Coagulation Factor X-Binding Protein from Deinagkistrodon acutus Venom Is a Gla Domain-Binding Protein

Abstract

Factor IX/factor X-binding protein (IX/X-bp) is an anticoagulant isolated from the venom of Trimeresurus flavoviridis (habu snake) and binds predominantly to factor IX. In this study, we isolated IX/X-bp-like proteins from the venom of Deinagkistrodon acutus (hundred pace snake) with binding characteristics different from those of IX/X-bp. The complete amino acid sequence and binding characteristics of the main anticoagulant protein, named X-bp, were investigated. The concentrations of X-bp at half-maximal binding to solid-phase factors X and IX were 0.4 and 3 nM, respectively. The binding of X-bp to solid-phase factor X was inhibited by 50% by 6- and 9-fold excess concentrations of factor X and Gla domain (GD) peptide 1-44, respectively, but was not influenced by GD peptide 1-41 and Gla domainless factor X. X-bp bound two Ca2+ ions per molecule with Kd values of 16 +/- 0.7 (mean +/- SE, n = 6) and 103 +/- 10 microM. X-bp was a heterodimer of C-type lectin-like subunits. The 16 kDa chain (A chain) consisted of 129 amino acid residues and was 68% identical to the sequence of the A chain of IX/X-bp. The 15 kDa chain (B chain) consisted of 123 amino acid residues and was 87% identical to IX/X-bp. Three-dimensional model construction from the known fold of IX/X-bp showed that amino acid residues different from those of IX/X-bp are mostly on the molecular surface. Some of these are concentrated on a part of the concave surface which is considered to be the coagulation factor-binding site, presumably acting as a discriminator for ligand binding. These results indicated that X-bp isolated from D. acutus venom was a GD-binding protein, and the C-terminal region of GD peptide was critical for folding of the peptide.

Crystal structure of an anticoagulant protein in complex with the Gla domain of factor X

“…Amino acid sequences are highly homologous, so that variable residues are concentrated only in the loop between the second ␣-helix and the second ␤-strand in subunit A (Fig. 1 A), suggesting that this is a discriminator site (6). This region and the C-terminal region of subunit A interact with XGD1-44 through water molecules.…”

“…X-bp was isolated and purified from the venom of Deinagkistrodon acutus (6). A Gla domain-containing peptide 1-44 (XGD1-44) was prepared and purified with factor X, which was isolated from bovine plasma (6).…”

“…X-bp was isolated and purified from the venom of Deinagkistrodon acutus (6). A Gla domain-containing peptide 1-44 (XGD1-44) was prepared and purified with factor X, which was isolated from bovine plasma (6). The mixture of X-bp and XGD1-44 was chromatographed on one column of Superdex 75 pg (1.6 ϫ 60 cm) by using 50 mM Tris⅐HCl, pH 8.0, containing 0.1 M NaCl and 5 mM CaCl 2 as the elution buffer to remove free XGD1-44.…”

Multifunctional Snake C-Type Lectins Affecting Platelets

“…A wide range of other snake C-type lectins has been described that activate coagulation factors or affect other plasma components involved in hemostasis. These include factor IX/factor X binding proteins from Trimeresurus flavoviridis [20] and Echis carinatus leucogaster [21] venom and coagulation factor X binding protein from Deinagkistrodon acutus venom [22].…”

Cloning of cDNAs and molecular characterisation of C-type lectin-like proteins from snake venoms

“…The binding of the CTLPs to GP receptors or vWF that activates platelets will cause thrombosis formation and CTLPs that act in this manner are crotacetin (CTC) and convulxin (CVX) from the venom of the tropical rattlesnake, Crotalus durissus terrificus, that act on the collagen receptors, GPVI and GPIa/IIa (Leduc and Bon, 1998;Rádis-Baptista et al, 2006), alboaggregin-A, with 2 α and 2 b subunits, from the venom of Trimeresurus albolabris, that acts on GPVI and GPIba (Dörmann et al, 2001) and botrocetin and bitiscetin, derived from Bothrops jararaca and Bitis arietans venom, respectively, that initiate vWF-dependent platelet aggregation by interaction with GPIb, in vitro (Matsui et al, 2010). Concerning the anticoagulant CTLPs, three coagulation factor binding targets have been determined as coagulation factor IX/X (Koo et al, 2002), coagulation factor IX (Zang et al, 2003a) and coagulation factor X (Atoda et al, 1998). Other CTLPs can block the platelet aggregation pathway by inhibiting binding between vWF and the platelet GPIb-factor/IX-factor V complex, examples being agkicetin from Agkistrodon acutus venom and mamushigin, from Agkistrodon halys blomohoffi venom (Ogawa et al, 2005).…”