INHIBITION OF PROTHROMBINASE BY HUMAN SECRETORY PHOSPHOLIPASE A(2) INVOLVES BINDING TO FACTOR XA

Human group II secretory phospholipase A(2) (hsPLA(2)) exhibits signif icant anticoagulant activity that does not require its enzymatic activ ity. We examined which coagulation factor was targeted by hsPLA(2) and analyzed which region of the protein may be involved in this inhibiti on. Prothrombin time coagulation assays indicated that hsPLA(2) did no t inhibit activated factor V (FVa) activity, whereas activated factor X (FXa) one-stage coagulation assays suggested that FXa was inhibited. The inhibitory effect of hsPLA(2) on prothrombinase activity of FXa, FV, phospholipids, and Ca2+ complex was markedly enhanced upon preincu bation of hsPLA(2) with FXa but not with FV, Prothrombinase activity w as also strongly inhibited by hsPLA(2) in the absence of FL. High conc entrations of FVa in the prothrombinase generation assay reversed the inhibitory effect of hsPLA(2). By using isothermal titration calorimet ry, we demonstrated that hsPLA(2) binds to FXa in solution with a 1:1 stoichiometry and a K-d of 230 nM. By using surface plasmon resonance we determined the rate constants, k(on) and k(off) of the FXaksPLA(2) interaction and analyzed the Ca2+ effect on these constants. When prei ncubated with FXa, synthetic peptides comprising residues 51-74 and 51 -62 of hsPLA(2) inhibited prothrombinase assays, providing evidence th at this part of the molecule, which shares similarities with a region of FVa that binds to FXa, is likely involved in the anticoagulant inte raction of hsPLA(2) with FXa. In conclusion, we propose that residues 51-62 of hsPLA(2) bind to FXa at a FVa-binding site and that hsPLA(2) decreases the prothrombinase generation by preventing FXa FVa complex formation.