The effect of calcium (II) on the binding of anticoagulation factor I withactivated coagulation factor X

Anticoagulation factor I (ACF I) from the venom of Agkistrodon acutus forms a 1:1 complex with activated coagulation factor X (FXa) in a Ca2+-dependen t fashion and thereby prolongs the clotting time. In the present study, the dependence of the binding of ACF I with FXa on the concentration of Ca2+ i ons was quantitatively analyzed by HIPLC, and the result showed that the ma ximal binding of ACF I to FXa occurred at concentration of Ca2+ ions of abo ut 1 mM. The binding of Ca2+ ions to ACF I was investigated by equilibrium dialysis and two Ca2+-binding sites with different affinities were identifi ed. At pH 7.6, the apparent association constants K-1 and K-2 for these two sites were (1.8 +/- 0.5) x 10(5) and (2.7 +/- 0.6) x 10(4) M-1 (mean +/- S E, n = 4), respectively. It was evident from the observation of Ca2+-induce d changes in the intrinsic fluorescence of ACF I that ACF I underwent a con formational change upon binding of Ca2+ ions. The occupation of both Ca2+-b inding sites in ACF I required a concentration of Ca2+ ions of about 1 mM, which is equal to the effective concentration of Ca2+ ions required both fo r maximal binding of ACF I to FXa and for the maximal enhancement of emissi on fluorescence of ACF I. It could be deduced from these results that the o ccupation of both Ca2+-binding sites in ACF I with Ca2+ ions and subsequent conformational rearrangement might be essential for the binding of ACF I t o FXa.