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Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:1738-1745
Published online before print May 25, 2006, doi: 10.1161/01.ATV.0000228844.65168.d1
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:1738.)
© 2006 American Heart Association, Inc.

Brief Reviews

Thrombin-Cofactor Interactions

Structural Insights Into Regulatory Mechanisms

Ty E. Adams; James A. Huntington

From the University of Cambridge, Department of Haematology, Division of Structural Medicine, Thrombosis Research Unit, Cambridge Institute for Medical Research, UK.

Correspondence to James A. Huntington, University of Cambridge, Division of Structural Medicine, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK. E-mail jah52{at}cam.ac.uk


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowGlycoprotein Ib{alpha}
 down arrowFibrin
 down arrowThrombomodulin
 down arrowHeparin and Other...
 down arrowSodium Ion
 down arrowConclusions
 down arrowReferences
 
Precise modulation of thrombin activity throughout the hemostatic response is essential for efficient cessation of bleeding while preventing inappropriate clot growth or dissemination which causes thrombosis. Regulating thrombin activity is made difficult by its ability to diffuse from the surface on which it was generated and its ability to cleave at least 12 substrates. To overcome this challenge, thrombin recognition of substrates is largely controlled by cofactors that act by localizing thrombin to various surfaces, blocking substrate binding to critical exosites, engendering new exosites for substrate recognition and by allosterically modulating the properties of the active site of thrombin. Thrombin cofactors can be classified as either pro- or anticoagulants, depending on how substrate preference is altered. The procoagulant cofactors include glycoprotein Ib{alpha}, fibrin, and Na+, and the anticoagulants are heparin and thrombomodulin. Over the last few years, crystal structures have been reported for all of the thrombin-cofactor complexes. The purpose of this article is to summarize the features of these structures and to discuss the mechanisms and physiological relevance of cofactor binding in thrombin regulation.

Substrate recognition by thrombin is largely controlled by cofactors, and crystal structures have now been reported for all of the thrombin-cofactor complexes. The purpose of this article is to summarize the features of these structures and to discuss the mechanisms and physiological relevance of cofactor binding in thrombin regulation.


Key Words: thrombin • hemostasis • regulation • cofactor • specificity


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowGlycoprotein Ib{alpha}
 down arrowFibrin
 down arrowThrombomodulin
 down arrowHeparin and Other...
 down arrowSodium Ion
 down arrowConclusions
 down arrowReferences
 
Damage to the blood vessel wall initiates the hemostatic response by exposing subendothelial proteins such as tissue factor and collagen.1,2 Crucial to the formation of the blood clot is the generation of the serine protease thrombin.3 Inability to generate sufficient amounts of thrombin in a timely manner results in hemorrhage, as observed in the bleeding disorders hemophilia A and B,4 whereas overproduction or failure to efficiently inhibit thrombin activity results in thrombosis. Thrombin has a broad range of diverse substrates that includes both pro- and anticoagulant functions (Figure 1). The activation of platelets through cleavage of protease-activated receptors (PARs) 1 and 4 and the cleavage of fibrinogen to fibrin are the primary procoagulant functions of thrombin,2,5 which serve to form the platelet plug and generate the fibrin meshwork that stabilizes the plug at the site of injury. The formation of a stable clot also depends on the ability of thrombin to stimulate its own generation through feedback activation of the protein cofactors V and VIII6,7 and through activation of the zymogen factor XI.8 Activation of the transglutaminase factor XIII by thrombin is also critical for stabilization of the clot through covalent cross-linking of the fibrin polymers.9 The von Willebrand factor (vWF) processing enzyme ADAMTS13 has also recently been identified as a procoagulant substrate of thrombin in vitro,10 the cleavage of which inactivates ADAMTS13 and may increase platelet adhesion at the site of vessel injury. In addition to these clot-promoting activities, thrombin also has the ability to downregulate its own generation through activation of the protein C pathway.11 Activated protein C (APC) inactivates cofactors Va and VIIIa, thereby blunting thrombin generation. Thrombin also participates directly in its final inhibition and clearance from the circulation by specifically recognizing the serpins antithrombin (AT) and heparin cofactor II (HCII).


Figure 1
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  		Figure 1. Thrombin activities. Schematic representation of thrombin activities in coagulation, with cofactors indicated in parentheses.

Thrombin is generated from its zymogen prothrombin through proteolytic cleavage at 2 sites (arginines 271 and 320) by factor Xa. The resulting 37-kDa serine protease thrombin is no longer associated to its Gla and 2 Kringle domains and is thus free to diffuse away from the surface on which it was generated, and its special structural features are used to recognize a broad spectrum of substrates.12–15 The first structure of thrombin, solved in 1989, revealed an overall protein fold very similar to other members of the chymotrypsin family of proteases, including the characteristic catalytic triad of histidine 57, aspartate 102, and serine 195 (chymotrypsin template numbering is used throughout) in an active site cleft.16 The structure of thrombin differs from that of its simpler cousins by the presence of extension loops that line the walls of the active site cleft, the 60- and {gamma}-loops, and 2 regions of positive electrostatic potential known as anion-binding exosites I and II (Figure 2). Biochemical and mutagenesis studies have established that thrombin recognition of substrates invariably involves engagement of the active site cleft and either one17–19 or both20–22 of the exosites (see Huntington23 for review). In addition to direct interaction with substrates, thrombin specificity is also regulated through interaction with several cofactors (Table). Cofactor binding regulates thrombin activity toward various substrates by mechanisms such as localization, competition for exosite binding, and allostery. Although much is still unknown about how thrombin specificity is determined, within the last few years all of the thrombin-cofactor crystal structures have been determined (Figure 3). This review summarizes the structural features of the thrombin-cofactor complexes and the significance of cofactors in directing the activity of thrombin.


