Nichols W L, Gastineau D A, Solberg L A, Mann K G
Blood. 1985 Jun;65(6):1396-406.
Specific monoclonal and polyclonal antibody reagents and a double antigen indirect immunofluorescence microscopy technique were used to visualize coagulation factor V in human bone marrow. Marrow aspirates were smeared directly on glass slides, or washed and cytospun onto glass slides, or processed and plated into a plasma/methylcellulose cell culture system. Morphologically identifiable colonies of megakaryocytes, erythrocytes, granulocytes, or monocytes/macrophages were removed from 14- to 18-day marrow culture dishes by micropipette and streaked onto glass slides. Smears of marrow cell preparations were air-dried, fixed, washed, and incubated sequentially with primary IgG antibody reagents and with secondary anti-IgG antibody reagents conjugated with either fluorescein or rhodamine. Preparations were examined and photographed through a microscope suitably equipped for two-color fluorescence and phase contrast analysis. Cells of megakaryocytic lineage were identified by their immunofluorescent reactivity with murine monoclonal antibody HP1-1D, specific for human platelet plasma membrane glycoprotein IIb/IIIa (GP IIb/IIIa), or by their immunofluorescent reactivity with monoclonal or polyclonal antibodies specific for von Willebrand factor (vWF) or for platelet factor 4 (PF4). Coagulation factor V in bone marrow was detected by simultaneous immunofluorescent staining with polyclonal burro anti-human factor V antibody or with a panel of murine monoclonal anti-human factor V antibodies. The double antigen immunofluorescence staining technique, incorporating appropriate controls, revealed that coagulation factor V was principally located in marrow cells simultaneously identified as megakaryocytes by antibodies to GP IIb/IIIa, vWF, or PF4. The specific immunofluorescence of factor V in megakaryocytes and platelets was eliminated when excess purified factor V antigen was preincubated with anti-factor V antibody. Our observations establish the presence of human megakaryocyte coagulation factor V, confirm the presence of human platelet factor V, and indicate that human megakaryocyte/platelet coagulation factor V is a lineage-associated protein.
使用特异性单克隆和多克隆抗体试剂以及双抗原间接免疫荧光显微镜技术来观察人骨髓中的凝血因子V。骨髓穿刺液直接涂抹在载玻片上，或洗涤后通过细胞离心涂片在载玻片上，或进行处理并接种到血浆/甲基纤维素细胞培养系统中。在14至18天的骨髓培养皿中，通过微量移液器去除形态学上可识别的巨核细胞、红细胞、粒细胞或单核细胞/巨噬细胞集落，并划线接种到载玻片上。骨髓细胞制备涂片经空气干燥、固定、洗涤，然后依次与原发性IgG抗体试剂以及与荧光素或罗丹明偶联的继发性抗IgG抗体试剂孵育。通过配备有双色荧光和相差分析功能的显微镜对标本进行检查和拍照。巨核细胞系细胞通过其与鼠单克隆抗体HP1-1D（对人血小板血浆膜糖蛋白IIb/IIIa（GP IIb/IIIa）具有特异性）的免疫荧光反应性，或通过其与对血管性血友病因子（vWF）或血小板因子4（PF4）具有特异性的单克隆或多克隆抗体的免疫荧光反应性来鉴定。通过用多克隆驴抗人因子V抗体或一组鼠单克隆抗人因子V抗体进行同步免疫荧光染色来检测骨髓中的凝血因子V。结合适当对照的双抗原免疫荧光染色技术表明，凝血因子V主要位于通过针对GP IIb/IIIa、vWF或PF4的抗体同时鉴定为巨核细胞的骨髓细胞中。当过量纯化的因子V抗原与抗因子V抗体预孵育时，巨核细胞和血小板中因子V的特异性免疫荧光消失。我们的观察结果证实了人巨核细胞凝血因子V的存在，确认了人血小板因子V的存在，并表明人巨核细胞/血小板凝血因子V是一种与谱系相关的蛋白质。
