EFFECTS OF CA2+ BINDING ON THE PROTEASE MODULE OF FACTOR-XA AND ITS INTERACTION WITH FACTOR-VA - EVIDENCE FOR 2 GLA-INDEPENDENT CA2+-BINDING SITES IN FACTOR-XA

The assembly of macromolecular complexes containing factors Xa and Va on suitable phospholipid surfaces is crucial for rapid activation of prothrombin. We have used quantitative affinity chromatography to characterize the interaction between factor Va and intact factor Xa on the one hand and between factor Va and factor Xa lacking the gamma-carboxyglutamic acid (Gla)-containing module on the other. The dissociation constants were found to be 1.0 +/- 0.1 and 9.5 +/- 1.8 muM, respectively. There was good agreement between these dissociation constants and the concentrations of active site-inhibited factor Xa and Gla-domainless factor Xa that caused half-maximal inhibition of prothrombin activation. To investigate whether the noncatalytic modules of factor Xa interacted directly with factor Va, intact modules were isolated from proteolytic digests of factor X and used as inhibitors of prothrombin activation. The inhibitory effect observed with the isolated Gla module in the absence of phospholipid was due to inhibition of the amidolytic activity of factor Xa rather than to an interaction with factor Va. The epidermal growth factor-like modules did not inhibit prothrombin activation. Using antibodies specific for calcium-dependent epitopes in the serine protease module of factor Xa we demonstrated that Ca2+ binding to the Gla module alters the conformation of the catalytic module. Half-maximal binding was observed at almost-equal-to 0.8 mM Ca2+. Evidence was also obtained for the presence of two Gla-independent Ca2+-binding sites in factor Xa. One of these sites, located in the NH-2-terminal epidermal growth factor-like module, was half-saturated at almost-equal-to 60 muM Ca2+ in intact factor Xa and at almost-equal-to 1.2 mM Ca2+ in Gla-domainless factor Xa. This site appeared not to influence the conformation of the protease module. The second site, which was half-saturated at almost-equal-to 0.16 mM Ca2+, appeared to reside in the serine protease module and to alter its conformation as judged by binding of antibodies specific for calcium-dependent epitopes.