombin generation by inactivating essential cofactors Va and VIIIa.

Download figure to PowerPoint

Protein C

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

How TM improves PC recognition by thrombin is illustrated in Fig. 4. It is clear that the tight interaction of EGF domains five and six with exosite I of thrombin effectively blocks the binding of any procoagulant substrate which depends on direct or cofactor dependent exosite I contacts (e.g. fibrinogen, FV, FVIII, FXI, FXIII, and PAR1). In addition, EGF domain four of TM directly interacts with a basic patch on PC [75], thus explaining why efficient recognition of PC requires EGF domains four to six [67,76]. Colocalization on the intact endothelial cell surface also contributes to PC recognition by thrombin [77–79]. Mutagenesis studies conclusively show that no direct thrombin–PC exosite contacts play a significant role in recognition [13] (Fig. 3I) [80].

TAFI

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

Thrombin-activated fibrinolysis inhibitor (TAFI) is a metallo-carboxypeptidase which cleaves C-terminal lysine residues off of fibrin, thus protecting the clot from fibrinolysis (for recent reviews see Refs. [81–83]). Its activation by thrombin is similar to that of PC, in that it is wholly dependent on TM and there are no important direct contacts with thrombin [80] (Fig. 3J, colored as in 3I). Rather, interactions between TAFI and TM EGF domain three appear to be critical for recognition of TAFI by TM-bound thrombin [84].

Heparin

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

Glycosaminoglycans (GAGs) (e.g. heparan sulfate, CS and dermatan sulfate) are sulfated polysaccharides which decorate the proteoglycans in the vascular and extravascular spaces. Heparin, a highly sulfated GAG produced by mast cells and used as a therapeutic anticoagulant, serves as a model compound for most protein-GAG-dependent processes. The heparin-binding site of thrombin was localized to exosite II through mutagenesis studies [69,85–87], and recently through a high-resolution crystal structure of a heparin octasaccharide bound to thrombin [88]. The interaction between heparin and thrombin is non-specific [89], fully ionic in nature, and involves residues R93, K236, K240, R101 and R233 (listed in order of importance). GAGs are thrombin cofactors for its interactions with TM (through CS, Fig. 3I,J) and the inhibitors AT (through heparan sulfate and therapeutic heparin) and heparin cofactor II (HCII) (through dermatan sulfate). GAG binding therefore participates exclusively in the antithrombotic reactions of thrombin.

Heparin cofactor II and antithrombin

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

Heparin cofactor II and AT are the only specific thrombin inhibitors in the circulation and, although they appear to have evolved in a convergent fashion, they are similar in many important ways (for review see Ref. [90]). Both circulate in the plasma at μm concentrations; both are members of the serpin family of protease inhibitors and thus share the irreversible serpin mechanism of inhibition [91]; both inhibit thrombin at enhanced rates in the presence of activating GAG such as heparin; and both can utilize thrombin exosites to efficiently recognize thrombin. Inhibition of thrombin by serpins requires efficient progression through the catalytic steps leading to the acyl-enzyme complex, and is thus dependent on the formation of a productive Michaelis complex. HCII lacks the otherwise conserved P1Arg in its reactive center (see Table 1), and would not be a physiological inhibitor of thrombin where it not for the presence of an 80 amino acid N-terminal insert which contains two highly acidic, hirudin-like repeats [92]. The structure of HCII in its Michaelis complex with thrombin revealed how the acidic tail is used to confer thrombin inhibitory specificity to HCII through interaction with exosite I [93] (Fig. 3K, active site and exosite I-interacting residues shown as white rods). As with other exosite I interactions defined by crystal structures, the interface between the N-terminal tail of HCII and thrombin is predominantly hydrophobic in nature, involving only one basic residue on thrombin (Lys110). Although it is unclear which GAG is responsible for the physiological activation of HCII toward thrombin, the absolute requirement of GAG brings into play exosite II of thrombin in the formation of the GAG-bridged HCII-thrombin encounter complex [94] (heparin shown as white rods bound to exosite II in Fig. 3K). Thus, both anion-binding exosites are exploited by HCII to efficiently recognize and terminate thrombin.

