Characterization of bothrojaracin interaction with human prothrombin

Abstract

The blood-clotting serine proteinase, thrombin, is formed by cleavage of two peptide bonds in prothrombin by blood coagulation factor Xa. Thrombin generation is accompanied by separation of the prothrombin activation fragment 1 and 2 domains and the protease domain (thrombin). The prothrombin fragment 1 domain functions in prothrombin activation by binding calcium and negatively charged phospholipid surfaces (Gitel et al. 1973; Dombrose et al. 1979). Binding of prothrombin to the cofactor protein, factor Va, which regulates the activation of prothrombin by factor Xa, is thought to involve fragment 2 and/or an exosite in the proteinase domain of prothrombin (Kotkow et al. 1995; Krishnaswamy and Walker 1997; Anderson et al. 2000b). During the conversion of prothrombin into thrombin, the catalytic site is activated and additional allosteric regulatory sites become expressed on the enzyme (Liu et al. 1991b; Wu et al. 1994). Anion-binding exosite I is a positively charged site on thrombin that interacts with fibrinogen (Naski et al. 1990), the platelet thrombin receptor (Liu et al. 1991a), thrombomodulin (Ye et al. 1992), heparin cofactor II (Hortin et al. 1989), factor V and Va (Dharmawardana et al. 1999), and with C-terminal hirudin peptides (Krstenansky and Mao 1987). Through these interactions, exosite I plays key roles in the specificity of thrombin towards macromolecular substrates, cofactors, and some inhibitors (Stubbs and Bode 1993; Guillin et al. 1995). Recently, exosite I has been shown to be partially exposed on prothrombin in a precursor state (proexosite I), and is fully expressed on thrombin, with a ∼100-fold increase in affinity for hirudin peptides (Anderson et al. 2000a). Another positively charged site on thrombin is anion-binding exosite II, which binds heparin (Sheehan and Sadler 1994) and fragment 2 (Walker and Esmon 1979). The crystal structure of the thrombin–fragment 2, domain–domain complex indicates that exosite II is not accessible on prothrombin (Arni et al. 1993).

Bothrojaracin (BJC) is a potent and highly specific thrombin inhibitor isolated from the venom of the jararaca snake common to Brazil (Bothrops jararaca) (Zingali et al. 1993). BJC is a 27-kD disulfide-linked heterodimer that belongs to the C-type (Ca^{-dependent) lectin-like related protein family (}Arocas et al. 1997). The mechanism of thrombin inhibition is unusual in that BJC interacts with both exosites I and II on thrombin (Arocas et al. 1996), forming a 1:1 noncovalent complex and inhibiting thrombin activity toward fibrinogen, platelets, protein C, and factor V (Zingali et al. 1993; Arocas et al. 1998), while the activity towards tripeptide chromogenic substrates is modulated but not impaired (Monteiro et al. 1999). In addition, BJC has been found to form a noncovalent complex with prothrombin (Arocas et al. 1996), and this was associated with inhibition by BJC of prothrombin activation by Oxyuranus scutellatus venom (Monteiro and Zingali 2000). However, the mechanism by which BJC binds to prothrombin is not known, and the binding site on prothrombin has not been identified.

In the present work we quantitatively characterize the binding of BJC to prothrombin for the first time, and identify the binding site on prothrombin as proexosite I in the catalytic domain of prothrombin. Competitive binding studies employing fluorescence probes showed that specific binding of sulfated hirudin ^(Hir[SO₃^{]) to proexosite I displaced BJC from the complex formed with prothrombin. BJC similarly disrupted the prothrombin complex with Hir(SO}₃^{), indicating that BJC and Hir(SO}₃^{) bind competitively to proexosite I on human prothrombin. The results for BJC represent the highest affinity interaction with proexosite I described thus far, and may therefore aid the development of novel anticoagulants based on exosite inhibitors.}

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Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/