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 sim ilar modular structure. The noncatalytic part of each protein consists of a gamma-carboxyglutamic acid (Gla)-containing module and two modul es 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 t he presence and absence of phospholipid and the cofactor, factor VIIIa . In contrast, the COOH-terminal EGF-like module has no such effect. O ur data indicate that the NH2-terminal EGF-like module of factor IXabe ta interacts either with the corresponding module or with the serine p rotease module in the substrate, factor X, without affecting the hydro lysis of low molecular weight substrates. Using antibodies as structur al probes, we found that Ca2+ binding to the Gla module of factor IXab eta 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 Ca 2+ binding to sites in this module induces a conformation in the serin e protease module that is commensurate with factor VIIIa interaction. In addition, the immunochemical approach revealed a Gla-independent Ca 2+ binding site in the serine protease module (apparent K(d) of almost -equal-to 120 gm) that also might influence its conformation. Antibodi es against the EGF-like modules of factor IX were used to probe Ca2+ b inding to these modules in intact and in Gla-domainless factor IXabeta . The data indicate a Ca2+ binding site with an apparent K(d) of almos t-equal-to 50 mum in the NH2-terminal EGF-like module of both factor I X species.