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.