THE GAMMA-CARBOXYGLUTAMIC ACID AND EPIDERMAL GROWTH FACTOR-LIKE MODULES OF FACTOR-IXA-BETA - EFFECTS ON THE SERINE-PROTEASE MODULE AND FACTOR-X ACTIVATION

Blood coagulation factors IX and X are two serine proteases with a similar modular structure. The noncatalytic part of each protein consists of a gamma-carboxyglutamic acid (Gla)-containing module and two modules homologous to the epidermal growth factor (EGF) precursor. We have now found that the NH2-terminal EGF-like module of both factors IX and X inhibits factor Xa formation in a Gla-independent manner, both in the presence and absence of phospholipid and the cofactor, factor VIIIa. In contrast, the COOH-terminal EGF-like module has no such effect. Our data indicate that the NH2-terminal EGF-like module of factor IXabeta interacts either with the corresponding module or with the serine protease module in the substrate, factor X, without affecting the hydrolysis of low molecular weight substrates. Using antibodies as structural probes, we found that Ca2+ binding to the Gla module of factor IXabeta induces a conformational transition in the serine protease module. No evidence was found for a direct interaction between the Gla module and factor VIIIa. We therefore propose that the Gla module in factor IXabeta is indirectly involved in the cofactor interaction, in that Ca2+ binding to sites in this module induces a conformation in the serine protease module that is commensurate with factor VIIIa interaction. In addition, the immunochemical approach revealed a Gla-independent Ca2+ binding site in the serine protease module (apparent K(d) of almost-equal-to 120 gm) that also might influence its conformation. Antibodies against the EGF-like modules of factor IX were used to probe Ca2+ binding to these modules in intact and in Gla-domainless factor IXabeta. The data indicate a Ca2+ binding site with an apparent K(d) of almost-equal-to 50 mum in the NH2-terminal EGF-like module of both factor IX species.