Jennings L K, Fox C F, Kouns W C, McKay C P, Ballou L R, Schultz H E
Department of Medicine, University of Tennessee, Memphis 38163.
J Biol Chem. 1990 Mar 5;265(7):3815-22.
Anti-human platelet p24/CD9 (p24/monoclonal antibody 7) causes the activation of platelets and in the presence of calcium induces platelet aggregation. Our studies suggest that platelet response to this antibody is mediated at least in part by the pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) that stimulate phosphoinositide hydrolysis and inhibit adenylate cyclase. Prior exposure of saponin-treated platelets to anti-p24/CD9 inhibited the [32P] ADP-ribosylation of the alpha 41 protein by pertussis toxin. Platelet aggregation induced by this antibody is preceded by and/or accompanied by accelerated phosphatidylinositol turnover, the generation of inositol phosphates and diacylglycerol (DAG), calcium mobilization, and protein phosphorylation. The production of inositol phosphate(s) was measurable within 15 s of either anti-p24/CD9 or thrombin addition. Within 10 s of antibody addition (10 micrograms/ml), the level of DAG was 200% over that of the control and similar to that observed with 2 units/ml thrombin (201% over that of the control). Therefore, as it appears to be true for thrombin, platelet response upon binding of anti-p24/CD9 is primarily mediated by the activation of phospholipase C. When platelets pretreated with aspirin (200 microM) and apyrase (1 mg/ml) were subsequently exposed to anti-p24/CD9, aggregation still occurred. This indicates that neither secreted ADP nor thromboxane generation is required for this aggregation response. Using indo-1 and ratio cytofluorometry, we observed that an increase in platelet cytosolic calcium is a relatively early event and occurs in either the presence or absence of calcium in the external media. Phosphorylation studies of platelet proteins showed that anti-p24/CD9 binding to platelets caused increased phosphorylation of four proteins with apparent molecular masses of 50,000, 47,000, 36,000, and 20,000 daltons. These studies suggest that platelet activation mediated by the surface protein p24/CD9 is mainly through the stimulation of a phospholipase C, the activation of which is responsible for the generation of second messengers inositol trisphosphate and DAG.
抗人血小板p24/CD9（p24单克隆抗体7）可引起血小板活化，且在有钙存在的情况下诱导血小板聚集。我们的研究表明，血小板对该抗体的反应至少部分是由百日咳毒素敏感的鸟嘌呤核苷酸结合蛋白（G蛋白）介导的，这些G蛋白可刺激磷酸肌醇水解并抑制腺苷酸环化酶。用皂苷处理过的血小板预先暴露于抗p24/CD9可抑制百日咳毒素对α41蛋白的[32P] ADP核糖基化作用。该抗体诱导的血小板聚集之前和/或伴随着磷脂酰肌醇周转加速、肌醇磷酸和二酰基甘油（DAG）的生成、钙动员以及蛋白质磷酸化。在加入抗p24/CD9或凝血酶后的15秒内即可检测到肌醇磷酸的产生。在加入抗体（10微克/毫升）后的10秒内，DAG水平比对照高出200%，与加入2单位/毫升凝血酶时观察到的情况相似（比对照高出201%）。因此，就像凝血酶的情况一样，抗p24/CD9结合后血小板的反应主要是由磷脂酶C的激活介导的。当用阿司匹林（200微摩尔）和腺苷三磷酸双磷酸酶（1毫克/毫升）预处理过的血小板随后暴露于抗p24/CD9时，仍会发生聚集。这表明这种聚集反应既不需要分泌的ADP也不需要血栓素的生成。使用indo-1和比率细胞荧光测定法，我们观察到血小板胞质钙的增加是一个相对较早的事件，且在细胞外介质中有钙或无钙的情况下均会发生。血小板蛋白的磷酸化研究表明，抗p24/CD9与血小板结合会导致四种表观分子量分别为50,000、47,000、36,000和20,000道尔顿的蛋白磷酸化增加。这些研究表明，由表面蛋白p24/CD9介导的血小板活化主要是通过刺激磷脂酶C，其激活负责第二信使肌醇三磷酸和DAG的生成。
