Differential contributions of Glu⁹⁶, Asp¹⁰² and Asp¹¹¹ to coagulation Factor V/Va metal ion binding and subunit stability

Blood coagulation FV (Factor V) is activated by thrombin-mediated excision of the B domain, resulting in a non-covalent heterodimer, FVa (activated FV). Previous studies implicated Glu⁹⁶, Asp¹⁰² and Asp¹¹¹ in the essential Ca²⁺-dependent FVa subunit interaction. In the present study, FV E96A, D102A and D111A were purified and evaluated for function, subunit dissociation and metal ion binding. Chromogenic and clotting assays in the presence of procoagulant vesicles showed that each variant was inhibited (∼20–40%). D111A was further inhibited (>90%) after cleavage by thrombin. Comparable function was observed on activated platelets. D111A inhibition correlated to spontaneous subunit dissociation and severely impaired Ca²⁺ binding. The Cu²⁺ interaction was also inhibited, suggesting interdependent Ca²⁺ and Cu²⁺ binding to FV. The parental FV (FV-810; wild-type human FV missing residues 811–1491) used here is fully active without proteolysis because the B domain is truncated. Therefore, a FVa-like functional configuration exists for intact D111A independent of normal metal ion interactions. Unlike D111A, the thrombin-mediated FVa derived from E96A and D102A had only moderately enhanced subunit dissociation upon chelation and had normal metal ion binding. For FV-810-, E96A- and D102A-derived FVa, loss of function after chelation significantly preceded subunit dissociation. This study defines the highly conserved segment spanning Glu⁹⁶–Asp¹¹¹ in FV as multifunctional. Of the three amino acids evaluated, Asp¹¹¹ is essential and probably functions through direct and indirect effects on Ca²⁺ and Cu²⁺ interactions. Glu⁹⁶ and Asp¹⁰² individually influence FV/FVa by more subtle effects, possibly at the metal ion-dependent subunit interface.