Symer D E, Paznekas W A, Shin H S
Department of Molecular Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Exp Med. 1993 Apr 1;177(4):937-47. doi: 10.1084/jem.177.4.937.
Platelets are potent antibody- and complement-dependent cytotoxic effector cells. We showed previously that a single platelet can lyse a target cell sensitized with immunoglobulin G (IgG) and complement components up to C3 (C integral of 3b denotes the target cell-bound fragment of complement up to C3; the precise nature of the bound C3 fragment has not been established), and that the complete cytotoxic system capable of specific recognition and lysis resides in platelet membranes. To define the components of platelet membranes required for cytotoxicity, a set of inhibitors of phospholipase A2 (PLA2) that act by different chemical mechanisms was tested. The lytic reaction is blocked at appropriate concentrations of bromophenacylbromide, mepacrine, and manoalide. When platelets are treated with bromophenacylbromide, inhibition of cytolytic activity and that of PLA2 enzymatic activity occur in parallel. Platelets release arachidonate when incubated with target cells bearing IgG and C integral of 3b, confirming that Fc gamma R and complement receptor trigger both PLA2 action and efficient lysis. Inhibition by thimerosal of a reverse reaction, i.e., reacylation catalyzed by acyltransferase, causes increased target cell lysis, presumably by increasing the products of PLA2 action. Platelet cytotoxicity is increased when platelets are pretreated with some products of PLA2: exogenous lysophospholipids and not free arachidonic acid increase cytotoxicity. Electron microscopy suggests that platelets and target cells may fuse, possibly as a result of the formation of lysophospholipids which are well-known membrane fusogens. Fixation with paraformaldehyde does not affect platelet cytotoxicity, suggesting that the complete cytotoxic system resides as a preformed complex in platelet membranes. The results indicate that platelet membrane-associated PLA2, together with receptors for Fc and complement, are required for platelet cytotoxicity.
血小板是强大的抗体和补体依赖性细胞毒性效应细胞。我们之前表明，单个血小板能够裂解用免疫球蛋白G（IgG）和补体成分直至C3致敏的靶细胞（C3b表示靶细胞结合的补体片段直至C3；结合的C3片段的确切性质尚未确定），并且能够进行特异性识别和裂解的完整细胞毒性系统存在于血小板膜中。为了确定细胞毒性所需的血小板膜成分，测试了一组通过不同化学机制起作用的磷脂酶A2（PLA2）抑制剂。在适当浓度的溴苯甲酰溴、米帕林和 manoalide 作用下，裂解反应被阻断。当血小板用溴苯甲酰溴处理时，细胞溶解活性的抑制和PLA2酶活性的抑制同时发生。当血小板与携带IgG和C3b的靶细胞一起孵育时，血小板会释放花生四烯酸，这证实FcγR和补体受体触发了PLA2的作用和有效的裂解。硫柳汞对反向反应（即由酰基转移酶催化的再酰化反应）的抑制作用会导致靶细胞裂解增加，这可能是通过增加PLA2作用的产物实现的。当血小板用PLA2的一些产物预处理时，血小板细胞毒性会增加：外源性溶血磷脂而不是游离花生四烯酸会增加细胞毒性。电子显微镜显示血小板和靶细胞可能会融合，这可能是由于形成了众所周知的膜融合剂溶血磷脂。用多聚甲醛固定不会影响血小板的细胞毒性，这表明完整的细胞毒性系统以预先形成的复合物形式存在于血小板膜中。结果表明，血小板膜相关的PLA2以及Fc和补体受体是血小板细胞毒性所必需的。