Figure 2
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  		Figure 2. Thrombin topography. Stereo view of surface representations of thrombin, shown in the standard orientation (Protein DataBank entry 1PPB), bound to the active site inhibitor d-Phe-Pro-Arg-ck (PPAck). The figure (displayed with a transparent surface and underlying ribbon structure) shows positions of relevant specificity-determining sites: the 60- and {gamma}-loops (green), residues that make up exosite I (blue), and residues in exosite II (red). The position of coordinated Na+ is indicated.


View this table:
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  		Thrombin-Cofactor Binding Affinities and Kinetic Effects


Figure 3
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  		Figure 3. Thrombin-cofactor exosite interactions. The surface representation of thrombin is shown in the standard orientation (left) for exosite I interactions (Protein DataBank entries: fibrin-1QVH, TM-1DX5) and rotated 90° (right) to show exosite II interactions (PDB entries: GPIb{alpha}-1P8V, heparin-1XMN). The thrombin residues involved at the cofactor interface (<4 Å distant) are colored as green.


*    Glycoprotein Ib{alpha}
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 up arrowAbstract
 up arrowIntroduction
 *Glycoprotein Ib{alpha}
down arrowFibrin
 down arrowThrombomodulin
 down arrowHeparin and Other...
 down arrowSodium Ion
 down arrowConclusions
 down arrowReferences
 
A fraction of thrombin generated during the early stages of coagulation binds to platelet glycoprotein (GP) Ib{alpha} (GpIb{alpha}), 1 of 4 proteins of the GpIb-IX-V platelet receptor complex. GpIb{alpha} is a heavily O-glycosylated transmembrane protein that has been hypothesized to be the high-affinity binding site for thrombin on the platelet surface.24,25 GpIb{alpha} acts as a cofactor by enhancing thrombin cleavage of the platelet receptor PAR-1, resulting in platelet activation.26,27 Through binding to the third apple domain of factor XI, GpIb{alpha} also stimulates the activation of factor XI by thrombin.8,28 Finally, GpIb{alpha} can additionally promote platelet activation through enhancing thrombin cleavage of GpV.29,30 Several studies using mutations31 and synthetic peptides32 identified the thrombin binding site on GpIb{alpha} as the negatively charged region 271 to 284, including 3 sulfated tyrosine residues, 276, 278, and 279.25,33 The binding site for GpIb{alpha} on thrombin has been localized to exosite II, with mutations in this exosite displaying a marked decrease in affinity for GpIb{alpha},34 whereas similar mutations in exosite I of thrombin showed little or no effect.35 Exosite II residue arginine 233 is critical for the interaction, with the R233A and the R233A/K236A/Q239A mutations reducing the affinity for GpIb{alpha} by 29- and 31-fold, respectively.8,34 A complementary study found that thrombin residues in exosite I are involved directly in binding to substrates factor XI8 and PAR-1,36,37 further supporting exosite II as the only cofactor binding site for GpIb{alpha}.

In 2003, 2 crystal structures of the N-terminal domain of GpIb{alpha} in complex with thrombin were solved.38,39 Both structures revealed an interaction between the negatively charged tail of GpIb{alpha} and exosite II of thrombin (Figure I in the online data supplement, available at http://atvb.ahajournals.org), with arginine 233 making contacts with several backbone oxygen atoms of the acidic region of GPIb{alpha}. Both structures also showed interactions involving GpIb{alpha} residues in the acidic region, 275 to 279, including 2 of the 3 sulfated tyrosines at positions 276 and 279. However, the 2 structures showed some important differences as to the precise binding interactions involved. The structure by Dumas et al38 contained several salt bridges in this region: the sulfate of tyrosine 276 interacts with thrombin residues arginine 126 and lysine 236, with lysine 235 in close proximity; sulfated tyrosine 279 forms a salt bridge with thrombin residue lysine 240, with arginine 93 nearby; and aspartate 277 of GPIb{alpha} interacts with arginine 101 of thrombin. Interestingly, in the structure by Dumas et al, sulfation of tyrosine 278 would have allowed an additional salt bridge with lysine 236 (tyrosine 278 was not sulfated in the construct used). The significance of these interactions is illustrated by the charge reversal mutation K236E on thrombin, which reduces affinity for GpIb{alpha} by 25-fold.35 By comparison, the structure by Celikel et al39 contains only 2 salt bridges: sulfated tyrosine 276 with arginine 126 of thrombin; and aspartate 277 of GpIb{alpha} with arginine 101. Additionally, lysine 236 hydrogen bonds to tyrosine 278 of GPIb{alpha}, yet does not interact with sulfated tyrosine 276. The dependence of affinity on ionic strength suggests the involvement of approximately 4 ionic interactions,35 consistent with the structure by Dumas et al more closely representing the interaction that takes place in solution. Although both structures also found contacts between the acidic tail of GpIb{alpha} and exosite I of thrombin, for the reasons described above, such an interaction is unlikely to be relevant to the cofactor activity of GpIb{alpha}.


