Nematode Antocoagulant Protein c2 (NAPc2) Interferes with Factor Xa Dimerization: Structural Alteration of Factor Xa Upon Dimerization.

Abstract 1128

Nematode Anticoagulant protein c2 (NAPc2) from the hematophagous hookworm, Ancyclostoma canimum, is a potent inhibitor of factor × activation to Xa by the Xase complex composed of VIIa and tissue factor. Factor Xa (FXa) plays a crucial role in blood coagulation; including formation of a prothrombin activating complex with its co-factor Va. NAPc2 also inhibits activation of prothrombin by this complex. FXa forms a functionally inactive dimer upon binding to either membrane bound or solution phosphatidylserine (PS) in the presence of calcium, and that the structure of FXa in the dimer is altered (Chattopadhyay et al, 2009; Koklic et al., 2009). The dimer interface predicted from mass spectroscopy involves a residue in the hirudin-binding site. NAPc2 interacts with two regions in FXa, one overlapping with the hirudin site and another in the substrate binding exosite (Murakami et al., 2007; Yegneswaran et al., 2003; Wilkens et al., 2002). We show that NAPc2 at low concentration does not interfere with dimerization, but at high concentration it destabilizes the dimer as documented with homoFRET studies with FEGRXa (Fluorescein-GLU-GLY-ARG-chloromethylketone-Xa). Proteolytic activity (prothrombin as substrate) suggested that the species Xa · NAPc2 (NAPc2 binding to FXa monomer) and FXa₂ · NAPc2 (NAPc2 binding to FXa dimer) have activity comparable to FXa dimer. A model is suggested in which initial NAPc2 binding to the substrate-binding region in FXa alters interactions in the hirudin-binding region and thus alters dimer formation. Supported by USPHS grant HL072827 to BRL.