Mechanism of factor Va inactivation by plasmin - Loss of A2 and A3 domainsfrom a Ca2+-dependent complex of fragments bound to phospholipid

The coagulation cofactor Va (FVa) is a noncovalent heterodimer consisting o f a heavy chain (FVaH) and a light chain (FVaL), Previously, the fibrinolyt ic effector plasmin (Pn) has been shown to inhibit FVa function. To underst and this mechanism, the fragmentation profile of human FVa by Pn and the no ncovalent association of the derived fragments were determined in the prese nce of Ca2+ using anionic phospholipid (aPL)-coated microtiter wells and la rge (1 mum) aPL micelles as affinity matrices. Following Pn inactivation of aPL-bound FVa, a total of 16 fragments were observed and their NH, termini sequenced. These had apparent molecular weights and starting residues as f ollows (single letter abbreviation is used): 50(L1766), 48(L1766), 43(Q1828 ), 46(Q1828), 30(S1546), 12(T1657), and 7(S1546) kDa from FVaL; and 65(A1), 50(A1), 45(A1), 34(S349), 30(L94), 30(M110), and 3 small <5(W457, W457, an d K365) kDa from FVaH. Of these, 50(L1766), 48(1766), 43(Q1828), and 40(Q18 28) spanning the C1/C2 domains, and 30(L94), but not the similar 30(M110), positioned within the A1 domain remained associated with aPL. These were de tected antigenically during Pn- or tissue plasminogen activator-mediated ly sis of fibrin clot formed in plasma. Chelation by EDTA dissociated the 30(L 94)-kDa fragment, which was observed to associate with intact FVaL upon rec alcification, indicating that the Leu-94 to Lys-109 region of the A1 domain plays a critical role in the FVaL and FVaH Ca2+ dependent association. By using domain-specific monoclonal antibodies and an assay for thrombin gener ation, loss of FVa prothrombinase function was coincident with proteolysis at sites in the A2 and A3 domains resulting in their dissociation. Inactiva tion of FV or FVa by Pn was independent of the thrombophilic R506Q mutation . These results identify the molecular composition of Pn-cleaved FVa that r emains bound to membrane as largely A1-C1/C2 in the presence of Ca2+ and su ggest that Pn inhibits FVa by a process involving A2 and A3 domain dissocia tion.