*    Fibrin
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 up arrowAbstract
 up arrowIntroduction
 up arrowGlycoprotein Ib{alpha}
 *Fibrin
down arrowThrombomodulin
 down arrowHeparin and Other...
 down arrowSodium Ion
 down arrowConclusions
 down arrowReferences
 
One of the most important roles of thrombin in blood coagulation is the cleavage of the plasma protein fibrinogen (for review see Mosesson et al40). Fibrinogen is a 340-kDa protein composed of 2 identical subunits of 3 polypeptide chains (A{alpha}, Bß, and G{gamma}) linked head-to-head, with the N-terminus of each subunit creating the central E domain (supplemental Figure II). On cleavage of fibrinopeptide A (FpA) by thrombin, the fibrin monomers assemble into protofibrils where subsequent cleavage of fibrinopeptide B (FpB) triggers lateral association into fibrils that provide the scaffold for the growing thrombus.17,41–44 These newly generated fibrin polymers can then act as a cofactor for the thrombin activation of the transglutaminase factor XIII, which cross-links the fibrin to strengthen the clot (supplemental Figure II).9,45–47 Factor XIII deficiency has been shown to greatly decrease clot strength and leads to severe bleeding diathesis.48,49 Fibrinogen/fibrin contains 2 thrombin binding sites.50 With a Kd of 2 to 5 µmol/L (Table), the low-affinity binding site has been shown by mutagenesis, peptide, and modeling studies to encompass residues at the N-terminal flexible tails of both the A{alpha} and Bß chains of fibrinogen.51–53 Analysis of the chicken fibrinogen structure revealed a negatively charged region composed of residues 35 to 40 of the A{alpha} chain and 68 to 71 of the Bß chain.54 Additionally, 2 mutagenesis studies identified residues within exosite I, the Na+-binding site, and active site of thrombin as potentially important for interaction with fibrinogen.55,56 The crystal structure of active site-inhibited human {alpha}-thrombin in complex with the central E domain fragment of human fibrin57 (Figure 3) confirmed the biochemical studies showing thrombin interacting with fibrin via exosite I residues phenylalanine 34, serine 36a, leucine 76, arginine 77a, isoleucine 82, and lysine 110. Although the interface juxtaposes complementary charged regions on thrombin and fibrin, hydrophobic interactions provide the bulk of the binding energy. The high affinity binding site (Kd{approx}0.1 µmol/L) on fibrin involves the engagement of thrombin exosite II with the fibrin {gamma}' chain (present in approximately 10% of fibrin monomers)58,59 along with the simultaneous binding of exosite I as described above.60 The C-terminal portion of the {gamma}' chain contains negatively charged residues, including sulfated tyrosine, and is assumed to bind equivalently to other exosite II ligands.53,59,61 Whether the binding of thrombin to the {gamma}' chain plays a role in the activation of factor XIII, which has also been shown to bind to the {gamma}' chain of fibrin,62 is unclear.


*    Thrombomodulin
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowGlycoprotein Ib{alpha}
 up arrowFibrin
 *Thrombomodulin
down arrowHeparin and Other...
 down arrowSodium Ion
 down arrowConclusions
 down arrowReferences
 
Thrombomodulin (TM) is expressed on the surface of endothelial cells and binds thrombin with high affinity (Table). Binding to TM alters thrombin specificity from procoagulant substrates toward activation of the anticoagulant protease protein C (Figure 1).63,64 When bound to TM, thrombin also activates the carboxypeptidase B-like enzyme thrombin activatable fibrinolysis inhibitor (TAFI).65 TM is organized into several distinct regions: an N-terminal lectin domain, 6 contiguous epidermal growth factor (EGF)-like domains, an O-glycosylated serine-threonine rich region, a transmembrane region, and a short cytoplasmic tail. The binding site for thrombin has been localized by alanine-scanning mutagenesis and peptide-binding studies to the final 2 EGF domains, EGF56.66–68 Thrombin interacts with TM primarily through exosite I, yet exosite II can also be involved in the presence of a chondroitin sulfate (CS) moiety found within the serine/threonine-rich region on approximately 20% to 35% of TM molecules.69 Although binding of the EGF56 fragment efficiently competes for thrombin binding to full-length TM, it fails to provide any cofactor activity. Addition of the preceding EGF4 domain is necessary for cofactor function67,70,71 and the 3 orders of magnitude increase in rate of activation of protein C.11 EGF4 of TM has been shown to interact directly with protein C,72 providing a supplementary exosite to bring about effective substrate recognition. Further addition of the EGF3 domain is required to stimulate activation of TAFI.73 TM induces a change in thrombin specificity significantly by blocking the binding of procoagulant substrates which depend on an interaction with exosite I. Protein C inhibitor (PCI) has recently been shown to efficiently inhibit thrombin in the presence of TM, presumably through direct interactions between PCI and TM.74