Although HCII is undoubtedly a specific inhibitor of thrombin, AT is the main circulating inhibitor of the coagulation proteases, including thrombin. Thrombin inhibition by AT is accelerated ∼1000-fold through GAG binding, however the uncatalyzed rate is appreciable [95] and is likely to be of physiological relevance. The mechanism by which endothelial cell surface heparan sulfate accelerates thrombin inhibition by AT can be understood in terms of improved diffusion, known as the template effect [96]; with AT binding with high affinity to specific pentasaccharide sequences as thrombin translates along the one-dimensional GAG chain through a weak (μm) interaction with exosite II [96]. The productive Michaelis complex between AT and thrombin is then stabilized by their co-occupation on the same heparin chain [97]. Thus, exosite II of thrombin is critically involved in its heparin-enhanced inhibition by AT, and it was hypothesized that the surface properties of AT and thrombin would make direct exosite interactions unlikely [90]. The recent structure of the ternary complex between AT, thrombin and a heparin mimetic [46] revealed a surprisingly intimate direct contact interface with the engagement of two additional exosites: the γ-loop and the Na+-binding site (Fig. 3L).

Exosite I or II?

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

It is clear from Fig. 3 that thrombin utilizes both exosites I and II in almost all of the molecular recognition events which govern thrombin function. One perplexing feature of thrombin exosite interactions is that anionic peptides seem to bind exclusively to one exosite or the other. Although in crystal structures one occasionally sees interactions with both exosites (e.g. GpIbα), solution studies support absolute exosite discrimination. How can it be that two anion-binding exosites discriminate between anionic peptide sequences? Enough structural information now exists of thrombin exosite interactions to compare sequences in an attempt to determine if exosite I- and II-binding peptides differ in any obvious features. In Table 2, thrombin-binding peptides are grouped according to their site of interaction, and the number of acidic, basic and hydrophobic residues counted. When the fraction of net negative charge is divided by the fraction of hydrophobic residues, a clear trend emerges: all exosite I-interacting peptides have a ratio below two, and all exosite II-interacting peptides have a ratio above two. This scheme reflects the observation that hydrophobic contacts provide the majority of the binding energy for exosite I interactions, with electrostatics mostly involved in orienting the complementary hydrophobic surfaces (so-called ‘electrostatic steering’) [98,99]. Exosite II interactions, on the other hand, are primarily ionic in nature, with hydrophobics contributing minimally to the overall binding energy. Indeed, it is thus more accurate to call exosite I the ‘apolar-binding exosite’, and exosite II the ‘anion-binding exosite’.

Table 2.  How does thrombin discriminate between exosite I- and II-binding peptides?
 Binding sequenceFractional negative chargeFraction hydrophobicRatio
  1. Fractional negative charge is calculated by subtracting the basic from acidic residues, and dividing by the number of all residues. Fraction hydrophobic is the ratio of hydrophobic residues (I, V, F, Y, W, P) divided by the number of all residues. *Indicates a sulfated tyrosine, which is considered as both acidic and hydrophobic.

Exosite I
 Fibrinogen AαDSDWPFCSDEDWNY0.360.361
 HirudinDFEEIPEEY*LQ0.550.451.2
 HCIIEGEEDDDY*LDLEKIFSEDDDY*IDIVD0.580.351.7
 PAR1DKYEPFWEDEEK0.330.331
 Average 0.45 ± 0.110.37 ± 0.051.2 ± 0.3
Exosite II
 HemadinEFEEFEIDEEE0.720.272.7
 GpIbαDEGDTDLY*DY*PEEDTEGD0.680.262.6
 Fibrinogen γETEY*DSLY*PEDD0.750.332.3
 Average 0.72 ± 0.030.29 ± 0.032.5 ± 0.2

Acknowledgements

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference

I would like to thank the Scientific Committee of ISTH 2005 for the invitation to contribute this manuscript. Funding was provided by the British Heart Foundation and the Medical Research Council (UK).

