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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.

Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH2-terminal domains of the Aα- and Bβ-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γA/γA isoform of fibrin(ogen) and the γA/γ' variant with an extended γ-chain. Thrombin binds to the γ'-chain and forms a higher affinity interaction with γA/γ'-fibrin(ogen) than γA/γA-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (Kd values of about 0.5 μM) even though it does not interact with the γ'-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γA/γA-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a Kd value of 2-5 μM, the α(17-51) and Bβ(1-42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.

Heparin binds fibrin and, by bridging thrombin onto fibrin, promotes the formation of a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin. Because thrombin binds γ(A)/γ'-fibrin, a variant with an extended γ-chain, with higher affinity than the bulk γ(A)/γ(A)-fibrin, γ(A)/γ'-fibrin affords bound thrombin more protection from inhibition by antithrombin-heparin. We examined the effect of Zn(2+) on heparin-thrombin-fibrin complex formation because Zn(2+) modulates heparin-protein interactions. Zn(2+) increased the affinity of heparin for γ(A)/γ(A)- and γ(A)/γ'-fibrin by 4.3- and 3.7-fold, respectively, but had no effect on the affinity of thrombin for either form of fibrin. In contrast, in the presence of heparin, Zn(2+) increased the affinity of thrombin for γ(A)/γ(A)-fibrin 4-fold (from a K(d) value of 0.8 to 0.2 μM) and slowed the rate of thrombin dissociation from γ(A)/γ(A)-fibrin clots. These findings suggest that Zn(2+) enhances the formation of ternary heparin-thrombin-fibrin complexes with γ(A)/γ(A)-fibrin but does not influence the already high affinity interaction of thrombin with γ(A)/γ'-fibrin. Consistent with this concept, in the presence of Zn(2+), γ(A)/γ(A)-fibrin protected thrombin from inhibition by antithrombin-heparin to a similar extent as γ(A)/γ'-fibrin. Therefore, by enhancing the binding of heparin to fibrin, physiological concentrations of Zn(2+) render fibrin-bound thrombin more protected from inhibition by antithrombin. Because fibrin-bound thrombin can trigger thrombus expansion, these findings help to explain why recurrent thrombosis can occur despite heparin treatment.

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.

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.

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.