Binding Properties of the Coagulation Factor IX/factor X‐binding Protein Isolated from the Venom of Trimeresurus flavoviridis

The binding properties of the coagulation factor IX/factor X‐binding anticoagulant protein (IW/X‐bp) isolated from the venom of Trimeresurus fluvoviridis (habu snake) were investigated with an enzyme‐linked immunosorbent assay. The half‐maximal binding and maximal binding of IX/X‐bp to both factors IX and X were observed at concentrations of Ca 2+ ions of 0.4 mM and 1 mM, respectively. Concentration of IX/X‐bp at half‐maximal binding to solid‐phase bovine factor IX and solid‐phase bovine factor X were 0.4 ± 0.1 nM and 1.1 ± 0.4 nM, respectively, in the presence of 1 mM Ca 2+ ions. The kinetics of binding activity of IX/X‐bp to bovine factors IXa and Xa and to human factors IX and X resembled those of the binding to bovine factors IX and X. IX/X‐bp did not bind to solid‐phase coagulation factors other than factor IX/IXa and factor X/Xa, for example, prothrombin, factor VII, protein C, and protein Z, under the conditions of the experiment. To localize the binding sites of IX/X‐bp on the coagulation factors, the ability of IX/X‐bp to bind to various fragments derived from factors IX and X was examined. The binding of IX/X‐bp to solid‐phase factor IX was inhibited by a peptide containing the 4‐carboxyglutamic acid (Gla) domain derived from factor IXaβ′ (residues 1–42) in the liquid phase, but the binding was not inhibited by Gla‐domainless factor IXaβ′. Half‐maximal binding of IX/X‐bp to solid‐phase Gla‐domain peptide of factor IX occurred at 9.2 ± 1.9 nM. Factor X was partially reduced and the S ‐carboxymethylated light and heavy chains of factor X were prepared. IX/X‐bp bound to the S ‐carboxymethylated light chain of factor X but not to the heavy chain. The binding of IX/X‐bp to solid‐phase factor X was inhibited by the Gla‐domain peptide of factor X (residues 1–44) but not by Gla‐domainless factor X. IX/X‐bp bound to PCGFX, a recombinant human protein C whose Gla‐domain region (residues 1–43) had been replaced by residues 1–43 of human factor X. The affinity of binding was about one tenth of that to intact human factor X. IX/X‐bp was unable to bind at all to human protein C. These data indicate that IX/X‐bp is a protein that binds to the Gla‐domain regions of factors IX and X in the presence of Ca 2+ ions.