Reference

  1. Top of page
  2. Abstract
  3. Multiple functions of thrombin in hemostasis
  4. Structural features of thrombin
  5. Method
  6. Fibrinogen
  7. Factors V and VIII
  8. Factor XIII
  9. GpIbα
  10. PARs
  11. Factor XI
  12. Thrombomodulin
  13. Protein C
  14. TAFI
  15. Heparin
  16. Heparin cofactor II and antithrombin
  17. Exosite I or II?
  18. Acknowledgements
  19. Reference
  • 1
    RatnoffOD, RatnoffOD, ForbesCD, eds. The evolution of knowledge about hemostasis. Disorders of Hemostasis. 3rd edn. Philadelphia: WB Saunders Company, 1996;
  • 2
    Mann KG, Butenas S, Brummel K. The dynamics of thrombin formation. Arterioscler Thromb Vasc Biol 2003; 23: 1725.
  • 3
    Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 1967; 27: 15762.
  • 4
    Bode W, Mayr I, Baumann U, Huber R, Stone SR, Hofsteenge J. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J 1989; 8: 346775.
  • 5
    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: 42671.
  • 6
    Stubbs MT, Bode W. A player of many parts: the spotlight falls on thrombin's structure. Thromb Res 1993; 69: 158.
  • 7
    Stubbs MT, Bode W. The clot thickens: clues provided by thrombin structure. Trends Biochem Sci 1995; 20: 238.
  • 8
    Kraulis PJ. Molscript – a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr 1991; 24: 94650.
  • 9
    Esnouf RM. An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. J Mol Graph Model 1997; 15: 132.
  • 10
    Merritt EA, Murphy MEP. Raster3d version-2.0 – A program for photorealistic molecular graphics. Acta Crystalogr 1994; D50: 86973.
  • 11
    Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features of fibrinogen and fibrin. Ann N Y Acad Sci 2001; 936: 1130.
  • 12
    Higgins DL, Lewis SD, Shafer JA. Steady state kinetic parameters for the thrombin-catalyzed conversion of human fibrinogen to fibrin. J Biol Chem 1983; 258: 927682.
  • 13
    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: 1685463.
  • 14
    Hall SW, Gibbs CS, Leung LL. Identification of critical residues on thrombin mediating its interaction with fibrin. Thromb Haemost 2001; 86: 146674.
  • 15
    Martin PD, Robertson W, Turk D, Huber R, Bode W, Edwards BF. The structure of residues 7–16 of the A alpha-chain of human fibrinogen bound to bovine thrombin at 2.3-A resolution. J Biol Chem 1992; 267: 791120.
  • 16
    Martin PD, Malkowski MG, DiMaio J, Konishi Y, Ni F, Edwards BF. Bovine thrombin complexed with an uncleavable analog of residues 7–19 of fibrinogen A alpha: geometry of the catalytic triad and interactions of the P1′, P2′, and P3′ substrate residues. Biochemistry 1996; 35: 130309.
  • 17
    Malkowski MG, Martin PD, Lord ST, Edwards BF. Crystal structure of fibrinogen-Aalpha peptide 1–23 (F8Y) bound to bovine thrombin explains why the mutation of Phe-8 to tyrosine strongly inhibits normal cleavage at Arg-16. Biochem J 1997; 326: 81522.
  • 18
    Maurer MC, Trosset JY, Lester CC, DiBella EE, Scheraga HA. New general approach for determining the solution structure of a ligand bound weakly to a receptor: structure of a fibrinogen Aalpha-like peptide bound to thrombin (S195A) obtained using NOE distance constraints and an ECEPP/3 flexible docking program. Proteins 1999; 34: 2948.
  • 19
    Krishnan R, Sadler JE, Tulinsky A. Structure of the Ser195Ala mutant of human alpha–thrombin complexed with fibrinopeptide A(7–16): evidence for residual catalytic activity. Acta Crystallogr D Biol Crystallogr 2000; 56: 40610.
  • 20
    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 USA 2004; 101: 271823.
  • 21
    Duga S, Asselta R, Tenchini ML. Coagulation factor V. Int J Biochem Cell Biol 2004; 36: 13939.
  • 22
    Fay PJ. Activation of factor VIII and mechanisms of cofactor action. Blood Rev 2004; 18: 115.
  • 23
    Esmon CT, Lollar P. Involvement of thrombin anion-binding exosites 1 and 2 in the activation of factor V and factor VIII. J Biol Chem 1996; 271: 138827.
