Biology of human megakaryocyte factor V

Abstract

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.

Factor‐V expression in platelets from human megakaryocytic culture

“…Human platelet-factor-V has been considered to derive either from megakaryocytic synthesis and/or from plasma uptake (Gewirtz et al, 1986(Gewirtz et al, , 1992Camire et al, 1998). To elucidate conclusively whether and when platelet-factor-V is synthesized by megakaryocytes, we utilized a serum-free liquid suspension culture for the megakaryocytic differentiation of haematopoietic progenitor cells.…”

“…Other studies suggested that megakaryocytes synthesize factor-V (Chiu et al, 1985;Gewirtz et al, 1986); in particular, factor-V mRNA was identified in human megakaryocytes and platelets by RT-PCR (Gewirtz et al, 1992). Recently, it was found out that murine platelet-factor-V is derived from primary biosynthesis in megakaryocytes, and that murine platelet and plasma-factor-V pools are biosynthetically distinct (Sun et al, 2003;Yang et al, 2003).…”

“…Plasma-factor-V is known to be synthesized in the liver (Wilson et al, 1984). Recently, the origin of human plateletfactor-V has been questioned again (Colman, 1999) in the controversy on the two theories of megakaryocytic synthesis (Gewirtz et al, 1986(Gewirtz et al, , 1992 and plasma uptake (Camire et al, 1998). Two recent studies (Sun et al, 2003;Yang et al, 2003), proved that murine platelet-factor-V is derived entirely from primary biosynthesis in megakaryocytes, with no evidence for significant uptake of factor-V from plasma.…”

Asp68His mutation in the A1 domain of human factor V causes impaired secretion and ineffective translocation

“…The FV protein is encoded by the F5 gene, which has been mapped to human chromosome 1q23 [8]. Previous studies have shown that FV is synthesized in human and guinea pig megakaryocytes [9,10], human Correspondences: (1) Tsun-Mei Lin, PhD, Department of Laboratory Medicine, E-DA Hospital/I-SHOU University, Kaohsiung County, Taiwan Tel: +886-7-6150011-2801; Fax: +886-7-6150925; e-mail: ltmei@mail.ncku.edu.tw (2) Ming-Ching Shen, MD, Hemophilia Treatment and Thrombosis Center, Changhua Christian Hospital, Changhua 500, Taiwan Tel: +886-4-7238589-7246; Fax: +886-4-7228289; e-mail: 111710@cch.org.tw macrophages [11], bovine aortic endothelial cells [12] and the hepatoma cell line, HepG2 [13]. Moreover, several posttranslational modifications are required for FV protein secretion and activity, including disulphide bond formation, N-linked glycosylation, O-linked glycosylation, tyrosine sulphation and phosphorylation [14].…”

“In vitro” correction of the severe factor V deficiency‐related coagulopathy by a novel plasma‐derived factor V concentrate

“…Platelet FV derives from megakaryocytes that can either synthetize 6 or internalize FV from plasma by clathrin-mediated endocytosis. [7][8][9][10] In contrast to plasma FV, platelet FV is stored in a partially activated form and is more resistant to inactivation by APC.…”