Abstract
The venom of the viper Echis carinatus contains a metalloprotease, ecarin, that is a potent prothrombin activator. We here show that the venom is also rich in another prothrombin activator, which does not belong to any known category of prothrombin activators. The novel enzyme, designated carinactivase-1 (CA-1), consists of two subunits held together non-covalently but very tightly. One subunit is a 62-kDa polypeptide that has metalloprotease activity and is homologous to the single-chain enzyme ecarin; the other subunit of 25 kDa consists of two disulfide-linked polypeptides of 17 and 14 kDa, and this subunit resembles the anticoagulant in the habu snake venom, IX/X-bp, that specifically binds the Gla domains of coagulation factors IX and X in a Ca2+-dependent fashion. The activation of prothrombin by CA-1 requires Ca2+ ions at millimolar concentrations and in the absence of Ca2+ ions this enzyme is virtually inactive. By contrast, activation by ecarin is completely independent of Ca2+ ions. CA-1, unlike ecarin, does not activate prothrombin derivatives, in which binding of Ca2+ ions has been perturbed, namely prethrombin-1 and acarboxyprothrombin. Furthermore, the isolated catalytic subunit, although its activity is greatly reduced as compared to that of the holoenzyme, no longer requires Ca2+ ions for the activation of prothrombin. Reconstitution with the non-catalytic 25-kDa subunit restores high level activity and the dependence on Ca2+ ions. Finally, prothrombin activation by CA-1 is inhibited by prothrombin fragment 1, and the isolated non-catalytic subunit is capable of binding fragment 1 in the presence of Ca2+ ions. From these observations, we postulate the following unique mechanism for the activation of prothrombin by CA-1. The enzyme primarily recognizes the Ca2+-bound conformation of the Gla domain in prothrombin via the 25-kDa regulatory subunit, and the subsequent conversion of prothrombin to active thrombin is catalyzed by the 62-kDa catalytic subunit.
Highlights
Activators have been reported [2]
Three types of prothrombin activator have been identified in venoms [3]: group 1 enzymes, which are metalloproteases whose actions on prothrombin are independent of any plasma or exogenous cofactors; group 2 enzymes, which are Gla-containing, factor Xa-like serine proteases that require factor Va, anionic phospholipids and Ca2ϩ ions, resembling in this respect the physiological activator factor Xa; and group 3 enzymes, which are hybrid proteins that consist of factor Xa-like catalytic subunits and factor Va-like regulatory subunits and require phospholipids and Ca2ϩ ions for their action
This enzyme, designated carinactivase-1 (CA-1),1,2 is strongly dependent on Ca2ϩ ions for the activation of prothrombin, in sharp contrast to ecarin, whose action is unaffected by exogenous Ca2ϩ ions
Summary
Materials—The venom of Malian E. carinatus leucogaster used for the isolation of CA-1 and ecarin was obtained from Latoxan (Rosans, France). Separation of the CA-1 Subunits—To a solution of purified CA-1, guanidine HCl was added to 4 M This mixture was subjected to gel filtration on Superdex 200pg preequilibrated with 50 mM Tris-HCl, pH 8.0, plus 4 M guanidine HCl. Two peaks were obtained; the first peak contained a 62-kDa component, and the second one contained a 25-kDa component. Fluorogenic Assay of Amidase Activity of CA-1—The design of the fluorogenic substrate, (7-methoxycoumarin-4-yl)acetyl-Ile-Asp-GlyArg-Ile-Val-Glu-Gly-(⑀-2,3-dinitrophenyl)Lys-amide, was based on the structure around the scissile site in human prothrombin (Ile317–Gly324) and was synthesized by the Peptide Institute (Osaka, Japan) This peptide was designed so that fluorescence derived from the N-terminal coumarin derivative (ex 328 nm, em 393 nm) was strongly quenched by the C-terminal dinitrophenyl group. Protein concentrations were determined with a BCA Protein Assay kit (Pierce) with bovine serum albumin as the standard
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