Ca²⁺ Binding to Both the Heavy and Light Chains of Factor VIII Is Required for Cofactor Activity

Abstract

Previously, we demonstrated that Ca(2+) was necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC) but that Ca(2+) did not affect HC-LC binding affinity (Wakabayashi et al. (2001) Biochemistry 40, 10293-10300). Titration of EDTA-treated factor VIII with Ca(2+) followed by factor Xa generation assay showed a two-site binding pattern, with indicated high-affinity (K(d) = 8.9 +/- 1.8 microM) and low-affinity (K(d) = 4.0 +/- 0.6 mM) sites. Analysis by equilibrium dialysis using (45)Ca and <400 microM free Ca(2+) verified a high-affinity binding (K(d) = 18.9 +/- 3.7 microM). Preincubation of either HC or LC with 6 mM Ca(2+) followed by reassociation with the untreated complementary chain in the presence of 0.12 mM Ca(2+) failed to generate significant cofactor activity (<0.5 nM min(-1) (nM LC)(-1)). However, pretreatment of both HC and LC with 6 mM Ca(2+) followed by reassociation (at 0.12 mM Ca(2+)) generated high activity (7.5 +/- 0.4 nM min(-1) (nM LC)(-1)). Progress curves for activity regain following factor VIII-Ca(2+) association kinetics fitted well to a series reaction scheme rather than one of simple association (p < 0.0001), suggesting a multistep process which may include a Ca(2+)-dependent conformational change. These results suggest that factor VIII contains two Ca(2+) binding sites with different affinities and that active factor VIII can be reconstituted from HC and LC only when both chains are preactivated by Ca(2+).

Factor VIII Lacking the C2 Domain Retains Cofactor Activity in Vitro

“…FXa Generation Assay-The rate of conversion of FX to FXa was monitored in a purified system (20) according to methods previously described (18,21). FVIII (1 nM) in buffer A, containing 20 M PSPCPE vesicles was activated with 20 nM ␣-thrombin for 10 s. The reaction was stopped by adding hirudin (10 units/ml) and the resulting FVIIIa was reacted with FIXa (40 nM) for 10 s. FX (300 nM) was added to initiate reactions, which were quenched after 1 min by the addition of 50 mM EDTA.…”

Identification of Residues Contributing to A2 Domain-dependent Structural Stability in Factor VIII and Factor VIIIa

“…Two-stage Chromogenic Factor Xa Generation Assay-The rate of conversion of factor X to factor Xa was monitored in a purified system (19) according to methods previously described (20,21). Briefly, factor VIII (1 nM) in buffer containing 20 mM HEPES, pH 7.2, 0.1 M NaCl, 0.01% Tween 20, 0.01% bovine serum albumin, 5 mM CaCl 2 , and 10 M PS/PC/PE vesicles (Buffer A) was activated with 20 nM ␣-thrombin for 1 min.…”

Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa

“…The rate of conversion of FX to FXa was monitored in a purified system (25) according to methods previously described (26,27). FVIII (1 nM) in 20 mM HEPES, 0.1 M NaCl, 5 mM CaCl 2 , 0.01% Tween 20, 0.01% BSA, pH 7.2 (HEPES buffer), containing 20 μM PSPCPE vesicles (PS:PC:PE = 3:2:5) was activated with 30 nM α-thrombin for 1 min.…”