Although platelets contain Factor V, localized primarily in the a-granules, the origin of this coagulation cofactor in these cells is not known. We therefore explored whether isolated megakaryocytes could biosynthesize Factor V. Guinea pig plasma Factor V coagulant activity was demonstrated to be neutralized by human monoclonal and rabbit polyclonal antibodies directed monospecifically against human Factor V. These antibodies had been used earlier to purify human Factor V and to quantify Factor V antigen concentration, respectively (1983. Chin, H. C., E. Whitaker, and R. W. Colman. J. Clin. Invest. 72:493-503). As determined by a competitive enzyme-linked immunosorbent assay with guinea pig plasma as a standard, Factor V solubilized from guinea pig megakaryocytes was present at 0.098±0.018 gg/105 cells. Each megakaryocyte contained about 500 times as much Factor V as is in a platelet (0.234±0.180 gig/108 platelets). The content of Factor V antigen in guinea pig plasma was greater (27.0±3.0 gig/ml) than that of Factor V antigen in human plasma (11.1±0.4 gig/ ml). In contrast, human platelets contain ninefold more Factor V antigen (2.01±1.09 gg/108 platelets) than do guinea pig platelets. The Factor V coagulant activities in the guinea pig were 2.85±030 U/ml plasma, 0.022±0.012 U/108 platelets, and 0.032±0.03 U/105 megakaryocytes, compared with human values of 0.98±0.02 U/ml plasma and 0.124±0.064 U/1O8 platelets. Isolated megakaryocytes were found to contain Factor V by cytoimmunofluorescence. The megakaryocytes were incubated with V35Sjmethionine, and radiolabeled intracellular proteins purified were on a human anti-Factor V immunoaffinity column. The purified protein exhibited Factor V coagulant activity and neutralized the inhibitory activity of a rabbit antihuman Factor V antibody, which suggests that megakaryocyte Factor V is functionally and antigenically intact. These results indicate that Factor V is synthesized by guinea pig megakaryocytes. Nonetheless, megakaryocyte Factor V was more slowly activated by thrombin and in the absence of calcium was more stable after activation than was plasma Factor Va. Electrophoresis in sodium dodecyl sulfate and autoradiography of the purified molecule showed a major band of Mr 380,000 and a minor band of Mr 350,000, as compared with guinea pig and human plasma Factor V, where the protein had an Mr of

A B S T R A C T Functional human Factor V has been purified using a rapid immunoaffinity method. Following barium citrate adsorption of plasma, Factor V was precipitated with polyethylene glycol at a concentration between 5 and 14%. The resulting preparation was applied to a column containing an immobilized immunoadsorbent consisting of an IgG fraction containing a naturally occurring human monoclonal (IgG4 X) antibody with inhibitory activity against human Factor V. The solid phase immunoglobulin quantitatively bound Factor V from human plasma. The bound Factor V was effectively eluted with a Tris buffer pH 7.2 containing 1.2 M NaCl and 1 M a-methyl-D-mannoside. The isolated native Factor V with high specific activity (92 U/mg) showed a single band (Mr, 350,000) on both reduced and nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Factor V was purified 5,100-fold over plasma with an overall yield of 77%. The purified Factor V when subjected to thrombin activation exhibited an 18-fold increase in coagulant activity.The isolated Factor V neutralized the inhibitory activities of the monoclonal antibody that was used to purify it, as well as the rabbit antibodies produced by immunizing the animals with the purified Factor V. Immunoelectrophoresis of purified Factor V against the polyclonal rabbit antiserum resulted in a single precipitin arc of identical mobility to the Factor V in normal human plasma. Analysis by double immunodiffusion showed a line of identity between plasma and purified Factor V and crossed immunoelectrophoresis showed a single species in normal plasma.A competitive enzyme-linked immunosorbent assay using the rabbit antibody against Factor V was applied to quantify Factor V antigen level in human plasma. Reconstitution of congenitally deficient or immunodepleted plasma with normal plasma or purified Factor V gave parallel dose-response curves. In 14 normal plasma the coagulant activity was 0.98±0.02 U/ml (mean±SEM) and antigen concentration was 11.1±0.4 ,lg/ml. A pool of 14 patients with congenital Factor V deficiency were studied. 10 patients had Factor V antigen ranging from 1.0 to 2.4 jg/ml with corresponding coagulant activities (0-0.17 U/ml) indicating a low concentration of normal Factor V, presumnably due to decreased synthesis or increased degradation. When these patient plasmas and the normal plasmas were analyzed together an excellent correlation (r = 0.97, P < 0.01) was obtained. However, four patients with coagulant activity (0-0.08 U/ml) had Factor V antigen concentrations ranging from 4.4 to 6.1 ,ug/ml, indicating the presence of a reduced concentration of abnormal Factor V protein. The presence of patients with antigen similar in concentration to coagulant activity and antigen in excess of Factor V activity indicates the heterogeneity of congenital Factor V deficiency.

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.

Thrombin stimulates prostacyclin formation in cultured human endothelial cells. However, a countervailing process that prevents prostacyclin overproduction has not been described previously. In this study, we demonstrate that Factor Xa can inhibit prostacyclin synthesis induced by thrombin or sodium arachidonate. The required concentration of Factor Xa represents activation of only 2% of the Factor X in plasma. The inhibition is reversed by a human monoclonal antibody directed against the light chain of Factor Va (Mr = 78,000), which suggests that Factor Va is required for this down-regulation of prostacyclin production. Confluent human endothelial cells (10(7)) contained 1.4 to 2.2 micrograms of Factor V antigen as measured by a competitive enzyme-linked immunosorbent assay. The results indicate that in endothelial cells Factor Xa may play a regulatory role in prostacyclin formation through interaction with Factor Va.

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