STRUCTURE AND FUNCTION OF PLATELET MEMBRANE GPIIB/IIIA

已知血小板聚集在止血和止血中起关键作用 血栓形成。相当多的文献表明，血小板聚集是 纤维蛋白原依赖的过程，以及血小板细胞表面 糖蛋白IIb和IIIa密切参与 血小板细胞表面纤维蛋白原受体。已经证明，当 这些糖蛋白是用非离子从血小板膜上提取出来的。 洗涤剂，它们以依赖钙的复合体的形式存在，可被 EDTA的作用。还有证据表明，GPIIb/GPIIIa 复杂的形成可以发生在血小板表面，而形成 这一复合体可能是一个受调控的事件。我们已经培育出4株杂交瘤。 与GPIIb和GPIIa反应的抗体。初步实验 强烈提示这些杂交瘤中有3个是针对新抗原的。 由GPIIb与GPIIIa在一个 钙依赖复合体，与任何一种解离的 糖蛋白。此外，这些杂交瘤中有两个完全抑制 ADP诱导的血小板聚集。鉴于这些潜在令人兴奋的事情 属性，我们将直接确定这3个杂交瘤是否 通过研究它们与GPIIb/GPIIa复合体结合的能力 通过交叉反应制备这些糖蛋白的解离或络合形式 免疫电泳法。如果它们是复杂特定的，我们将使用它们作为 研究血小板表面GPIIb/IIIa复合体形态的探讨 通过检测杂交瘤细胞与静息和 激活血小板。我们还将确定GPIIb/GPIIa是否通过使用纯化 我们的杂交瘤能够结合纤维蛋白原。我们还将确定 GPIIb/GPIIa复合体的哪些亚区可能调节复合体 通过蛋白水解性产生的片段形成或结合纤维蛋白原 构建了GPIIb/GPIIa复合体，并鉴定了能与其结合的片段 复杂的特异性杂交瘤或纤维蛋白原。最后，用这些杂交瘤 我们已经开发出一种化验方法，证明了 部分ITP患者血浆中抗GPIIb/GPIIa抗体的检测我们 将利用这一检测方法来表征这种 抗体。

Platelet aggregation is known to play a crucial role in hemostasis and thrombosis. Considerable literature suggests that platelet aggregation is a fibrinogen-dependent process, and that platelet cell-surface glycoproteins IIb and IIIa are intimately involved in the formation of the platelet cell-surface fibrinogen receptor. It has been shown that when these glycoproteins are extracted from the platelet membrane with nonionic detergents, they exist as a calcium-dependent complex, dissociable by the action of EDTA. There is also evidence to suggest that GPIIb/GPIIIa complex formation can occur on the platelet surface, and that formation of this complex may be a regulated event. We have produced 4 hybridoma antibodies reactive with GPIIb and GPIIIa. Preliminary experiments strongly suggest that 3 of these hybridoma are specific for new antigenic determinants created by the association of GPIIb with GPIIIa in a calcium-dependent complex, being unreactive with either dissociated glycoprotein. Additionally, 2 of these hybridomas totally inhibit ADP-induced platelet aggregation. In view of these potentially exciting properties, we will directly determine if these 3 hybridomas are GPIIb/GPIIIa complex-specific by studying their ability to bind to the dissociated or complexed forms of these glycoproteins prepared by crossed immunoelectrophoresis. If they are complex specific, we will use them as probes to study the appearance of the GPIIb/IIIa complex on the platelet surface by examining the ability of the hybridomas to bind to resting and activate platelets. We will also determine if GPIIb/GPIIIa purified by use of our hybridomas are able to bind fibrinogen. We will also determine which subregions of the GPIIb/GPIIIa complex might mediate complex formation or fibrinogen binding by proteolytically generating fragments of the GPIIb/GPIIIa complex, and characterize the fragments able to bind to complex-specific hybridomas or fibrinogen. Finally, with these hybridomas we have developed an assay which has demonstrated the presence of antibodies to GPIIb/GPIIIa in the plasma of some patients with ITP. We will use this assay to characterize the frequency and specificity of such antibodies.