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Rivaroxaban and apixaban are both small molecules that reversibly inhibit factor Xa. Compared with rivaroxaban, apixaban has minimal effects on the prothrombin time and activated partial thromboplastin time. To investigate this phenomenon, we used a factor Xa-directed substrate in a buffer system. Although rivaroxaban and apixaban inhibited factor Xa with similar Ki values at equilibrium, kinetic measurements revealed that rivaroxaban inhibited factor Xa up to 4-fold faster than apixaban (p

Factor (F) XII, a key component of the contact system, triggers clotting via the intrinsic pathway, and is implicated in propagating thrombosis. Although nucleic acids are potent activators, it is unclear how the contact system is regulated to prevent uncontrolled clotting. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa and attenuates its capacity to trigger coagulation. To investigate the role of HRG as a regulator of the intrinsic pathway, we compared RNA- and DNA-induced thrombin generation in plasma from HRG-deficient and wild-type mice. Thrombin generation was enhanced in plasma from HRG-deficient mice, and accelerated clotting was restored to normal with HRG reconstitution. Although blood loss after tail tip amputation was similar in HRG-deficient and wild-type mice, carotid artery occlusion after FeCl3 injury was accelerated in HRG-deficient mice, and HRG administration abrogated this effect. To confirm that HRG modulates the contact system, we used DNase, RNase, and antisense oligonucleotides to characterize the FeCl3 model. Whereas DNase or FVII knockdown had no effect, carotid occlusion was abrogated with RNase or FXII knockdown, confirming that FeCl3-induced thrombosis is triggered by RNA in a FXII-dependent fashion. Therefore, in a nucleic acid-driven model, HRG inhibits thrombosis by modulating the intrinsic pathway of coagulation.

Fibrin (Fn) clots formed from γ'-fibrinogen (γ'-Fg), a variant with an elongated γ-chain, are resistant to lysis when compared with clots formed from the predominant γA-Fg, a finding previously attributed to differences in clot structure due to delayed thrombin-mediated fibrinopeptide (FP) B release or impaired cross-linking by factor XIIIa. We investigated whether slower lysis of γ'-Fn reflects delayed plasminogen (Pg) binding and/or activation by tissue plasminogen activator (tPA), reduced plasmin-mediated proteolysis of γ'-Fn, and/or altered cross-linking. Clots formed from γ'-Fg lysed more slowly than those formed from γA-Fg when lysis was initiated with tPA/Pg when FPA and FPB were both released, but not when lysis was initiated with plasmin, or when only FPA was released. Pg bound to γ'-Fn with an association rate constant 22% lower than that to γA-Fn, and the lag time for initiation of Pg activation by tPA was longer with γ'-Fn than with γA-Fn. Once initiated, however, Pg activation kinetics were similar. Factor XIIIa had similar effects on clots formed from both Fg isoforms. Therefore, slower lysis of γ'-Fn clots reflects delayed FPB release, which results in delayed binding and activation of Pg. When clots were formed from Fg mixtures containing more than 20% γ'-Fg, the upper limit of the normal level, the delay in lysis was magnified. These data suggest that circulating levels of γ'-Fg modulate the susceptibility of clots to lysis by slowing Pg activation by tPA and provide another example of the intimate connections between coagulation and fibrinolysis.

The nonspecific binding of heparin to plasma proteins compromises its anticoagulant activity by reducing the amount of heparin available to bind antithrombin. In addition, interaction of heparin with fibrin promotes formation of a ternary heparin-thrombin-fibrin complex that protects fibrin-bound thrombin from inhibition by the heparin-antithrombin complex. Previous studies have shown that heparin binds the E domain of fibrinogen. The current investigation examines the role of Zn(2+) in this interaction because Zn(2+) is released locally by platelets and both heparin and fibrinogen bind the cation, resulting in greater protection from inhibition by antithrombin. Zn(2+) promotes heparin binding to fibrinogen, as determined by chromatography, fluorescence, and surface plasmon resonance. Compared with intact fibrinogen, there is reduced heparin binding to fragment X, a clottable plasmin degradation product of fibrinogen. A monoclonal antibody directed against a portion of the fibrinogen αC domain removed by plasmin attenuates binding of heparin to fibrinogen and a peptide analog of this region binds heparin in a Zn(2+)-dependent fashion. These results indicate that the αC domain of fibrinogen harbors a Zn(2+)-dependent heparin binding site. As a consequence, heparin-catalyzed inhibition of factor Xa by antithrombin is compromised by fibrinogen to a greater extent when Zn(2+) is present. These results reveal the mechanism by which Zn(2+) augments the capacity of fibrinogen to impair the anticoagulant activity of heparin.

