MOLECULAR BIOLOGY OF THE THROMBOCYTE FIBRINOGEN RECEPTOR

这项研究计划的长期目标是获得 进一步了解识别位点的身份和性质 对RGD肽和H12的IIb-IIIa，以表征相互作用 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，并进一步了解的性质， 由血小板引发的IIb-IIIa的构象变化 刺激. 为了解决这些问题，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， 确定特异性、亲和性和 依赖于IIb-IIIa激活。 Op-G2和CP 3与PAC-1一样，含有 H3中的序列赖德，从而模拟含RGD的蛋白质配体。 与PAC-1不同，OP-G2和CP 3与未活化血小板中的IIb-IIIa结合。 比较这三种抗体的序列和结构， 以及许多其他含有类似序列的抗体， H3但不能与IIb-IIIa结合是结构模型的基础， 可用于研究IIb识别RGD或H12的机制- 三 其他抗体，例如，AP 2使用替代结合基序， 抑制纤维蛋白原结合和血小板聚集。 分子 这种抗体的特异性的表征可以阐明 IIb-IIIa上的替代识别位点。 2)为了表征由AP 2、OP-G2和其他蛋白识别的表位， 在IIb-IIIa识别处或附近结合的鼠单克隆抗体 网站. 我们认为这种构象的鉴定- 受限制的“复合物依赖性”表位将提供重要的 关于IIb-IIIa复合体的地形和 它的RGD和H12识别“口袋”的结构。“这个具体的 由于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 诱变以确认反应位点的鉴定。 我们提出 以适当的表达形式表达β 3整联蛋白构建体 系统 重组整联蛋白将用作抗原来源， 表位同一性的确认。 这些研究的成功完成将有助于我们 了解特异性、亲和力和 与RGD和十二肽结合的配体的活化依赖性 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.