In 2000, the crystal structure of the complex between human thrombin and the minimal cofactor fragment of TM, EGF456, revealed the features of the TM-thrombin interaction and corroborated earlier biochemical information (Figure 3).75 A reasonably small contact interface of {approx}900 Å2 is seen between exosite I of thrombin and EGF domains 5 and 6 of TM. Although the surfaces at the interface display charge complementarity, which may aid in steering the 2 fragments together,76 only a single salt bridge exists between lysine 110 of thrombin and aspartate 461 of TM. The majority of binding energy is supplied by hydrophobic contacts, exemplified by isoleucine 414 and 424 on TM; isoleucine 414 inserts into a hydrophobic cavity on thrombin surrounded by phenylalanine 34, tyrosine 76, and isoleucine 82, and isoleucine 424 interacts with leucine 65 of thrombin. Confirming the importance of these hydrophobic contacts, mutation of isoleucine 414 and 424 to alanine reduced the affinity of TM for thrombin by 25- to 30-fold.68 The TM-thrombin complex crystal structure revealed no obvious conformational changes within the active site of thrombin, but the presence of a bound active-site inhibitor clouds this issue. However, allostery may play a role in switching the substrate preference of thrombin, as several biochemical studies, including fluorescence and amide exchange studies, suggest a tightening of the active site in response to TM binding.77–79 The cofactor effect of TM may also be partially attributable to an improved accessibility of the activation peptide of TM-bound protein C.80,81


*    Heparin and Other Glycosaminoglycans
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 up arrowAbstract
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 up arrowFibrin
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 *Heparin and Other...
down arrowSodium Ion
 down arrowConclusions
 down arrowReferences
 
Final inhibition of thrombin is primarily the responsibility of members of the serpin family of serine protease inhibitors.82 One common feature of these serpins that inhibit thrombin is the ability to bind to and be activated by heparin-like glycosaminoglycans (GAGs). GAGs, such as heparan sulfate, chondroitin sulfate, and dermatan sulfate, are long-chain sulfated polysaccharides attached to proteoglycans that coat the surface of cells lining the vascular and extravascular spaces. Heparin, the widely used anticoagulant drug, is a relatively small and uniform GAG secreted by mast cells. GAGs bind to thrombin with micromolar affinity and have been shown to significantly accelerate the inhibition of thrombin by serpins through a bridging mechanism.83,84 The 2 principal circulating thrombin inhibitors are the serpins antithrombin (AT) and heparin cofactor II (HCII). The rate of thrombin inhibition by AT is enhanced by {approx}1000-fold in the presence of heparin and heparan sulfates,85,86 and inhibition by HCII is accelerated to a similar degree by either heparin or dermatan sulfate.87,88 Mutagenesis studies have identified the heparin binding site of thrombin as exosite II and includes (in order of importance) arginine 93, lysine 236, lysine 240, arginine 101, and arginine 233.89–91 Biochemical work has also shown a minimal heparin binding site size of 6 monosaccharide residues, with binding strongly dependent on ionic strength, suggesting a nonspecific electrostatic association of 5 to 6 ionic interactions.92 Whereas smaller heparins can activate AT toward factors IXa and Xa, activation of thrombin inhibition is only realized for a heparin molecule of at least 18 monosaccharide units in length.93 This is attributable to the requirement that a single heparin molecule bridge AT and thrombin simultaneously. A similar bridging mechanism is in operation for the GAG acceleration of HCII inhibition of thrombin94; however, allosteric activation of HCII also plays an important role.95,96

The recent crystal structure of human thrombin bound to an 8-monosaccharide heparin fragment revealed the molecular basis of the interaction (Figure 3).97 The structure shows how thrombin engages heparin through the interposing of positively charged residues (histidine 91, arginine 93, arginine 101, arginine 126, arginine 165, lysine 236, and lysine 240) with the negatively charged sulfate groups of heparin in a predominantly ionic interaction. A length of 6 monosaccharide units is necessary to achieve full occupancy of the heparin binding site,92 and only 6 of the 8 monosaccharide units can be seen in the crystal structure. In 2004, 2 crystal structures of the thrombin-AT-heparin ternary complex were solved, using catalytically inert thrombin.98,99 Both structures were crystallized using a heparin mimetic containing 3 fully sulfated glucose units as the thrombin binding site. The structure by Li et al98 showed binding of the heparin mimetic to thrombin consistent with that seen using the natural fragment and with the mutagenesis data described above. Specifically arginine 93 is of crucial importance making 3 ionic interactions to sulfate oxygens as well as several other hydrogen bonds to the GAG. Lysine 236 participates in 2 interactions with sulfate moieties, and arginine 101 and 233 each make 1 interaction. The partially modeled lysine 240 could potentially provide an additional 3 interactions. Incomplete density for the heparin mimetic in the structure by Dementiev et al99 makes direct comparison difficult, but no sulfate density was observed on the single monosaccharide unit modeled in the heparin binding region. Also, the structure by Dementiev et al suggests histidine 230 contributes significantly to the interaction with heparin, whereas, in the thrombin-heparin structure by Carter et al97 described above, only a single water-mediated interaction between histidine 230 and heparin is observed. The chondroitin sulfate moiety (CS) found on the serine/threonine-rich domain of TM is likely to bind thrombin in a similar manner to that observed in the crystal structure of thrombin bound to heparin.100