We show that the nonlinear mechanical response of networks formed from un–cross-linked fibrin or collagen type I continually changes in response to repeated large-strain loading. We demonstrate that this dynamic evolution of the mechanical response arises from a shift of a characteristic nonlinear stress–strain relationship to higher strains. Therefore, the imposed loading does not weaken the underlying matrices but instead delays the occurrence of the strain stiffening. Using confocal microscopy, we present direct evidence that this behavior results from persistent lengthening of individual fibers caused by an interplay between fiber stretching and fiber buckling when the networks are repeatedly strained. Moreover, we show that covalent cross-linking of fibrin or collagen inhibits the shift of the nonlinear material response, suggesting that the molecular origin of individual fiber lengthening may be slip of monomers within the fibers. Thus, a fibrous architecture in combination with constituents that exhibit internal plasticity creates a material whose mechanical response adapts to external loading conditions. This design principle may be useful to engineer novel materials with this capability.

Thrombin is a highly plastic molecule whose activity and specificity are regulated by exosites 1 and 2, positively-charged domains that flank the active site. Exosite binding by substrates and cofactors regulates thrombin activity by localizing thrombin, guiding substrates, and by inducing allosteric changes at the active site. Although inter-exosite and exosite-to-active-site allostery have been demonstrated, the impact of active site ligation on exosite function has not been examined. To address this gap, we used surface plasmon resonance to determine the effects of dabigatran and argatroban, active site-directed inhibitors, on thrombin binding to immobilized γA/γA-fibrin or glycoprotein Ibα peptide via exosite 1 and 2, respectively, and thrombin binding to γA/γ'-fibrin or factor Va, which is mediated by both exosites. Whereas dabigatran attenuated binding, argatroban increased thrombin binding to γA/γA- and γA/γ'-fibrin and to factor Va. The results with immobilized fibrin were confirmed by examining the binding of radiolabeled thrombin to fibrin clots. Thus, dabigatran modestly accelerated the dissociation of thrombin from γA/γA-fibrin clots, whereas argatroban attenuated dissociation. Dabigatran had no effect on thrombin binding to glycoprotein Ibα peptide, whereas argatroban promoted binding. These findings not only highlight functional effects of thrombin allostery, but also suggest that individual active site-directed thrombin inhibitors uniquely modulate exosite function, thereby identifying potential novel mechanisms of action.

Background —Thrombolytic therapy induces a procoagulant state characterized by elevated plasma levels of fibrinopeptide A (FPA), but the responsible mechanism is uncertain. Methods and Results —Washed plasma clots were incubated in citrated plasma in the presence or absence of tissue plasminogen activator (t-PA), and FPA generation was monitored as an index of unopposed thrombin activity. FPA levels are almost twofold higher in the presence of t-PA than in its absence. This primarily reflects the action of thrombin bound to soluble fibrin degradation products because (a) there is progressive FPA generation even after clots are removed from t-PA–containing plasma, and (b) clot lysates produce concentration-dependent FPA generation when incubated in citrated plasma. Using thrombin-agarose affinity chromatography, (DD)E and fragment E but not d -dimer were identified as the thrombin-binding fibrin fragments, indicating that the thrombin-binding site is located within the E domain. Heparin inhibits thrombin bound to fibrin degradation products less effectively than free thrombin. In contrast, D-Phe-Pro-ArgCH 2 Cl, hirudin and hirugen inhibit free thrombin and thrombin bound to fibrin degradation products equally well. Conclusions —Thrombin bound to soluble fibrin degradation products is primarily responsible for the increase in FPA levels that occurs when a clot undergoes t-PA–induced lysis. Like clot-bound thrombin, thrombin bound to fibrin derivatives is protected from inhibition by heparin but susceptible to inactivation by direct thrombin inhibitors. These findings help to explain the superiority of direct thrombin inhibitors over heparin as adjuncts to thrombolytic therapy.

Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn(2+)-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn(2+)-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn(2+), HRG binds the predominant γ(A)/γ(A)-fibrinogen and the γ-chain elongated isoform, γ(A)/γ'-fibrinogen, with K(d) values of 9 nm. Likewise, (125)I-labeled HRG binds γ(A)/γ(A)- or γ(A)/γ'-fibrin clots with similar K(d) values when Zn(2+) is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ'-peptide, an analog of the COOH terminus of the γ'-chain that mediates the high affinity interaction of thrombin with γ(A)/γ'-fibrin. Thrombin competes with HRG for γ'-peptide binding and displaces (125)I-HRG from γ(A)/γ'-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for γ(A)/γ'-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation.

Thrombin exosite 1 binds the predominant gamma(A)/gamma(A)-fibrin form with low affinity. A subpopulation of fibrin molecules, gamma(A)/gamma'-fibrin, has an extended COOH terminus gamma'-chain that binds exosite 2 of thrombin. Bivalent binding to gamma(A)/gamma'-fibrin increases the affinity of thrombin 10-fold, as determined by surface plasmon resonance. Because of its higher affinity, thrombin dissociates 7-fold more slowly from gamma(A)/gamma'-fibrin clots than from gamma(A)/gamma(A)-fibrin clots. After 24 h of washing, however, both gamma(A)/gamma'- and gamma(A)/gamma(A)-fibrin clots generate fibrinopeptide A when incubated with fibrinogen, indicating the retention of active thrombin. Previous studies demonstrated that heparin heightens the affinity of thrombin for fibrin by simultaneously binding to fibrin and exosite 2 on thrombin to generate a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin and heparin cofactor II. In contrast, dermatan sulfate does not promote ternary complex formation because it does not bind to fibrin. Heparin-catalyzed rates of thrombin inhibition by antithrombin were 5-fold slower in gamma(A)/gamma'-fibrin clots than they were in gamma(A)/gamma(A)-fibrin clots. This difference reflects bivalent binding of thrombin to gamma(A)/gamma'-fibrin because (a) it is abolished by addition of a gamma'-chain-directed antibody that blocks exosite 2-mediated binding of thrombin to the gamma'-chain and (b) the dermatan sulfate-catalyzed rate of thrombin inhibition by heparin cofactor II also is lower with gamma(A)/gamma'-fibrin than with gamma(A)/gamma(A)-fibrin clots. Thus, bivalent binding of thrombin to gamma(A)/gamma'-fibrin protects thrombin from inhibition, raising the possibility that gamma(A)/gamma'-fibrin serves as a reservoir of active thrombin that renders thrombi thrombogenic.

Although exosites 1 and 2 regulate thrombin activity by binding substrates and cofactors and by allosterically modulating the active site, it is unclear whether there is direct allosteric linkage between the two exosites. To begin to address this, we first titrated a thrombin variant fluorescently labeled at exosite 1 with exosite 2 ligands, HD22 (a DNA aptamer), gamma'-peptide (an analog of the COOH terminus of the gamma'-chain of fibrinogen) or heparin. Concentration-dependent and saturable changes in fluorescence were elicited, supporting inter-exosite linkage. To explore the functional consequences of this phenomenon, we evaluated the capacity of exosite 2 ligands to inhibit thrombin binding to gamma(A)/gamma(A)-fibrin, an interaction mediated solely by exosite 1. When gamma(A)/gamma(A)-fibrinogen was clotted with thrombin in the presence of HD22, gamma'-peptide, or prothrombin fragment 2 there was a dose-dependent and saturable decrease in thrombin binding to the resultant fibrin clots. Furthermore, HD22 reduced the affinity of thrombin for gamma(A)/gamma(A)-fibrin 6-fold and accelerated the dissociation of thrombin from preformed gamma(A)/gamma(A)-fibrin clots. Similar responses were obtained when surface plasmon resonance was used to monitor the interaction of thrombin with gamma(A)/gamma(A)-fibrinogen or fibrin. There is bidirectional communication between the exosites, because exosite 1 ligands, HD1 (a DNA aptamer) or hirudin-(54-65) (an analog of the COOH terminus of hirudin), inhibited the exosite 2-mediated interaction of thrombin with immobilized gamma'-peptide. These findings provide evidence for long range allosteric linkage between exosites 1 and 2 on thrombin, revealing further complexity to the mechanisms of thrombin regulation.

