Synthesis and ultrastructural localization of protein C inhibitor in human platelets and megakaryocytes
Synthesis and ultrastructural localization of protein C inhibitor in human platelets and megakaryocytes
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DOI：
10.1182/blood.v94.4.1300.416k26_1300_1312
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发表时间：
1999-08-15
期刊：
影响因子：
20.3
通讯作者：
Geiger, M
中科院分区：
文献类型：
--
作者：
Prendes, MJ;Bielek, E;Geiger, M
分析了人血小板和巨核细胞中蛋白C抑制剂（PCI）的发生情况。通过酶联免疫吸附试验（elisa）判断，PCI以160 ng/(2) × 10(9)个细胞的浓度存在于血小板中。单克隆抗PCI- igg免疫沉淀血小板裂解液，用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western blotting分析血小板裂解液时，主要检测到57-kD形式（活性PCI）和一些高分子量（M-r,）形式，但未检测到与裂解PCI对应的条带。通过免疫荧光组织化学和免疫透射电镜研究PCI在血小板中的定位：PCI在颗粒、开放小管系统和质膜中检测到。在这些位点，可见与纤溶酶原激活物抑制剂-1共定位。进行研究以澄清血小板PCI是内源性合成还是从血浆中吸收。采用血小板悬液和金标记链霉亲和素可视化方法分析生物素化pci的内化情况。发现PCI的剂量和时间依赖性摄取。通过逆转录聚合酶链反应（RT-PCR）和Southern印迹法在血小板中检测到PCI mRNA，并通过人骨髓冷冻原位杂交在巨核细胞中检测到PCI mRNA。因此，我们得出结论，血小板含有一个功能活跃的PCI库，该库来源于内源性合成和内化。(C) 1999年由美国血液病学会出版。
The occurrence of protein C inhibitor (PCI) in human platelets and megakaryocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELISAs), PCI was present in platelets at a concentration of 160 ng/(2) x 10(9) cells. Its specific activity was 5 times higher than that of plasma PCI, Consistently, mainly the 57-kD form (active PCI) and some high molecular weight (M-r,) forms, but no bands corresponding to cleaved PCI, were detected when platelet lysates were immunoprecipitated with monoclonal anti-PCI-IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The localization of PCI in platelets was studied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in a granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminogen activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internalization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- and time-dependent uptake of PCI was found. PCI mRNA was detected in platelets by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as In megakaryocytes by in situ hybridization of human bone marrow cryosections, We therefore conclude that platelets contain a functionally active PCI pool that is derived from both endogenous synthesis as well as internalization. (C) 1999 by The American Society of Hematology.