Definition of a factor Va binding site in factor Xa

We reported previously that residue 347 in activated fX (fXa) contributes t o binding of the cofactor, factor Va (fVa) (Rudolph, A. E., Porche-Sorbet, R. and Miletich, J. P. (2000) Biochemistry 39, 2861-2867). Four additional residues that participate in Na binding have now been identified by mutagen esis. All five resulting fX species, fX(R306A), fX(E310N), fX(R347N), fX(K3 51A), and fX(K414A), are activated and inhibited normally. However, the rat e of inhibition by antithrombin III in the presence of submaximal concentra tions of heparin is reduced for all the enzymes. In the absence of fVa, all of the enzymes bind and activate prothrombin similarly except fXa(E310N), which has a reduced apparent affinity (similar to3-fold) for prothrombin co mpared with wild type fXa (fXa(WT)). In the absence of phospholipid, fVa en hances the catalytic activity of fXa(WT) significantly, but the response of the variant enzymes was greatly diminished. On addition of 100 nm PC:PS (3 :1) vesicles, fVa enhanced fXa(WT), fXa(R306A), and fXa(E310N) similarly, w hereas fXa(R347N), fXa(K351A), and fXa(K414A) demonstrated near-normal cata lytic activity but reduced apparent affinity for fVa under these conditions . All enzymes function similarly to fXa(WT) on activated platelets, which p rovide saturating fVa on an ideal surface. Loss of binding affinity for fVa as a result of the substitutions in residues Arg-347, Lys-351, and Lys-414 was verified by a competition binding assay. Thus, Arg-347, Lys-351, and L ys-414 are likely part of a core fVa binding site, whereas Arg-306 and Glu- 310 serve a less critical role.