Gewirtz A M, Keefer M, Doshi K, Annamalai A E, Chiu H C, Colman R W
Blood. 1986 Jun;67(6):1639-48.
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.
为了更深入了解人巨核细胞凝血辅因子V（FV），我们研究了该蛋白在正常骨髓巨核细胞以及在FV缺失的血浆凝块培养物中从其集落形成单位克隆而来的巨核细胞中的表达情况。针对人FV轻链或激活肽的小鼠单克隆抗体以及兔多克隆、单特异性FV抗血清与多种免疫化学检测技术结合用作这些实验的探针。所有形态上可识别的巨核细胞均显示含有FV。该蛋白的来源似乎既来自与细胞结合的FV，也来自大多数所检测细胞中的内源性FV。还注意到存在一群同时表达血小板糖蛋白和FV的小骨髓单核细胞。这类细胞约占所有血小板糖蛋白阳性小细胞的70%。相比之下，在FV缺陷培养基中克隆的巨核细胞集落中，只有约40%的细胞含有免疫化学可检测到的FV。FV表达在集落中的大细胞中最为明显，而较小的、可能不太成熟的细胞标记较弱或根本不标记。使用在含35S - 甲硫氨酸的培养基中孵育的淘析富集细胞记录了人巨核细胞对FV的合成。这些细胞的巨核细胞裂解物和条件培养基经过免疫亲和柱纯化。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和放射自显影分析柱洗脱物，发现放射性条带与凝血酶激活的FV的重链和轻链共迁移。这些研究表明人巨核细胞既能结合又能合成FV。这些特性的表达似乎与细胞成熟有关，结合能力比合成该蛋白的能力出现得更早。最后，虽然结合FV的能力在巨核细胞中似乎普遍存在，但我们的培养数据表明合成可能是这些细胞的一种受限或组成性表达的特性。
