Kornecki E, Walkowiak B, Naik U P, Ehrlich Y H
Department of Anatomy and Cell Biology, State University of New York Health Science Center, Brooklyn 11203.
J Biol Chem. 1990 Jun 15;265(17):10042-8.
The clinical significance of the interaction of antibodies with circulating platelets is well documented, but the mechanisms underlying these interactions are not fully known. Here we describe the characterization of anti-human platelet membrane protein monoclonal antibody (mAb) termed F11. Interaction of mAb F11 with human platelets resulted in dose-dependent granular secretion, measured by [14C]serotonin and ATP release, fibrinogen binding and aggregation. Analysis of the specific binding of mAb F11 to platelets revealed a high affinity site with 8,067 +/- 1,307 sites per platelet with a dissociation constant (Kd) of 2.7 +/- 0.9 x 10(-8) M. Two membrane proteins of 32,000 and 35,000 daltons, identified by Western blotting, were recognized by mAb F11. Incubation of 32Pi-labeled platelets with mAb F11 resulted in rapid phosphorylation of intracellular 40,000- and 20,000-dalton proteins, followed by dephosphorylation of these proteins. Monovalent Fab fragments or Fc fragments of mAb F11 IgG did not induce platelet aggregation or secretion; however, Fab fragments of mAb F11 IgG blocked mAb F11-induced platelet aggregation and the binding of 125I-mAb F11 to platelets. The addition of an anti-GPIIIa monoclonal antibody (mAb G10), which inhibits 125I-fibrinogen binding and platelet aggregation, completely blocked mAb F11-induced [14C]serotonin secretion and aggregation but not the binding of 125I-mAb F11 to platelets. mAb G10 also inhibited the increase in the phosphorylation of the 40,000- and 20,000-dalton proteins induced by mAb F11. These results implicate the involvement of the GPIIIa molecule in the chain of biochemical events involved in the induction of granular secretion.
抗体与循环血小板相互作用的临床意义已有充分记载，但这些相互作用的潜在机制尚不完全清楚。在此，我们描述了一种名为F11的抗人血小板膜蛋白单克隆抗体（mAb）的特性。mAb F11与人血小板的相互作用导致剂量依赖性颗粒分泌，通过[14C]血清素和ATP释放、纤维蛋白原结合和聚集来衡量。对mAb F11与血小板特异性结合的分析显示，每个血小板有8,067±1,307个高亲和力位点，解离常数（Kd）为2.7±0.9×10（-8）M。通过蛋白质印迹法鉴定出的两种分子量分别为32,000和35,000道尔顿的膜蛋白可被mAb F11识别。用mAb F11孵育32Pi标记的血小板导致细胞内40,000道尔顿和20,000道尔顿蛋白快速磷酸化，随后这些蛋白发生去磷酸化。mAb F11 IgG的单价Fab片段或Fc片段不诱导血小板聚集或分泌；然而，mAb F11 IgG的Fab片段可阻断mAb F11诱导的血小板聚集以及125I-mAb F11与血小板的结合。添加一种抑制125I-纤维蛋白原结合和血小板聚集的抗GPIIIa单克隆抗体（mAb G10），可完全阻断mAb F11诱导的[14C]血清素分泌和聚集，但不影响125I-mAb F11与血小板的结合。mAb G10还抑制了mAb F11诱导的40,000道尔顿和20,000道尔顿蛋白磷酸化的增加。这些结果表明，GPIIIa分子参与了颗粒分泌诱导过程中涉及的一系列生化事件。
