Evaluation of the efficacy and safety of rivaroxaban using a computer model for blood coagulation

Rivaroxaban is an oral, direct Factor Xa inhibitor approved in the European Union and several other countries for the prevention of venous thromboembolism in adult patients undergoing elective hip or knee replacement surgery and is in advanced clinical development for the treatment of thromboembolic disorders. Its mechanism of action is antithrombin independent and differs from that of other anticoagulants, such as warfarin (a vitamin K antagonist), enoxaparin (an indirect thrombin/Factor Xa inhibitor) and dabigatran (a direct thrombin inhibitor). A blood coagulation computer model has been developed, based on several published models and preclinical and clinical data. Unlike previous models, the current model takes into account both the intrinsic and extrinsic pathways of the coagulation cascade, and possesses some unique features, including a blood flow component and a portfolio of drug action mechanisms. This study aimed to use the model to compare the mechanism of action of rivaroxaban with that of warfarin, and to evaluate the efficacy and safety of different rivaroxaban doses with other anticoagulants included in the model. Rather than reproducing known standard clinical measurements, such as the prothrombin time and activated partial thromboplastin time clotting tests, the anticoagulant benchmarking was based on a simulation of physiologically plausible clotting scenarios. Compared with warfarin, rivaroxaban showed a favourable sensitivity for tissue factor concentration inducing clotting, and a steep concentration-effect relationship, rapidly flattening towards higher inhibitor concentrations, both suggesting a broad therapeutic window. The predicted dosing window is highly accordant with the final dose recommendation based upon extensive clinical studies.     

Coagulation proteases and human cancer

Tumours are capable of activating blood coagulation through the expression of procoagulant molecules such as tissue factor, cancer procoagulant and hepsin. Initiation of the clotting cascade results in the generation of the activated serine proteases factor VIIa, factor Xa and thrombin. These proteases act via protease-activated receptors and tissue factor to alter gene expression, thereby modulating tumour cell growth, invasion, metastasis and angiogenesis.     

Intratracheal gene transfer of tissue factor pathway inhibitor attenuates pulmonary fibrosis

Activation of the coagulation system and increased expression of tissue factor (TF) in pulmonary fibrosis associated with acute and chronic lung injury have been previously documented. In the present study, we evaluated the effect of TF inhibition with intratracheal gene transfer of tissue factor pathway inhibitor (TFPI), a potent and highly specific endogenous inhibitor of TF-dependent coagulation activation, in a rat model of bleomycin-induced lung fibrosis. Significant lung fibrotic changes as assessed by histologic findings and hydroxyproline content, and increased procoagulant activity and thrombin generation in bronchoalveolar lavage fluid were detected in rats after intratracheal injection of bleomycin. Intratracheal administration of an adenovirus vector expressing TFPI significantly decreased bleomycin-induced procoagulant and thrombin generation resulting in a strong inhibition of pulmonary fibrosis. TFPI-overexpression in the lung was associated with a significant reduction in gene expression of the connective tissue growth factor, a potent profibrotic growth factor. This is the first report showing that direct inhibition of TF-mediated coagulation activation abrogates bleomycin-induced pulmonary fibrosis.     

Coagulation cascade proteases and tissue fibrosis

Fibrotic disorders of the liver, kidney and lung are associated with excessive deposition of extracellular matrix proteins and ongoing coagulation-cascade activity. In addition to their critical roles in blood coagulation, thrombin and the immediate upstream coagulation proteases, Factors Xa and VIIa, influence numerous cellular responses that may play critical roles in subsequent inflammatory and tissue repair processes in vascular and extravascular compartments. The cellular effects of these proteases are mediated via proteolytic activation of a novel family of cell-surface receptors, the protease-activated receptors (PAR-1, -2, -3 and -4). Although thrombin is capable of activating PAR-1, -3 and -4, there is accumulating in vitro evidence that the profibrotic effects of thrombin are predominantly mediated via PAR-1. Factor Xa is capable of activating PAR-1 and PAR-2, but its mitogenic effects for fibroblasts are similarly mediated via PAR-1. These proteases do not exert their profibrotic effects directly, but act via the induction of potent fibrogenic mediators, such as platelet-derived growth factor and connective tissue growth factor. In vivo studies using proteolytic inhibitors, PAR-1 antagonists and PAR-1-deficient mice have provided evidence that coagulation proteases play a key role in tissue inflammation and in a number of vascular pathologies associated with hyperproliferation of smooth muscle cells. More recently, coagulation proteases have also been shown to play a role in the pathogenesis of fibrosis but the relative contribution of their cellular versus their procoagulant effects awaits urgent evaluation in vivo. These studies will be informative in determining the potential application of PAR-1 antagonists as antifibrotic agents.     

