The binding properties of the coagulation factor IX/factor X-binding anticoagulant protein (W X-bp) isolated from the venom of Trimeresurus flavoviridis (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 Ca2+ 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 Ca2+ 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 IXa beta' (residues 1-42) in the liquid phase, but the binding was not inhibited by Gla-domainless factor IXa beta'. 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factor X. IX/X-bp bound to PCGFX, a recombinant human protein C whose Gla-domain region (residues 1-43) had been replaced by residues 1-43 of human factor X. The affinity of binding was about one tenth of that to intact human factor X. IX/X-bp was unable to bind at all to human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 M and X in the presence of Ca2+ ions.
