Na+ Site in Blood Coagulation Factor IXa: Effect on Catalysis and Factor VIIIa Binding

During blood coagulation, factor IXa (FIXa) activates factor X (FX) requiring Ca2+, phospholipid, and factor VIIIa (FVIIIa). The serine protease domain of FIXa contains a Ca2+ site and is predicted to contain a Na+ site. Comparative homology analysis revealed that Na+ in FIXa coordinates to the carbonyl groups of residues 184A, 185, 221A, and 224 (chymotrypsin numbering). Kinetic data obtained at several concentrations of Na+ and Ca2+ with increasing concentrations of a synthetic substrate (CH3-SO2-d-Leu-Gly-Arg-p-nitroanilide) were fit globally, assuming rapid equilibrium conditions. Occupancy by Na+ increased the affinity of FIXa for the synthetic substrate, whereas occupancy by Ca2+ decreased this affinity but increased kcat dramatically. Thus, Na+-FIXa-Ca2+ is catalytically more active than free FIXa. FIXaY225P, a Na+ site mutant, was severely impaired in Na+ potentiation of its catalytic activity and in binding to p-aminobenzamidine (S1 site probe) validating that substrate binding in FIXa is linked positively to Na+ binding. Moreover, the rate of carbamylation of NH2 of Val16, which forms a salt-bridge with Asp194 in serine proteases, was faster for FIXaY225P and addition of Ca2+ overcame this impairment only partially. Further studies were aimed at delineating the role of the FIXa Na+ site in macromolecular catalysis. In the presence of Ca2+ and phospholipid, with or without saturating FVIIIa, FIXaY225P activated FX with similar Km but threefold reduced kcat. Further, interaction of FVIIIa:FIXaY225P was impaired fourfold. Our previous data revealed that Ca2+ binding to the protease domain increases the affinity of FIXa for FVIIIa ∼15-fold. The present data indicate that occupancy of the Na+ site further increases the affinity of FIXa for FVIIIa fourfold and kcat threefold. Thus, in the presence of Ca2+, phospholipid, and FVIIIa, binding of Na+ to FIXa increases its biologic activity by ∼12-fold, implicating its role in physiologic coagulation.