Factor Xa and thrombin evoke additive calcium and proinflammatory responses in endothelial cells subjected to coagulation

Endothelial cells react to factor Xa and thrombin by proinflammatory responses. It is unclear how these cells respond under physiological conditions, where the serine proteases factor VIIa, factor Xa and thrombin are all simultaneously generated, as in tissue factor-driven blood coagulation. We studied the Ca(2+) signaling and downstream release of interleukins (ILs), induced by these proteases in monolayers of human umbilical vein endothelial cells. In single cells, factor Xa, but not factor VIIa, complexed with tissue factor, evoked a greatly delayed, oscillatory Ca(2+) response, which relied on its catalytic activity and resembled that of SLIGRL, a peptide specifically activating the protease-activated receptor 2 (PAR2). Thrombin even at low concentrations evoked a rapid, mostly non-oscillating Ca(2+) response through activation of PAR1, which reinforced the factor Xa response. The additive Ca(2+) signals persisted, when factor X and prothrombin were activated in situ, or in the presence of plasma that was triggered to coagulate with tissue factor. Further, thrombin reinforced the factor Xa-induced production of IL-8, but not of IL-6. Both interleukins were produced in the presence of coagulating plasma. In conclusion, under coagulant conditions, factor Xa and thrombin appear to contribute in different and additive ways to the Ca(2+)-mobilizing and proinflammatory reactions of endothelial cells. These data provide first evidence that these serine proteases trigger distinct signaling modules in endothelium that is activated by plasma coagulation.     

The role of the COOH-terminal region of antithrombin III. Evidence that the COOH-terminal region of the inhibitor enhances the reactivity of thrombin and factor Xa with the inhibitor.

To elucidate the role of the COOH-terminal region of antithrombin III, we studied the effects of synthetic peptides corresponding to its sequence on the amidolytic and proteolytic activities of thrombin and Factor Xa in the presence or absence of the inhibitor, antithrombin III. The peptides ANRPFLVFI and IIFMGRVANP corresponding to residues Ala404 to Ile412 and Ile420 to Pro429, respectively, blocked the inhibition by antithrombin III. The effect of IIFMGRVANP was reduced in the presence of heparin. Both peptides at a concentration of 1 mM blocked complex formation between antithrombin III and thrombin or Factor Xa. The two peptides, particularly IIFMGRVANP, directly enhanced the amidolytic activity of thrombin and Factor Xa on the synthetic substrate Boc-Ala-Gly-Arg-MCA (where Boc is t-butoxycarbonyl and MCA is 4-methylcoumarin), which corresponds to residues P3-P1 of the reactive site of antithrombin III, and also on other substrates due to increased Vmax. IIFMGRVANP also shortened the thrombin-induced fibrinogen clotting time, whereas ANRPFLVFI inhibited the thrombin-catalyzed activation of protein C both in the presence and absence of thrombomodulin. The direct effect of ANRPFLVFI and IIFMGRVANP on thrombin was confirmed by enhancement of the incorporation of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide into thrombin. These findings suggest that the COOH-terminal region of antithrombin III interacts with thrombin and Factor Xa to increase the reactivity of the enzyme, which may enhance acyl-bond formation between the inhibitor and the enzyme.     