TNK-tissue plasminogen activator (TNK-t-PA), a bioengineered variant of tissue-type plasminogen activator (t-PA), has a longer half-life than t-PA because the glycosylation site at amino acid 117 (N117Q, abbreviated N) has been shifted to amino acid 103 (T103N, abbreviated T) and is resistant to inactivation by plasminogen activator inhibitor 1 because of a tetra-alanine substitution in the protease domain (K296A/H297A/R298A/R299A, abbreviated K). TNK-t-PA is more fibrin-specific than t-PA for reasons that are poorly understood. Previously, we demonstrated that the fibrin specificity of t-PA is compromised because t-PA binds to (DD)E, the major degradation product of cross-linked fibrin, with an affinity similar to that for fibrin. To investigate the enhanced fibrin specificity of TNK-t-PA, we compared the kinetics of plasminogen activation for t-PA, TNK-, T-, K-, TK-, and NK-t-PA in the presence of fibrin, (DD)E or fibrinogen. Although the activators have similar catalytic efficiencies in the presence of fibrin, the catalytic efficiency of TNK-t-PA is 15-fold lower than that for t-PA in the presence of (DD)E or fibrinogen. The T and K mutations combine to produce this reduction via distinct mechanisms because T-containing variants have a higher K(M), whereas K-containing variants have a lower k(cat) than t-PA. These results are supported by data indicating that T-containing variants bind (DD)E and fibrinogen with lower affinities than t-PA, whereas the K and N mutations have no effect on binding. Reduced efficiency of plasminogen activation in the presence of (DD)E and fibrinogen but equivalent efficiency in the presence of fibrin explain why TNK-t-PA is more fibrin-specific than t-PA.

Tissue plasminogen activator (t-PA) causes fibrinogen proteolysis when alpha 2-antiplasmin levels fall, and this may contribute to t-PA-induced hemorrhage. Because clot-bound plasmin is protected from alpha 2-antiplasmin inhibition, we tested the possibility that alpha 2-antiplasmin supplementation would block t-PA-induced fibrinogenolysis and bleeding without affecting thrombolysis. When added to human or rabbit plasma, alpha 2-antiplasmin inhibits t-PA-induced fibrinogenolysis, but hat little effect on the lysis of 125I-fibrin clots. To examine its effect in vivo, rabbits with preformed 125I-labeled-jugular vein thrombi were randomized to receive t-PA, t-PA and alpha 2-antiplasmin, or saline. alpha 2-Antiplasmin infusion produced a modest decrease in t-PA-induced thrombolysis (from 40.2% to 30.1%, P = 0.12), but reduced fibrinogen consumption from 87% to 27% (P = 0.0001), and decreased blood loss from standardized ear incisions from 5,594 to 656 microliter (P < 0.0001). We hypothesize that alpha 2-antiplasmin limits t-PA-induced hemorrhage by inhibiting fibrinogenolysis and subsequent fragment X formation because (a) SDS-PAGE and immunoblot analysis indicate less fragment X formation in alpha 2-antiplasmin treated animals, and (b) when added to a solution of fibrinogen and plasminogen clotted with thrombin in the presence of t-PA, fragment X shortens the lysis time in a concentration-dependent fashion. These findings suggest that fragment X incorporation into hemostatic plugs contributes to t-PA-induced bleeding. By blocking t-PA-mediated fibrinogenolysis, alpha 2-antiplasmin supplementation may improve the safety of fibrin-specific plasminogen activators.

