Function and structure of anionic phospholipid-binding proteins: I Factor Va, a coagulation cofactor II p135, a novel protein

Abstract

Coagulation cofactor factor Va (FVa) is an essential component of the prothrombinase complex, and is both produced and inactivated by proteolytic events. The cofactor is a heterodimer noncovalently associated by divalent cations with unknown contact sites. The mechanism of inactivation by the fibrinolytic effector plasmin (Pn) and the Ca2+ binding site in the FVa heavy chain (FVaH) was studied. Following Pn inactivation of aPL-bound FVa, a total of 16 fragments were observed. Of these, the 50(L1766), 48(1766), 43(Q1828), 40(Q1828) kDa fragments spanning the C-terminal C1/C2 domains, and 30(L94) kDa fragment, but not the similar 30(M110) kDa fragment, positioned within the N-terminal A1 domain remained associated with anionic phosphilipid (aPL). These data identify the molecular composition of the Pn-cleaved FVa which remains bound to membrane in the presence of Ca2+ as largely A1-C1/C2. A speculative model that describes the process of inactivation of FVa by Pn was produced. Chelation by EDTA dissociated the 30(L94) kDa fragment, which then associated with intact FVaL in the presence of Ca2+, indicating that the Leu94-Lys109 region of the A1 domain plays a critical role in the FVaL and FVaH Ca2+-dependent association. To identify the residues involved in the Ca2+-binding, conserved amino acids in a recombinant FV (AFV) were replaced with Ala. The following thrombin generating activities were observed: DeltaFV (100%), E96A (19%), D111A (30%), D102A (40%), T104A (65%), E108A (70%), D112A (75%), Y100A (84%), D79A (93%), and E119A (100%). D111A resulted in spontaneous dissociation of FVaH and FVaL. Conversely, E96A or D102A had no apparent effect on Ca2+-dependent subunit interactions, but greatly enhanced chelator-induced dissociation. In addition to the FVa project, a novel human protein (p135) with two synaptotagmin-like C2-domains (sC2) suggestive of Ca2+-dependent phospholipid binding is described. After cloning, sequencing, expression and monoclonal antibody production a preliminary characterization of p135 was conducted. Strong expression of p135 mRNA was observed in some leukemia and adenocarcinoma cell lines. Northern and Western analysis on sixteen non-pathological human tissues suggested a role of p135 in the immune system. The results indicated that the recombinant protein mediate Ca2+-dependent interactions with phosphatidylserine but not with phosphatidylcholine. Thus, p135 is the first member of the important sC2-family of proteins that appears to be preferentially expressed by blood cells.