Identification of basic residues in the heparin-binding exosite of factor Xa critical for heparin and factor Va binding

Abstract

We recently demonstrated that a template mechanism makes a significant contribution to the heparin-accelerated inactivation of factor Xa (FXa) by antithrombin at physiologic Ca2+, suggesting that FXa has a potential heparin-binding site. Structural data indicate that 7 of the 11 basic residues of the heparin-binding exosite of thrombin are conserved at similar three-dimensional locations in FXa. These residues, Arg93, Lys96, Arg125, Arg165, Lys169, Lys236, and Arg240 were substituted with Ala in separate constructs in Gla domainless forms. It was found that all derivatives cleave Spectrozyme FXa with similar catalytic efficiencies. Antithrombin inactivated FXa derivatives with a similar second-order association rate constant (k2) in both the absence and presence of pentasaccharide. In the presence of heparin, however, k2 with certain mutants were impaired up to 25-fold. Moreover, these mutants bound to heparin-Sepharose with lower affinities. Heparin concentration dependence of the inactivation revealed that only the template portion of the cofactor effect of heparin was affected by the mutagenesis. The order of importance of these residues for binding heparin was as follows: Arg240 Lys236 > Lys169 > Arg165 > Lys96 > Arg93 ≥ Arg125. Interestingly, further study suggested that certain basic residues of this site, particularly Arg165 and Lys169, play key roles in factor Va and/or prothrombin recognition by FXa in prothrombinase.