  • 24
    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: 251439.
  • 25
    Myles T, Yun TH, Leung LL. Structural requirements for the activation of human factor VIII by thrombin. Blood 2002; 100: 28206.
  • 26
    Ariens RA, Lai TS, Weisel JW, Greenberg CS, Grant PJ. Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms. Blood 2002; 100: 74354.
  • 27
    Janus TJ, Lewis SD, Lorand L, Shafer JA. Promotion of thrombin-catalyzed activation of factor XIII by fibrinogen. Biochemistry 1983; 22: 626972.
  • 28
    Philippou H, Rance J, Myles T, Hall SW, Ariens RA, Grant PJ, Leung L, Lane DA. 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: 320206.
  • 29
    Sadasivan C, Yee VC. Interaction of the factor XIII activation peptide with alpha -thrombin. Crystal structure of its enzyme-substrate analog complex. J Biol Chem 2000; 275: 369428.
  • 30
    Andrews RK, Gardiner EE, Shen Y, Whisstock JC, Berndt MC. Glycoprotein Ib-IX-V. Int J Biochem Cell Biol 2003; 35: 11704.
  • 31
    Brass LF. Thrombin and platelet activation. Chest 2003; 124: 18S25S.
  • 32
    Dumas JJ, Kumar R, Seehra J, Somers WS, Mosyak L. Crystal structure of the GpIbalpha-thrombin complex essential for platelet aggregation. Science 2003; 301: 2226.
  • 33
    Celikel R, McClintock RA, Roberts JR, Mendolicchio GL, Ware J, Varughese KI, Ruggeri ZM. Modulation of alpha-thrombin function by distinct interactions with platelet glycoprotein Ibalpha. Science 2003; 301: 21821.
  • 34
    Sadler JE. Structural biology. A menage a trois in two configurations. Science 2003; 301: 1779.
  • 35
    Vanhoorelbeke K, Ulrichts H, Romijn RA, Huizinga EG, Deckmyn H. The GPIbalpha-thrombin interaction: far from crystal clear. Trends Mol Med 2004; 10: 339.
  • 36
    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: 61618.
  • 37
    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: 1326873.
  • 38
    Marchese P, Murata M, Mazzucato M, Pradella P, De Marco L, Ware J, Ruggeri ZM. Identification of three tyrosine residues of glycoprotein Ib alpha with distinct roles in von Willebrand factor and alpha-thrombin binding. J Biol Chem 1995; 270: 95718.
  • 39
    De Marco L, Mazzucato M, Masotti A, Ruggeri ZM. Localization and characterization of an alpha-thrombin-binding site on platelet glycoprotein Ib alpha. J Biol Chem 1994; 269: 647884.
  • 40
    Ofosu FA. Protease activated receptors 1 and 4 govern the responses of human platelets to thrombin. Transfus Apheresis Sci 2003; 28: 2658.
  • 41
    Liu LW, Vu TK, Esmon CT, Coughlin SR. The region of the thrombin receptor resembling hirudin binds to thrombin and alters enzyme specificity. J Biol Chem 1991; 266: 1697780.
  • 42
    Jacques SL, LeMasurier M, Sheridan PJ, Seeley SK, Kuliopulos A. Substrate-assisted catalysis of the PAR1 thrombin receptor. Enhancement of macromolecular association and cleavage. J Biol Chem 2000; 275: 406718.
  • 43
    Vu TK, Wheaton VI, Hung DT, Charo I, Coughlin SR. Domains specifying thrombin-receptor interaction. Nature 1991; 353: 6747.
  • 44
    Ayala YM, Cantwell AM, Rose T, Bush LA, Arosio D, Di Cera E. Molecular mapping of thrombin-receptor interactions. Proteins 2001; 45: 10716.
  • 45
    Mathews II, Padmanabhan KP, Ganesh V, Tulinsky A, Ishii M, Chen J, Turck CW, Coughlin SR, Fenton JW. Crystallographic structures of thrombin complexed with thrombin receptor peptides: existence of expected and novel binding modes. Biochemistry 1994; 33: 326679.
  • 46
    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: 85762.
  • 47
    Brunger AT, Adams PD, Clore GM, Delano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Cryst 1998; D54: 90521.
  • 48
    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: 46928.
  • 49
    Gailani D, Broze GJJ. Factor XI activation in a revised model of blood coagulation. Science 1991; 253: 90912.
  • 50
