Metal ion binding to anticoagulation factor II from the venom of <Emphasis Type="Italic">Agkistrodon acutus</Emphasis>: stabilization of the structure and regulation of the binding affinity to activated coagulation factor X

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X (FXa)-binding protein with both anticoagulant and hypotensive activities. The thermodynamics of the binding of alkaline earth metal ions to ACF II and their effects on the stability of ACF II and the binding of ACF II to FXa were investigated by isothermal titration calorimetry, fluorescence, differential scanning calorimetry, and surface plasmon resonance. The binding of ACF II to FXa does not have an absolute requirement for Ca²⁺. Mg²⁺, Sr²⁺, and Ba²⁺ can induce the binding of ACF II to FXa. The radii of the cations bound in ACF II crucially affect the binding affinity of ACF II for cations and the structural stability of ACF II against guanidine hydrochloride and thermal denaturation, whereas the radii of cations bound in FXa markedly affect the binding affinity between ACF II and FXa. The binding affinities of ACF II for cations and the capacities of metal-induced stabilization of ACF II follow the same trend: Ca²⁺ > Sr²⁺ > Ba²⁺. The metal-induced binding affinities of ACF II for FXa follow the trend Mg²⁺ > Ca²⁺ > Sr²⁺ > Ba²⁺. Although Mg²⁺ shows significantly low binding affinity with ACF II, Mg²⁺ is the most effective to induce the binding of ACF II with FXa. Our observations suggest that in blood the bindings of Ca²⁺ in two sites of ACF II increase the structural stability of ACF II, but these bindings are not essential for the binding of ACF II with FXa, and that the binding of Mg²⁺ and Ca²⁺ to FXa may be essential for the recognition between FXa and ACF II. Like Ca²⁺, the abundant Mg²⁺ in blood also plays an important role in the anticoagulation of ACF II.