RANKL induces components of the extrinsic coagulation pathway in osteoclasts

Prothrombin is converted to thrombin by factor Xa in the cell-associated prothrombinase complex. Prothrombin is present in calcified bone matrix and thrombin exerts effects on osteoblasts as well as on bone resorption by osteoclasts.We investigated whether (1) osteoclasts display factor Xa-dependent prothrombinase activity and (2) osteoclasts express critical regulatory components upstream of the prothrombinase complex.The osteoclast differentiation factor RANKL induced formation of multinucleated TRAP positive cells concomitant with induction of prothrombinase activity in cultures of RAW 264.7 cells and bone marrow osteoclast progenitors.Expression analysis of extrinsic coagulation factors revealed that RANKL enhanced protein levels of factor Xa as well as of coagulation factor III (tissue factor). Inhibition assays indicated that factor Xa and tissue factor were involved in the control of prothrombinase activity in RANKL-differentiated osteoclasts, presumably at two stages (1) conversion of prothrombin to thrombin and (2) conversion of factor X to factor Xa, respectively.Activation of the extrinsic coagulation pathway during osteoclast differentiation through induction of tissue factor and factor Xa by a RANKL-dependent pathway indicates a novel role for osteoclasts in converting prothrombin to thrombin.     

Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation

Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR1). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR1-deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR1-specific agonists and inhibitors were used to demonstrate that PAR1 mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR1 and not PAR2. These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis.     

A model for the stoichiometric regulation of blood coagulation

We have developed a model of the extrinsic blood coagulation system that includes the stoichiometric anticoagulants. The model accounts for the formation, expression, and propagation of the vitamin K-dependent procoagulant complexes and extends our previous model by including: (a) the tissue factor pathway inhibitor (TFPI)-mediated inactivation of tissue factor (TF).VIIa and its product complexes; (b) the antithrombin-III (AT-III)-mediated inactivation of IIa, mIIa, factor VIIa, factor IXa, and factor Xa; (c) the initial activation of factor V and factor VIII by thrombin generated by factor Xa-membrane; (d) factor VIIIa dissociation/activity loss; (e) the binding competition and kinetic activation steps that exist between TF and factors VII and VIIa; and (f) the activation of factor VII by IIa, factor Xa, and factor IXa. These additions to our earlier model generate a model consisting of 34 differential equations with 42 rate constants that together describe the 27 independent equilibrium expressions, which describe the fates of 34 species. Simulations are initiated by "exposing" picomolar concentrations of TF to an electronic milieu consisting of factors II, IX, X, VII, VIIa, V, and VIIII, and the anticoagulants TFPI and AT-III at concentrations found in normal plasma or associated with coagulation pathology. The reaction followed in terms of thrombin generation, proceeds through phases that can be operationally defined as initiation, propagation, and termination. The generation of thrombin displays a nonlinear dependence upon TF, AT-III, and TFPI and the combination of these latter inhibitors displays kinetic thresholds. At subthreshold TF, thrombin production/expression is suppressed by the combination of TFPI and AT-III; for concentrations above the TF threshold, the bolus of thrombin produced is quantitatively equivalent. A comparison of the model with empirical laboratory data illustrates that most experimentally observable parameters are captured, and the pathology that results in enhanced or deficient thrombin generation is accurately described.     

Fibroblasts, glial, and neuronal cells are involved in extravascular prothrombin activation.

