MOLECULAR BIOLOGY OF THE THROMBOCYTE FIBRINOGEN RECEPTOR

这项研究计划的长期目标是获得 进一步洞察表彰现场的身份和性质(S) RGD多肽与H12相互作用的IIb-IIIa研究 在这些可能支持纤维蛋白原结合的识别位点之间 和血小板聚集，并进一步了解 由血小板引发的IIb-IIIa构象变化(S) 刺激。 针对这些问题，我们制定了三个具体目标： 1)开发和表征基于抗体的配体的原型和 确定特定、亲和力和 依赖IIb-IIIa激活。OP-G2和CP3和PAC-1一样，含有 序列Ryd在H3中，从而模仿含有RGD的蛋白质配体。 与PAC-1不同，OP-G2和CP3在未激活的血小板中与IIb-IIIa结合。 这三种抗体的序列和结构比较 以及许多其他包含相似序列的抗体 H3但未能绑定到IIb-IIIa是结构模型的基础 可用于研究IIb-识别RGD或H12的机制 IIIA。其他抗体，例如AP2，使用替代结合基序来 抑制纤维蛋白原结合和血小板聚集。分子 对这些抗体的特异性进行表征可能会阐明 IIb-IIIa上的替代识别站点。 2)鉴定AP2、OP-G2等识别的表位 结合在IIb-IIIa识别或接近IIb-IIIa识别的鼠单抗 网站。我们认为这种构象的识别- 受限制的、“依赖于络合物的”表位将提供重要的 对IIb-IIIa建筑群的地形和 其架构的RGD和H12识别“口袋”。这个特定的 AIM现在是可行的，因为我们的重组抗体表达 系统。 3)利用重组IIb-IIIa表达系统和定点 诱变以确认活性部位的鉴定。我们建议 在适当的表达中表达Beta3整合素结构 系统。重组整合素将作为抗原来源用于 表位身份的确认。 这些研究的成功完成将有助于我们 了解其特异性、亲和力和亲和力的分子基础 RGD和DODACEP结合配体的激活依赖性 整合素IIb-IIIa的识别位点。

The long-term objectives of this research proposal are to acquire further insight into the identity and nature of the recognition site(s) on IIb-IIIa for RGD peptides and H12, to characterize interactions between these recognition sites that might support fibrinogen binding and platelet aggregation, and to further understand the nature of the conformational change(s) of IIb-IIIa that is initiated by platelet stimulation. To address these questions, we have formulated three specific aims: 1) To develop and characterize prototypes of antibody-based ligands and to identify key structural requirements for specific, affinity and dependence upon IIb-IIIa activation. Op-G2 and CP3, like PAC-1, contain the sequence RYD in H3 and thus mimic RGD-containing protein ligands. Unlike PAC-1, OP-G2 and CP3 bind to IIb-IIIa in nonactivated platelets. A comparison of the sequence and structure of these three antibodies as well as a number of other antibodies which contain similar sequences in H3 but fail to bind to IIb-IIIa is a basis for structural models that can be used to study the mechanics of RGD or H12 recognition by IIb- IIIa. Other antibodies, e.g., AP2, uses alternative binding motifs to inhibit fibrinogen binding and platelet aggregation. The molecular characterization of the specificity of such antibodies may elucidate alternative recognition sites on IIb-IIIa. 2) To characterize the epitopes recognized by AP2, OP-G2 and other murine monoclonal antibodies that bind at or near IIb-IIIa recognition sites. We feel that the identification of such conformationally- constrained, "complex-dependent" epitopes will provide important insights about the topography of the IIb-IIIa complex and the architecture of its RGD and H12 recognition "pockets." This specific aim is now feasible because of our recombinant antibody expression system. 3) To utilize recombinant IIb-IIIa expression systems and site-directed mutagenesis to confirm the identification of reactive sites. We propose to express beta3 integrin constructs in an appropriate expression system. Recombinant integrins will be used as a source of antigens for confirmation of epitope identity. The successful completion of these studies will contribute to our understanding of the molecular basis for the specificity, affinity and activation-dependence of ligands which bind to RGD and dodacepeptide recognition sites of the integrin IIb-IIIa.