*    Sodium Ion
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 up arrowAbstract
 up arrowIntroduction
 up arrowGlycoprotein Ib{alpha}
 up arrowFibrin
 up arrowThrombomodulin
 up arrowHeparin and Other...
 *Sodium Ion
down arrowConclusions
 down arrowReferences
 
Another potential cofactor for the modulation of thrombin activity is the monovalent cation sodium (Na+). The binding site for Na+ was first identified in 1995 by Di Cera et al by substituting the more electron-rich rubidium into crystals grown in the presence of Na+, and it was concluded that most of the preceding crystal structures of thrombin had placed a water molecule into the position now known to be occupied by Na+.101 Na+ was found coordinated with octahedral geometry by the backbone carbonyl oxygens of arginine 221a and lysine 224 and 4 conserved water molecules (supplemental Figure III). When bound to Na+, thrombin exists in a prothrombotic state capable of efficient cleavage of procoagulant substrates like fibrinogen and PAR-1.21,22,102,103 In the absence of Na+, thrombin displays a generally reduced catalytic efficiency for these and other substrates yet maintains the ability to effectively activate protein C in the presence of TM.103 It has been assumed that the 2 states, Na+-bound and Na+-free, represent 2 distinct thrombin conformations, termed the "fast" and "slow" forms, respectively. Several biochemical and crystallographic studies have attempted to clarify the precise nature of the conformational change responsible for the activation of thrombin on Na+ binding.104–113 As many structures of Na+-bound thrombin have already been deposited in the protein data bank, crystallization efforts have recently focused on the Na+-free slow form. In 2002, a report by Pineda et al described a structure of thrombin crystallized in the absence of Na+ that displayed minor changes in the side chain positions of residues in the active site.106 Disappointingly, the conformation of the Na+-binding site and of other regions previously shown by mutagenesis studies to undergo conformational rearrangement on Na+ binding remained unchanged relative to the fast form (eg, tryptophan 215, which is known to undergo a significant change in environment105). Subsequent crystallization of wild-type and variants of thrombin in the presence of different monovalent cations such as choline, potassium, and lithium have resulted in an collection of thrombin structures displaying an array of conformational alterations with respect to the Na+-bound form.107–110,112,114,115 Just a few of these conformational changes include disruption of the Na+-binding loop, constriction and blockage of the active site cleft, reorientation of the catalytic serine 195, and loss of the oxyanion hole caused by flipping of glycine 193. Whether some or all of these changes are critical for the transition between the slow and fast forms is still unclear; however, release of Na+ appears to lead to an increase in conformational flexibility within thrombin. This suggests that in the absence of Na+ coordination, thrombin exists as an ensemble of conformational states whose collective activities have traditionally been taken to be that of a single slow form.115 Whatever the Na+-induced conformational transition may be, the physiological relevance of Na+ as an allosteric effector molecule depends on differential binding in the plasma. Accordingly, it has been reported that the affinity of thrombin for Na+ is highly temperature dependent, with a Kd of {approx}25 mmol/L at room temperature and near the physiological Na+ concentration of {approx}140 mmol/L at 37°C.116 However, with the plasma concentration of Na+ highly regulated, it is difficult to imagine how its interaction with thrombin could serve a true cofactor function in the regulation of hemostasis.


*    Conclusions
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowGlycoprotein Ib{alpha}
 up arrowFibrin
 up arrowThrombomodulin
 up arrowHeparin and Other...
 up arrowSodium Ion
 *Conclusions
down arrowReferences
 
This review focuses on how thrombin interacts with a number of different cofactors throughout a "lifecycle," which begins with its generation in response to vascular injury and concludes with its irreversible inhibition by serpins after the formation of a stable clot. The cofactors described in this report play a critical role in steering thrombin specificity throughout its lifecycle. The lifecycle of thrombin was nicely outlined in the recent review by Lane et al.15 In the early stages of the hemostatic response, thrombin activity is heavily weighted toward procoagulation. Thrombin, via exosite II interactions, preferentially binds GpIb{alpha} on the surface of platelets, cleaving PARs to activate platelets and release partially activated factor V from {alpha} granules. This causes several rounds of amplification of thrombin generation followed by positive feedback stimulation through activation of additional factor V and factor VIII. As more thrombin is produced, it begins to associate with the amassing fibrinogen within the platelet plug. Because of its weak affinity for fibrinogen (Kd{approx}7 µmol/L) thrombin relies on the high concentration of fibrinogen in plasma (15 µmol/L) and the increased local concentration of fibrinogen near the site of injury. Thrombin remains bound to fibrin through exosite interactions after cleavage of fibrinogen. Fibrin then acts as a cofactor for thrombin activation of factor XIII, as well as providing the substrate for factor XIIIa cross-linking. Once the growing thrombus spreads beyond the site of injury, the relative abundance of cofactors shifts from those of procoagulant (GpIb{alpha} and fibrin) to those of anticoagulant (TM and GAGs) activities. The tight-binding (Kd{approx}3 nmol/L) TM expressed on the surface of the endothelium effectively competes with the weak-binding fibrinogen for exosite I of thrombin. Heparan sulfate, which coats the endothelium, can also effectively compete with GpIb{alpha} for exosite II and assist in clearance of active thrombin through inhibition by AT and HCII. In this way, cofactors have evolved to use both exosites of thrombin and direct the enzyme toward procoagulant activity, anticoagulant activity, or inhibition. The thrombin-cofactor interactions, now fully revealed by crystallographic structures, are of critical importance for directing the activity of thrombin throughout its brief, but eventful, existence and thus help to maintain the hemostatic balance.