A membrane-associated prothrombin activator (MAPA) was found on various cultured cells derived from non-hematopoietic cells [Sekiya, F. et al. (1994) J. Biol. Chem. 269, 32441-32445]. In this study, we investigated the enzymatic properties of this enzyme using protease inhibitors. While the metalloproteinase inhibitor, o-phenanthroline, had no effect, some Kunitz type serine protease inhibitors attenuated MAPA activity. Recombinant tissue factor pathway inhibitor (rTFPI) also markedly reduced the activity (IC(50), 1. 3+/-0.6 x 10(-10) M). MAPA activity is, therefore, most likely to be due to factor Xa. We evaluated the effect of exogenous factor Xa on MAPA activity. Factor Xa-dependent prothrombin activation was observed on fibroblast cells (apparent K(d), 1.47+/-0.72 nM). Activation was also observed on glial and neuronal cells, which expressed MAPA activity. These results imply that membrane-bound factor Xa results in MAPA activity on these cells. Therefore, we considered the involvement of factor Va, a component of prothrombinase, in this activity. We examined whether or not the prothrombinase complex is assembled on these cells. Prothrombin was activated in a manner dependent on both exogenous factor Xa and factor Va (apparent K(d) of 0.51-1.81 nM for factor Va). These results indicate that the prothrombinase complex forms specifically on various extravascular cells. Although the prothrombinase complex can be assembled on monocytes and lymphocytes, it is not known why these cells can activate prothrombin specifically. These cells which have the capacity for prothrombin activator activity could also activate factor X; i.e. cells with factor X activation activity were able to convert prothrombin. These observations suggest that thrombin was generated via two procoagulant activities; factor X activation and subsequent prothrombinase complex formation on the surface of these cells. This mechanism may explain the various pathological states involving or resulting from extravascular thrombin and fibrin formation.     

Novel approach for preparation of heparins specific to factor Xa using affinity chromatography coupled with synthetic antithrombin III-related peptides

In the blood coagulation cascade, heparin activates human plasma antithrombin III (hAT III), resulting in the inhibition of factor Xa. This polysaccharide also exhibits hemorrhagic tendency mediated by the inhibition of thrombin in heparinotherapy. Therefore, attention has focused on the development of low molecular weight heparins (LMW-heparins) that inhibit factor Xa but not thrombin. In this investigation, we examined the biochemical and physicochemical properties of hAT III-derived heparin-binding peptides (HBPs). Of all the tested HBPs, hAT III (123-139) exhibited the highest affinity with heparin and showed an inhibitory effect on the heparin-induced enhancement of hAT III activity toward factor Xa, indicating that hAT III (123-139) specifically interacts with the active region in heparin. We prepared a synthetic hAT III (123-139)-coupled affinity chromatography system, and demonstrated that this novel affinity chromatography is useful for fractionation of highly active moieties in LMW-heparins.     

Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6-P3' proteinase binding sequence excluding P1-P1' to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2' residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6-P3' residues flanking the primary P1-P1' recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6-P3' region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.     

Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement.

The synthetic antithrombin-binding heparin pentasaccharide and a full-length heparin of approximately 26 saccharides containing this specific sequence have been compared with respect to their interactions with antithrombin and their ability to promote inhibition and substrate reactions of antithrombin with thrombin and factor Xa. The aim of these studies was to elucidate the pentasaccharide contribution to heparin's accelerating effect on antithrombin-proteinase reactions. Pentasaccharide and full-length heparins bound antithrombin with comparable high affinities (KD values of 36 +/- 11 and 10 +/- 3 nM, respectively, at I 0.15) and induced highly similar protein fluorescence, ultraviolet and circular dichroism changes in the inhibitor. Stopped-flow fluorescence kinetic studies of the heparin binding interactions at I 0.15 were consistent with a two-step binding process for both heparins, involving an initial weak encounter complex interaction formed with similar affinities (KD 20-30 microM), followed by an inhibitor conformational change with indistinguishable forward rate constants of 520-700 s-1 but dissimilar reverse rate constants of approximately 1 s-1 for the pentasaccharide and approximately 0.2 s-1 for the full-length heparin. Second order rate constants for antithrombin reactions with thrombin and factor Xa were maximally enhanced by the pentasaccharide only 1.7-fold for thrombin, but a substantial 270-fold for factor Xa, in an ionic strength-independent manner at saturating oligosaccharide. In contrast, the full-length heparin produced large ionic strength-dependent enhancements in second order rate constants for both antithrombin reactions of 4,300-fold for thrombin and 580-fold for factor Xa at I 0.15. These enhancements were resolvable into a nonionic component ascribable to the pentasaccharide and an ionic component responsible for the additional rate increase of the larger heparin. Stoichiometric titrations of thrombin and factor Xa inactivation by antithrombin, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of these reactions, indicated that pentasaccharide and full-length heparins similarly promoted the formation of proteolytically modified inhibitor during the inactivation of factor Xa by antithrombin, whereas only the full-length heparin was effective in promoting this substrate reaction of antithrombin during the reaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of cysteine proteinase inhibitors on platelet and plasma components of blood coagulation system