Despite its affinity for fibrin, tissue plasminogen activator (t-PA) administration causes systemic fibrinogenolysis. To investigate the mechanism, t-PA was incubated with plasma in the presence or absence of a fibrin clot, and the extent of fibrinogenolysis was determined by measuring B beta 1-42. In the presence of fibrin, there is a 21-fold increase in B beta 1-42 levels. The potentiation of fibrinogenolysis in the presence of fibrin is mediated by soluble fibrin degradation products because (a) the extent of t-PA induced fibrinogenolysis and clot lysis are directly related, (b) once clot lysis has been initiated, fibrinogenolysis continues even after the clot is removed, and (c) lysates of cross-linked fibrin clots potentiate t-PA-mediated fibrinogenolysis. Fibrin degradation products stimulate fibrinogenolysis by binding t-PA and plasminogen because approximately 70% of the labeled material in the clot lysates binds to both t-PA- and plasminogen-Sepharose, and only the bound fractions have potentiating activity. The binding site for t-PA and plasminogen is on the E domain because characterization of the potentiating fragments using gel filtration followed by PAGE and immunoblotting indicates that the major species is (DD)E complex, whereas minor components include high-molecular weight derivatives containing the (DD)E complex and fragment E. In contrast, D-dimer is the predominant species found in the fractions that do not bind to the adsorbants, and it has no potentiating activity. Thus, soluble products of t-PA-induced lysis of cross-linked fibrin potentiate t-PA-mediated fibrinogenolysis by providing a surface for t-PA and plasminogen binding thereby promoting plasmin generation. The occurrence of this phenomenon after therapeutic thrombolysis may explain the limited clot selectivity of t-PA.

Introduction: Unfractionated heparin (UFH) binds to thrombin (IIa), which bridges IIa to antithrombin (AT) and promotes inhibition. UFH also binds fibrin (Fn) and, by bridging IIa onto Fn, promotes the formation of a ternary UFH-IIa-Fn complex that protects IIa from AT inactivation. Within this ternary complex, UFH binds to exosite 2 on IIa, whereas Fn binds to exosite 1. γ′-fibrinogen (γ′-Fg), which comprises about 10% of circulating Fg, is a variant with an extended γ-chain. Because IIa binds to the γ′ chain via exosite 2, IIa binds γ′-Fn with higher affinity than it does to the bulk γA-Fn. Zinc (Zn) binds both Fn and UFH. Accordingly, we examined the possibility that Zn promotes the formation of the UFH-IIa-Fn complex, thereby increasing the protection of IIa from AT inhibition. Methods: The affinity of 125I-labelled UFH for γ A-Fn was obtained by measuring unbound UFH in the supernatant after γ A-fg was clotted with IIa in the absence or presence of Zn. Similarly, the affinity of IIa for γA- or γ′-Fn was determined by quantifying the chromogenic activity of unbound IIa in the supernatants of clots prepared from γA- or γ′-Fg with or without Zn and UFH. Rate constants of IIa inhibition by AT were determined under pseudo-first order conditions in the absence or presence of soluble fibrin monomer (Fm) prepared from γA- or γ′-Fg. Results: UFH binds γA-fibrin with 4-fold higher affinity in the presence of 12.5 μM Zn than in its absence (Kd values of 0.4 and 1.5 μM, respectively). Therefore, Zn enhances the affinity of UFH for γA-Fn. Likewise, the affinity of IIa for γA-Fn was 3-fold higher in the presence of UFH than in its absence (Kd values of 0.8 and 2.6 μM, respectively). Zn produced an additional 4-fold increase in affinity in the presence of UFH (Kd value of 0.2 μM), but had no effect on IIa binding in its absence. These data suggest that, by heightening the affinity of UFH for γA-Fn, Zn enhances the formation of a UFH-IIa complex with γA-Fn. In contrast, IIa binds γ′-Fn with high affinity (Kd 0.1 μM), and this interaction was unaffected by UFH or Zn. The heparin-catalyzed rate of IIa inhibition by AT was 2 ± 0.2 × 108 M−1 min−1. Using a concentration of γA-Fm that reduced this rate by 25%, the addition of Zn produced an additional 30% reduction in the rate. Contrarily, an equivalent concentration of γ′-Fm reduced the rate by 60%, and there was no further reduction with the addition of Zn. Conclusion: Physiological concentrations of Zn enhance the formation of the UFH-IIa-Fn complex with γA-Fn, thereby increasing the protection of Fn-bound IIa from inhibition by AT. Zn has no effect on ternary complex formation with γ′-Fn, which already binds IIa with high affinity and affords IIa with greater protection than γA-Fn. These findings suggest that the extent of protection of Fn-bound IIa from inhibition by the AT-UFH complex is greater than previously suspected. Because Fn-bound IIa can trigger thrombus growth, its resistance to inhibition helps to explain the limitations of UFH in patients with arterial thrombosis.

