Human platelet factor V is crosslinked to actin by FXIIIa during platelet activation by thrombin.

Although it has been established that factor V (FV) becomes associated irreversibly with the platelet cytoskeleton after stimulation with thrombin, the chemical nature of this complex is not known. Factor V has recently been demonstrated to be a substrate for factor XIIIa and to form factor V oligomers. We now show that thrombin-activated 125I-FV specifically links to a single protein (43 kDa) of the solubilized platelet membrane in a reaction which requires Ca++ and factor XIIIa. In a purified system, FV, activated by thrombin, forms covalently linked high molecular complexes with 125I-actin catalyzed by factor XIIIa. The site of crosslinking of actin was the factor V fragments, 150 kDa (connecting peptide, C1) and its parent molecule 200 kDa (B). Using radiolabeled actin and unlabeled FV, factor XIIIa catalysed the formation of both homopolymers and heteropolymers. Unlabeled actin was found to compete with radiolabeled FV as a substrate for FXIIIa. To evaluate the biological significance of the crosslinking of factor V to actin, intact platelets were treated with B10 (monoclonal antibody to C1), or monospecific polyclonal antibodies to actin or FXIII. After stimulation with thrombin, the cytoskeleton (material insoluble in Triton X-100) showed markedly decreased 125I-FV in the crosslinked complexes. FV coagulant activity associated with platelet cytoskeleton was also diminished following incubation with an antibody to actin, factor XIII, or B10. These data suggest that FV, through the C1 domain, is crosslinked to actin in the cytoskeleton of thrombin-treated platelets. Activated factor XIII may play a role in plasma FV-platelet interaction as well as the expression of FV derived from the alpha-granules on the cytoskeleton during platelet stimulation.

Biology of human megakaryocyte factor V.

To learn more about human megakaryocyte coagulation cofactor V (FV), we studied the expression of this protein in normal bone marrow megakaryocytes and in megakaryocytes cloned from their colony-forming unit in FV-depleted plasma clot cultures. Mouse monoclonal antibodies directed against either the light chain or an activation peptide of human FV and a rabbit polyclonal, monospecific FV antiserum were used as probes for these experiments in conjunction with a variety of immunochemical detection techniques. All morphologically recognizable megakaryocytes were shown to contain FV. The origin of this protein appeared to be both from FV bound to the cell as well as from endogenous FV in the majority of cells examined. The existence of a population of small bone marrow mononuclear cells that simultaneously expressed platelet glycoproteins and FV was also noted. Such cells represented approximately 70% of all small cells positive for platelet glycoproteins. In contrast, only about 40% of megakaryocyte colonies cloned in FV-deficient medium contained cells with immunochemically detectable FV. FV expression was most clearly demonstrated in large cells in the colonies, whereas smaller, presumably less mature cells labeled weakly or not at all. Synthesis of FV by human megakaryocytes was documented using elutriation-enriched cells incubated in 35S-methionine-containing medium. Megakaryocyte lysates and medium conditioned by these cells were subjected to immunoaffinity column purification. Column eluates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed radioactive bands comigrating with the heavy and light chains of thrombin-activated FV. These studies suggest that human megakaryocytes both bind and synthesize FV. Expression of these traits appears to be related to cell maturation, with binding ability appearing earlier than the ability to synthesize this protein. Finally, although the ability to bind FV appears to be universal among megakaryocytes, our culture data suggest that synthesis may be a restricted, or constitutively expressed property of these cells.

Epitope mapping of functional domains of human factor Va with human and murine monoclonal antibodies. Evidence for the interaction of heavy chain with factor Xa and calcium.

We have purified a unique neutralizing IgG1, kappa monoclonal antibody (MAb) against factor V (F-V) from a patient's plasma. This MAb (H2) demonstrated specificity for human F-V heavy chain (D), mol wt 105,000. Using an enzyme-linked immunosorbent assay (ELISA) we assessed the competitive binding to F-Va of H2, H1 (human MAb directed to light chain, F1F2), and two murine MAbs, B38 (to F1F2) and B10 (to activation peptide C1). All four antibodies are of high affinity with KD varying from 0.17 to 1.17 X 10(-10) mol/L. They recognized distinct epitopes in F-V. F-Xa competed in a concentration-dependent fashion for binding of H1, H2, and B38 but not B10 to F-V/Va in the absence of phospholipids or platelets. Thus both F1F2 and D polypeptides of F-Va but not C1 interacted with F-Xa. All MAbs bound to F-V/Va in the absence of Ca++. However, free Ca++ (0.1 to 4.0 mmol/L) increased the amount of H1 and H2 bound to factor V/Va, 1.65-fold and 3.65-fold, respectively but had little effect on the binding of either murine MAbs. Prothrombin (20 micrograms/mL to 400 micrograms/mL) in the absence of phospholipid did not inhibit the binding of MAbs. These studies provide evidence for the first time for a direct interaction between human F-Va heavy chain and F-Xa and Ca++ and for the direct binding of F-Xa to F-Va in the absence of phospholipids or platelets and enhance our understanding of functional F-V domains.

Expression of factor V on human umbilical vein endothelial cells is modulated by cell injury.

Since human endothelial cells synthesize Factor V but do not secrete it into the medium, we studied the effects of cell injury on the availability of Factor V at the surface of these cells. Human umbilical vein endothelial cells (HUVEC), grown to confluency and incubated with human 125I Factor Va, specifically bound 5000 to 7000 molecules per cell. In the absence of added Va, no antigen was detected on adherent HUVEC with either labeled anti-V(Va) monoclonal or polyclonal IgG. However, exogenous Va, not V, prebound to these cells allows binding of labeled 125I anti-V(Va). Immunodectectibility of bovine Factor V contributed by fetal calf serum in the concentration used in cultures is less than 0.1% of that detected in human plasma. HUVEC, suspended by scraping from dishes, specifically bound 4000 molecules/cell of 125/I monoclonal IgG against V(Va). Although undisturbed cells excluded trypan blue, dye uptake by many of the suspended HUVEC indicated cell injury. Quantitation of injury by 51Cr release after scraping followed by multiple passages through an 18 g needle showed that 51Cr release increased with number of manipulations up to 60% and was observed almost immediately after manipulation. We suggest that little Factor V(Va) is present on the surface of intact adherent HUVEC. However, mechanical injury to HUVEC released or exposed endogenous Factor V(Va), resulting in expression of V that might mediate Factor Xa binding as well as activation of protein C by thrombin. Thus, injured, but not intact, HUVEC could participate in both promoting and limiting blood coagulation.