Factor XIIIa plays an important role in stabilization of formed fibrin clot during blood coagulation. Recent studies proved that factor XIIIa affects formation of coated platelets, which are highly procoagulant and characterized by a high level of alpha-granular proteins on their surface and expose surface phosphatidylserine after platelet activation. The ability of newly found cysteine proteinase inhibitors (CPIs) from plants to affect thiol group of the factor XIIIa active centre was recently discovered. Here, the effect of CPIs on the formation of coated platelets and activity of plasma components during blood coagulation process was investigated. It was found that CPIs dose-dependently decreased the fraction of coated platelets in the total platelet population during platelet activation and decreased endogenous thrombin potential (ETP) by 40% for thrombin generation in platelet-rich as well as in platelet-poor plasma. Such decrease of ETP could not be explained by the CPIs influence on factor XIIIa. Investigation of the effects of these inhibitors on factor Xa and thrombin activity has shown that CPIs dose-dependently inhibited their activity and might cause an ETP decrease. Thus, the obtained data indicated that CPIs affected both platelet and plasma components of blood coagulation system.     

Deletion of P1 arginine in a novel antithrombin variant (antithrombin London) abolishes inhibitory activity but enhances heparin affinity and is associated with early onset thrombosis

A novel variant of antithrombin, the major serpin inhibitor of coagulation proteases, has been identified in a patient with early onset thrombosis and abnormal plasma antithrombin activity. Sequencing of the antithrombin genes of the patient revealed that one of the two alleles was abnormal due to an in-frame deletion of the codon for the P1 arginine residue. The abnormal antithrombin was separated from the normal inhibitor by complexing the latter with thrombin followed by heparin-agarose affinity chromatography. The purified variant, antithrombin London, was completely inactive as a thrombin or factor Xa inhibitor even after heparin activation. Surprisingly, the variant bound heparin with a K(D) reflecting an approximately 10-fold greater affinity than the normal inhibitor. Stopped-flow kinetic analysis showed that this was almost entirely due to a more favorable conformational activation of the variant than the normal inhibitor, as reflected by a decreased rate constant for reversal of the activation. Consistent with its higher than normal heparin affinity, the inactive antithrombin variant was a potent competitive antagonist of the heparin-catalyzed reaction of normal antithrombin with thrombin but did not affect the uncatalyzed reaction. These results suggest that deletion of the antithrombin P1 residue partially activates the serpin by inducing strain in the reactive center loop, which destabilizes the native loop-buried state and favors the activated loop-exposed state with high heparin affinity. The unusually severe thrombosis associated with the heterozygous mutation may be explained by the ability of antithrombin London to bind endogenous heparan sulfate or heparin molecules with high affinity and to thereby block activation of the normal inhibitor.     

Importance of the P2 glycine of antithrombin in target proteinase specificity, heparin activation, and the efficiency of proteinase trapping as revealed by a P2 Gly --> Pro mutation.

A sequence-specific heparin pentasaccharide activates the serpin, antithrombin, to inhibit factor Xa through an allosteric mechanism, whereas full-length heparin chains containing this sequence further activate the serpin to inhibit thrombin by an alternative bridging mechanism. To test whether the factor Xa specificity of allosterically activated antithrombin is encoded in the serpin reactive center loop, we mutated the factor Xa-preferred P2 Gly to the thrombin-preferred P2 Pro. Kinetic studies revealed that the mutation maximally enhanced the reactivity of antithrombin with thrombin 15-fold and decreased its reactivity toward factor Xa 2-fold when the serpin was activated by heparin pentasaccharide, thereby transforming antithrombin into an allosterically activated inhibitor of both factor Xa and thrombin. Surprisingly, the enhanced thrombin specificity of the mutant antithrombin was attenuated when a full-length bridging heparin was the activator, due both to a reduced rate of covalent reaction of the mutant serpin and thrombin and preferred reaction of the mutant serpin as a substrate. These results demonstrate that the reactive center loop sequence determines the specificity of allosterically activated antithrombin for factor Xa and that the conformational flexibility of the P2 Gly may be critical for optimal bridging of antithrombin and thrombin by physiologic heparin and for preventing antithrombin from reacting as a substrate in the bridging complex.     