*    Acknowledgments
 
Sources of Funding

Funding was provided by the British Heart Foundation, the Sir Isaac Newton Trust, and the Medical Research Council (UK).

Disclosure(s)

None.


*    Footnotes
 
Original received February 14, 2006; final version accepted May 10, 2006.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowGlycoprotein Ib{alpha}
 up arrowFibrin
 up arrowThrombomodulin
 up arrowHeparin and Other...
 up arrowSodium Ion
 up arrowConclusions
 *References
 

  1. Davie EW, Ratnoff OD. Waterfall sequence for intrinsic blood clotting. Science. 1964; 145: 1310–1312.[Abstract/Free Full Text]
  2. Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry. 1991; 30: 10363–10370.[CrossRef][Medline] [Order article via Infotrieve]
  3. Mann KG, Butenas S, Brummel K. The dynamics of thrombin formation. Arterioscler Thromb Vasc Biol. 2003; 23: 17–25.[Abstract/Free Full Text]
  4. Repke D, Gemmell CH, Guha A, Turitto VT, Broze GJ Jr, Nemerson Y. Hemophilia as a defect of the tissue factor pathway of blood coagulation: effect of factors VIII and IX on factor X activation in a continuous-flow reactor. Proc Natl Acad Sci U S A. 1990; 87: 7623–7627.[Abstract/Free Full Text]
  5. Kahn ML, Nakanishi-Matsui M, Shapiro MJ, Ishihara H, Coughlin SR. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest. 1999; 103: 879–887.[Medline] [Order article via Infotrieve]
  6. Monkovic DD, Tracy PB. Activation of human factor V by factor Xa and thrombin. Biochemistry. 1990; 29: 1118–1128.[CrossRef][Medline] [Order article via Infotrieve]
  7. Fay PJ. Activation of factor VIII and mechanisms of cofactor action. Blood Rev. 2004; 18: 1–15.[CrossRef][Medline] [Order article via Infotrieve]
  8. 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: 48112–48119.[Abstract/Free Full Text]
  9. Kanaide H, Shainoff JR. Cross-linking of fibrinogen and fibrin by fibrin-stablizing factor (factor XIIIa). J Lab Clin Med. 1975; 85: 574–597.[Medline] [Order article via Infotrieve]
  10. 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]
  11. Esmon CT. Molecular events that control the protein C anticoagulant pathway. Thromb Haemost. 1993; 70: 29–35.[Medline] [Order article via Infotrieve]
  12. Mann KG, Elion J, Butkowski RJ, Downing M, Nesheim ME. Prothrombin. Methods Enzymol. 1981; 80 (pt C): 286–302.[Medline] [Order article via Infotrieve]
  13. Krishnaswamy S, Church WR, Nesheim ME, Mann KG. Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism. J Biol Chem. 1987; 262: 3291–3299.[Abstract/Free Full Text]
  14. Boskovic DS, Krishnaswamy S. Exosite binding tethers the macromolecular substrate to the prothrombinase complex and directs cleavage at two spatially distinct sites. J Biol Chem. 2000; 275: 38561–38570.[Abstract/Free Full Text]
  15. Lane DA, Philippou H, Huntington JA. Directing thrombin. Blood. 2005; 106: 2605–2612.[Abstract/Free Full Text]
  16. 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: 3467–3475.[Medline] [Order article via Infotrieve]
  17. 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: 9276–9282.[Abstract/Free Full Text]
  18. 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]
  19. Di Cera E. Thrombin interactions. Chest. 2003; 124: 11S–17S.[CrossRef][Medline] [Order article via Infotrieve]
  20. 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: 13882–13887.[Abstract/Free Full Text]
  21. 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]
  22. 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]
  23. Huntington JA. Molecular recognition mechanisms of thrombin. J Thromb Haemost. 2005; 3: 1861–1872.[CrossRef][Medline] [Order article via Infotrieve]
  24. Berndt MC, Phillips DR. Purification and preliminary physicochemical characterization of human platelet membrane glycoprotein V. J Biol Chem. 1981; 256: 59–65.[Abstract/Free Full Text]
  25. Ward CM, Andrews RK, Smith AI, Berndt MC. Mocarhagin, a novel cobra venom metalloproteinase, cleaves the platelet von Willebrand factor receptor glycoprotein Ibalpha. Identification of the sulfated tyrosine/anionic sequence Tyr-276-Glu-282 of glycoprotein Ibalpha as a binding site for von Willebrand factor and alpha-thrombin. Biochemistry. 1996; 35: 4929–4938.[CrossRef][Medline] [Order article via Infotrieve]
  26. 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]
  27. Coughlin SR. Thrombin signalling and protease-activated receptors. Nature. 2000; 407: 258–264.[CrossRef][Medline] [Order article via Infotrieve]
  28. 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]
  29. McGowan EB, Ding A, Detwiler TC. Correlation of thrombin-induced glycoprotein V hydrolysis and platelet activation. J Biol Chem. 1983; 258: 11243–11248.[Abstract/Free Full Text]
  30. 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]
  31. 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: 9571–9578.[Abstract/Free Full Text]