Binding of heparin to fibrinogen and fibrin can compromise its anticoagulant function in two ways. First, binding to fibrinogen renders less heparin available to interact with antithrombin. Second, fibrin-bound heparin can sequester thrombin and protect it from inhibition by antithrombin. Protection results from the formation of a ternary heparin-thrombin-fibrin complex mediated by binary heparin-thrombin, heparin-fibrin, and thrombin-fibrin interactions. Thrombin within the ternary complex is protected from inhibition by antithrombin because exosite 2 is occupied by fibrin-bound heparin, which impairs binding of antithrombin-bound heparin. Because heparin binding to fibrin compromises the anticoagulant functions of heparin, it was of interest to further investigate this interaction. Previous studies have not considered the role of Zn2+, a divalent cation that binds both fibrinogen and heparin and promotes their interactions with numerous proteins. The effect of Zn2+ on binding of heparin to fibrin(ogen) and the subsequent effect on formation of the ternary heparin-thrombin-fibrin complex were investigated. Clots prepared with fibrinogen, 125I-heparin, 2 mM CaCl2, and increasing concentrations of Zn2+ were compacted by centrifugation and aliquots of the supernatant were removed to quantify 125I-heparin bound to fibrin. Titration of Zn2+ showed a saturable 3-fold increase in 125I-heparin bound to fibrin, with maximal binding observed at physiological Zn2+ concentration of 12 μM. When 125I-heparin was titrated with fibrinogen and the samples were clotted in the presence of 12 μM Zn2+, 125I-heparin bound to fibrin with 5-fold higher affinity in the presence of Zn2+ than its absence (Kd values of 0.3 and 1.5 μM, respectively). Comparable results were obtained regardless of whether clots were formed with thrombin or batroxobin, demonstrating that the increased affinity of heparin for fibrin was not the result of formation of thrombin-fibrin-heparin complexes. Investigating ternary complex formation, Zn2+ promoted a saturable 60% increase in thrombin binding to fibrin in the presence of heparin and the Zn2+ dose response mirrored that of the heparin-fibrin interaction. In the absence of heparin, Zn2+ had little effect on thrombin binding to fibrin. To further examine the effect of Zn2+ on heparin-fibrinogen interaction, surface plasmon resonance studies were performed. Varying concentrations of fibrinogen were applied to a biotinylated medium molecular weight heparin (6700 kDa) that was bound to a streptavidin sensor chip. Fibrinogen bound immobilized heparin with a Kd of 379 nM in the presence of EDTA. In the presence 2 mM CaCl2 or 12 μM Zn2+, the Kd values were 10 nM and 8 nM, respectively. However, the mass of heparin bound was ten-fold higher in the presence of Zn2+ than it was with CaCl2 or EDTA. These data reveal that Zn2+ promotes the interaction of heparin with fibrinogen and that this effect is maintained when fibrinogen is converted to fibrin. Because the increased heparin that binds to fibrin in the presence of Zn2+ has greater capacity to bind thrombin, this phenomenon may augment the protection of thrombin from inhibition by antithrombin. Therefore, the extent to which fibrin-bound thrombin is protected from inhibition is likely to be underestimated in studies that do not include physiological concentrations of Zn2+. This gives further emphasis for the need to develop heparin-derived anticoagulants that resist formation of the protective ternary complex.