Are factor Xa inhibitors superior to thrombin inhibitors in anticoagulation?

Protease inhibitors are useful tools for increasing the inhibitor potential of plasma. In this context, thrombin inhibitors attracted special interest. However, other clotting enzymes, especially factor Xa, are target enzymes of protease inhibitors besides thrombin. Our studies on structure-activity relationships of benzamidine derivatives resulted in selective inhibitors of thrombin and factor Xa. The use of these inhibitors enabled us to clarify whether the antithrombin activity or the anti-factor Xa activity of a compound is more efficient in anticoagulation. We assessed the concentration-dependent inhibition of the activated partial thromboplastin time by these compounds. If one correlates the inhibitor concentration, which prolonged the clotting time by 60 s, with the dissociation constants one will realize that thrombin inhibition is significantly more efficient in anticoagulation than inhibition of factor Xa.     

Antithrombin in vertebrate species: conservation of the heparin-dependent anticoagulant mechanism

Heparin is thought to regulate the rate of mammalian blood clotting by enhancing the activity of antithrombin, an inhibitor of coagulation enzymes. The present study establishes that this same inhibitor is present in the blood plasma of each of the terrestrial vertebrate groups including mammals, birds, reptiles, and amphibians. In each case, an inhibitor with remarkably similar properties to human antithrombin was isolated by affinity chromatography on immobilized porcine heparin. The purified vertebrate inhibitors all show the following physical and functional homologies to human antithrombin: (i) heparin-enhanced inhibition of both bovine thrombin and human Factor Xa, (ii) molecular masses of approximately 60,000, and (iii) heparin-induced increases in ultraviolet fluorescence. Also, the heparin-binding interaction of vertebrate antithrombins is highly selective with each demonstrating the same rigid specificity for heparin species fractionated on the basis of their affinity for human antithrombin. This common ability of vertebrate antithrombins to discriminate among heparins is accomplished by a nearly unvarying equilibrium binding constant for the high-affinity heparin species. Thus, the present results suggest that the anticoagulant relationship of heparin and antithrombin was established at an early point in the evolution of the coagulation system and has been highly conserved since that time.     

Evolution of Serpin Specificity: Cooperative Interactions in the Reactive-Site Loop Sequence of Antithrombin Specifically Restrict the Inhibition of Activated Protein C

Protease cascades and their inhibitors are a common feature of many biological regulatory systems, and the various components of such cascades have been subjected to a long and concerted evolution. We present here evidence that in the coagulation cascade, the sequence of the protease-binding reactive-site loop of antithrombin has evolved such that the majority of its residues has been acquired not for the efficient inhibition of its target proteases, thrombin and factor Xa, but to avoid the inhibition of activated protein C (APC). We substituted residues of the reactive-site loop of antithrombin into α₁-antitrypsin and tested the chimeras against thrombin, factor Xa, and APC. With respect to factor Xa and thrombin, the difference in association rate between the fastest and the slowest inhibitors was 5.5- and 88-fold, respectively. However, with respect to APC the difference was 12,500-fold. While most of the variation in the inhibition rates of thrombin could be accounted for by P2 Gly-to-Pro substitutions, for APC almost every residue had an effect on inhibition. In 22 of 25 direct comparisons of antitrypsin residues with antithrombin residues, either singly or in blocs, the antithrombin residues caused a decrease in the rate of inhibition of APC. The antithrombin residue Asn393, at position P′3, emerged as particularly important for avoiding the inhibition of APC, however, its 190-fold effect was seen only when in conjunction with antithrombin P7 to P′2 residues. Cooperative effects among residues of the reactive-site loop thus emerged as critical for restricting the activity of this sequence against APC. Received: 15 November 1999 / Accepted: 2 August 2000