    Naito K, Fujikawa K. Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces. J Biol Chem 1991; 266: 73538.
  • 51
    Baglia FA, Walsh PN. Prothrombin is a cofactor for the binding of factor XI to the platelet surface and for platelet-mediated factor XI activation by thrombin. Biochemistry 1998; 37: 227181.
  • 52
    Baglia FA, Walsh PN. Thrombin-mediated feedback activation of factor XI on the activated platelet surface is preferred over contact activation by factor XIIa or factor XIa. J Biol Chem 2000; 275: 205149.
  • 53
    Walsh PN. Roles of factor XI, platelets and tissue factor-initiated blood coagulation. J Thromb Haemost 2003; 1: 20816.
  • 54
    Yun TH, Baglia FA, Myles T, Navaneetham D, Lopez JA, Walsh PN, Leung LL. 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: 481129.
  • 55
    Baglia FA, Walsh PN. A binding site for thrombin in the apple 1 domain of factor XI. J Biol Chem 1996; 271: 36528.
  • 56
    Esmon NL, Owen WG, Esmon CT. Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem 1982; 257: 85964.
  • 57
    Esmon CT. The protein C pathway. Chest 2003; 124: 26S32S.
  • 58
    Dahlback B, Villoutreix BO. Molecular recognition in the protein C anticoagulant pathway. J Thromb Haemost 2003; 1: 152534.
  • 59
    Weiler H, Isermann BH. Thrombomodulin. J Thromb Haemost 2003; 1: 151524.
  • 60
    Zushi M, Gomi K, Yamamoto S, Maruyama I, Hayashi T, Suzuki K. The last three consecutive epidermal growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C-activating cofactor activity and anticoagulant activity. J Biol Chem 1989; 264: 103513.
  • 61
    Fuentes-Prior P, Iwanaga Y, Huber R, Pagila R, Rumennik G, Seto M, Morser J, Light DR, Bode W. Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex. Nature 2000; 404: 51825.
  • 62
    Kurosawa S, Stearns DJ, Jackson KW, Esmon CT. A 10-kDa cyanogen bromide fragment from the epidermal growth factor homology domain of rabbit thrombomodulin contains the primary thrombin binding site. J Biol Chem 1988; 263: 59936.
  • 63
    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: 110238.
  • 64
    Nagashima M, Lundh E, Leonard JC, Morser J, Parkinson JF. Alanine-scanning mutagenesis of the epidermal growth factor-like domains of human thrombomodulin identifies critical residues for its cofactor activity. J Biol Chem 1993; 268: 288892.
  • 65
    Tolkatchev D, Ng A, Zhu B, Ni F. Identification of a thrombin-binding region in the sixth epidermal growth factor-like repeat of human thrombomodulin. Biochemistry 2000; 39: 1036572.
  • 66
    Baerga-Ortiz A, Rezaie AR, Komives EA. Electrostatic dependence of the thrombin-thrombomodulin interaction. J Mol Biol 2000; 296: 6518.
  • 67
    Tsiang M, Lentz SR, Sadler JE. Functional domains of membrane-bound human thrombomodulin. EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity. J Biol Chem 1992; 267: 616470.
  • 68
    Lin JH, McLean K, Morser J, Young TA, Wydro RM, Andrews WH, Light DR. Modulation of glycosaminoglycan addition in naturally expressed and recombinant human thrombomodulin. J Biol Chem 1994; 269: 2502130.
  • 69
    Ye J, Rezaie AR, Esmon CT. Glycosaminoglycan contributions to both protein C activation and thrombin inhibition involve a common arginine-rich site in thrombin that includes residues arginine 93, 97, and 101. J Biol Chem 1994; 269: 1796570.
  • 70
    Vindigni A, White CE, Komives EA, Di Cera E. Energetics of thrombin-thrombomodulin interaction. Biochemistry 1997; 36: 667481.
  • 71
    Nawa K, Sakano K, Fujiwara H, Sato Y, Sugiyama N, Teruuchi T, Iwamoto M, Marumoto Y. Presence and function of chondroitin-4-sulfate on recombinant human soluble thrombomodulin. Biochem Biophys Res Commun 1990; 171: 72937.
  • 72
    Hofsteenge J, Taguchi H, Stone SR. Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors. Biochem J 1986; 237: 24351.
  • 73
    Esmon CT, Esmon NL, Harris KW. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem 1982; 257: 79447.
  • 74