  32. Gralnick HR, Williams S, McKeown LP, Hansmann K, Fenton JW, Krutzsch H. High-affinity alpha-thrombin binding to platelet glycoprotein Ib alpha: identification of two binding domains. Proc Natl Acad Sci U S A. 1994; 91: 6334–6338.[Abstract/Free Full Text]
  33. Dong JF, Li CQ, Lopez JA. Tyrosine sulfation of the glycoprotein Ib-IX complex: identification of sulfated residues and effect on ligand binding. Biochemistry. 1994; 33: 13946–13953.[CrossRef][Medline] [Order article via Infotrieve]
  34. 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]
  35. 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]
  36. Vu TK, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991; 64: 1057–1068.[CrossRef][Medline] [Order article via Infotrieve]
  37. Vu TK, Wheaton VI, Hung DT, Charo I, Coughlin SR. Domains specifying thrombin-receptor interaction. Nature. 1991; 353: 674–677.[CrossRef][Medline] [Order article via Infotrieve]
  38. 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]
  39. 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: 218–221.[Abstract/Free Full Text]
  40. Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features of fibrinogen and fibrin. Ann N Y Acad Sci. 2001; 936: 11–30.[Abstract/Free Full Text]
  41. Ruf W, Bender A, Lane DA, Preissner KT, Selmayr E, Muller-Berghaus G. Thrombin-induced fibrinopeptide B release from normal and variant fibrinogens: influence of inhibitors of fibrin polymerization. Biochim Biophys Acta. 1988; 965: 169–175.[Medline] [Order article via Infotrieve]
  42. Weisel JW, Veklich Y, Gorkun O. The sequence of cleavage of fibrinopeptides from fibrinogen is important for protofibril formation and enhancement of lateral aggregation in fibrin clots. J Mol Biol. 1993; 232: 285–297.[CrossRef][Medline] [Order article via Infotrieve]
  43. Li X, Galanakis D, Gabriel DA. Transient intermediates in the thrombin activation of fibrinogen. Evidence for only the desAA species. J Biol Chem. 1996; 271: 11767–11771.[Abstract/Free Full Text]
  44. Mosesson MW. Fibrinogen and fibrin polymerization: appraisal of the binding events that accompany fibrin generation and fibrin clot assembly. Blood Coagul Fibrinolysis. 1997; 8: 257–267.[Medline] [Order article via Infotrieve]
  45. Lorand L, Konishi K. Activation of the fibrin stabilizing factor of plasma by thrombin. Arch Biochem Biophys. 1964; 105: 58–67.[CrossRef][Medline] [Order article via Infotrieve]
  46. Becker DL, Fredenburgh JC, Stafford AR, Weitz JI. Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II. J Biol Chem. 1999; 274: 6226–6233.[Abstract/Free Full Text]
  47. 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]
  48. Board PG, Losowsky MS, Miloszewski KJ. Factor XIII: inherited and acquired deficiency. Blood Rev. 1993; 7: 229–242.[CrossRef][Medline] [Order article via Infotrieve]
  49. Nielsen VG, Gurley WQ Jr, Burch TM. The impact of factor XIII on coagulation kinetics and clot strength determined by thrombelastography. Anesth Analg. 2004; 99: 120–123.[Abstract/Free Full Text]
  50. Meh DA, Siebenlist KR, Mosesson MW. Identification and characterization of the thrombin binding sites on fibrin. J Biol Chem. 1996; 271: 23121–23125.[Abstract/Free Full Text]
  51. Binnie CG, Lord ST. The fibrinogen sequences that interact with thrombin. Blood. 1993; 81: 3186–3192.[Free Full Text]
  52. Hogg PJ, Bock PE. Modulation of thrombin and heparin activities by fibrin. Thromb Haemost. 1997; 77: 424–433.[Medline] [Order article via Infotrieve]
  53. Rose T, Di Cera E. Three-dimensional modeling of thrombin-fibrinogen interaction. J Biol Chem. 2002; 277: 18875–18880.[Abstract/Free Full Text]
  54. Yang Z, Kollman JM, Pandi L, Doolittle RF. Crystal structure of native chicken fibrinogen at 2.7 A resolution. Biochemistry. 2001; 40: 12515–12523.[CrossRef][Medline] [Order article via Infotrieve]
  55. 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]
  56. 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]
  57. 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]
  58. Mosesson MW. Fibrinogen gamma chain functions. J Thromb Haemost. 2003; 1: 231–238.[CrossRef][Medline] [Order article via Infotrieve]
  59. Lovely RS, Moaddel M, Farrell DH. Fibrinogen gamma’ chain binds thrombin exosite II. J Thromb Haemost. 2003; 1: 124–131.[CrossRef][Medline] [Order article via Infotrieve]
  60. Pospisil CH, Stafford AR, Fredenburgh JC, Weitz JI. Evidence that both exosites on thrombin participate in its high affinity interaction with fibrin. J Biol Chem. 2003; 278: 21584–21591.[Abstract/Free Full Text]
  61. Meh DA, Siebenlist KR, Brennan SO, Holyst T, Mosesson MW. The amino acid sequence in fibrin responsible for high affinity thrombin binding. Thromb Haemost. 2001; 85: 470–474.[Medline] [Order article via Infotrieve]