    Esmon NL, Carroll RC, Esmon CT. Thrombomodulin blocks the ability of thrombin to activate platelets. J Biol Chem 1983; 258: 1223842.
  • 75
    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: 1048490.
  • 76
    Hayashi T, Zushi M, Yamamoto S, Suzuki K. Further localization of binding sites for thrombin and protein C in human thrombomodulin. J Biol Chem 1990; 265: 201569.
  • 77
    Fukudome K, Esmon CT. Identification, cloning, and regulation of a novel endothelial cell protein C/activated protein C receptor. J Biol Chem 1994; 269: 2648691.
  • 78
    Fukudome K, Kurosawa S, Stearns-Kurosawa DJ, He X, Rezaie AR, Esmon CT. The endothelial cell protein C receptor. Cell surface expression and direct ligand binding by the soluble receptor. J Biol Chem 1996; 271: 174918.
  • 79
    Stearns-Kurosawa DJ, Kurosawa S, Mollica JS, Ferrell GL, Esmon CT. The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex. Proc Natl Acad Sci USA 1996; 93: 102126.
  • 80
    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: 255106.
  • 81
    Bajzar L. Thrombin activatable fibrinolysis inhibitor and an antifibrinolytic pathway. Arterioscler Thromb Vasc Biol 2000; 20: 25118.
  • 82
    Marx PF. Thrombin-activatable fibrinolysis inhibitor. Curr Med Chem 2004; 11: 233548.
  • 83
    Nesheim M. Thrombin and fibrinolysis. Chest 2003; 124: 33S9S.
  • 84
    Kokame K, Zheng X, Sadler JE. Activation of thrombin-activable fibrinolysis inhibitor requires epidermal growth factor-like domain 3 of thrombomodulin and is inhibited competitively by protein C. J Biol Chem 1998; 273: 121359.
  • 85
    Sheehan JP, Sadler JE. Molecular mapping of the heparin-binding exosite of thrombin. Proc Natl Acad Sci USA 1994; 91: 551822.
  • 86
    Gan ZR, Li Y, Chen Z, Lewis SD, Shafer JA. Identification of basic amino acid residues in thrombin essential for heparin-catalyzed inactivation by antithrombin III. J Biol Chem 1994; 269: 13015.
  • 87
    Tsiang M, Jain AK, Gibbs CS. Functional requirements for inhibition of thrombin by antithrombin III in the presence and absence of heparin. J Biol Chem 1997; 272: 120249.
  • 88
    Carter WJ, Cama E, Huntington JA. Crystal structure of thrombin bound to heparin. J Biol Chem 2005; 280: 27459.
  • 89
    Olson ST, Halvorson HR, Bjork I. Quantitative characterization of the thrombin-heparin interaction. Discrimination between specific and nonspecific binding models. J Biol Chem 1991; 266: 634252.
  • 90
    Huntington JA. Mechanisms of glycosaminoglycan activation of the serpins in hemostasis. J Thromb Haemost 2003; 1: 153549.
  • 91
    Huntington JA, Read RJ, Carrell RW. Structure of a serpin-protease complex shows inhibition by deformation. Nature 2000; 407: 9236.
  • 92
    Tollefsen DM. Heparin cofactor II. Adv Exp Med Biol 1997; 425: 3544.
  • 93
    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 USA 2002; 99: 1107984.
  • 94
    Verhamme IM, Bock PE, Jackson CM. The preferred pathway of glycosaminoglycan-accelerated inactivation of thrombin by heparin cofactor II. J Biol Chem 2004; 279: 978595.
  • 95
    Olson ST, Bjork I, Shore JD. Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin. Methods Enzymol 1993; 222: 52559.
  • 96
    Olson ST, Bjork I. Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. Elucidation from salt concentration effects. J Biol Chem 1991; 266: 635364.
  • 97
    Stone SR, Le Bonniec BF. Inhibitory mechanism of serpins. Identification of steps involving the active-site serine residue of the protease. J Mol Biol 1997; 265: 34462.
  • 98
    Karshikov A, Bode W, Tulinsky A, Stone SR. Electrostatic interactions in the association of proteins: an analysis of the thrombin-hirudin complex. Protein Sci 1992; 1: 72735.
  • 99
    Myles T, Le Bonniec BF, Betz A, Stone SR. Electrostatic steering and ionic tethering in the formation of thrombin-hirudin complexes: the role of the thrombin anion-binding exosite-I. Biochemistry 2001; 40: 49729.