  62. Siebenlist KR, Meh DA, Mosesson MW. Plasma factor XIII binds specifically to fibrinogen molecules containing gamma chains. Biochemistry. 1996; 35: 10448–10453.[CrossRef][Medline] [Order article via Infotrieve]
  63. Esmon CT, Owen WG. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci U S A. 1981; 78: 2249–2252.[Abstract/Free Full Text]
  64. 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: 243–251.[Medline] [Order article via Infotrieve]
  65. Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. J Biol Chem. 1996; 271: 16603–16608.[Abstract/Free Full Text]
  66. 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: 5993–5996.[Abstract/Free Full Text]
  67. 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]
  68. 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: 2888–2892.[Abstract/Free Full Text]
  69. 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: 6164–6170.[Abstract/Free Full Text]
  70. Zushi M, Gomi K, Honda G, Kondo S, Yamamoto S, Hayashi T, Suzuki K. Aspartic acid 349 in the fourth epidermal growth factor-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C. J Biol Chem. 1991; 266: 19886–19889.[Abstract/Free Full Text]
  71. White CE, Hunter MJ, Meininger DP, White LR, Komives EA. Large-scale expression, purification and characterization of small fragments of thrombomodulin: the roles of the sixth domain and of methionine 388. Protein Eng. 1995; 8: 1177–1187.[Abstract/Free Full Text]
  72. 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]
  73. 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: 12135–12139.[Abstract/Free Full Text]
  74. 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]
  75. 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: 518–525.[CrossRef][Medline] [Order article via Infotrieve]
  76. 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: 4972–4979.[CrossRef][Medline] [Order article via Infotrieve]
  77. Ye J, Esmon NL, Esmon CT, Johnson AE. The active site of thrombin is altered upon binding to thrombomodulin. Two distinct structural changes are detected by fluorescence, but only one correlates with protein C activation. J Biol Chem. 1991; 266: 23016–23021.[Abstract/Free Full Text]
  78. Ye J, Esmon CT, Johnson AE. The chondroitin sulfate moiety of thrombomodulin binds a second molecule of thrombin. J Biol Chem. 1993; 268: 2373–2379.[Abstract/Free Full Text]
  79. Koeppe JR, Seitova A, Mather T, Komives EA. Thrombomodulin tightens the thrombin active site loops to promote protein C activation. Biochemistry. 2005; 44: 14784–14791.[CrossRef][Medline] [Order article via Infotrieve]
  80. Vindigni A, White CE, Komives EA, Di Cera E. Energetics of thrombin-thrombomodulin interaction. Biochemistry. 1997; 36: 6674–6681.[CrossRef][Medline] [Order article via Infotrieve]
  81. Yang L, Manithody C, Rezaie AR. Activation of protein C by the thrombin-thrombomodulin complex: cooperative roles of Arg-35 of thrombin and Arg-67 of protein C. Proc Natl Acad Sci U S A. 2006; 103: 879–884.[Abstract/Free Full Text]
  82. Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, Gettins PG, Irving JA, Lomas DA, Luke CJ, Moyer RW, Pemberton PA, Remold-O’Donnell E, Salvesen GS, Travis J, Whisstock JC. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem. 2001; 276: 33293–33296.[Free Full Text]
  83. Olson ST, Bjork I. Regulation of thrombin activity by antithrombin and heparin. Semin Thromb Hemost. 1994; 20: 373–409.[Medline] [Order article via Infotrieve]
  84. Huntington JA. Heparin activation of serpins. In: Garg HG, Linhardt RJ, Hales CA, eds. Chemistry and Biology of Heparin and Heparan Sulfate. Oxford: Elsevier Ltd; 2005: 367–398.
  85. Olson ST, Bjork I, Sheffer R, Craig PA, Shore JD, Choay J. Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement. J Biol Chem. 1992; 267: 12528–12538.[Abstract/Free Full Text]
  86. Olson ST, Bjork I, Shore JD. Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin. Methods Enzymol. 1993; 222: 525–559.[Medline] [Order article via Infotrieve]
  87. Pratt CW, Whinna HC, Meade JB, Treanor RE, Church FC. Physicochemical aspects of heparin cofactor II. Ann N Y Acad Sci. 1989; 556: 104–115.[Medline] [Order article via Infotrieve]
  88. Tollefsen DM. Heparin cofactor II. Adv Exp Med Biol. 1997; 425: 35–44.[Medline] [Order article via Infotrieve]
  89. Sheehan JP, Sadler JE. Molecular mapping of the heparin-binding exosite of thrombin. Proc Natl Acad Sci U S A. 1994; 91: 5518–5522.[Abstract/Free Full Text]
  90. 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: 1301–1305.[Abstract/Free Full Text]
  91. 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: 12024–12029.[Abstract/Free Full Text]
  92. 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: 6342–6352.[Abstract/Free Full Text]
  93. Olson ST, Shore JD. Demonstration of a two-step reaction mechanism for inhibition of alpha-thrombin by antithrombin III and identification of