MOLECULAR BIOLOGY OF THE THROMBOCYTE FIBRINOGEN RECEPTOR

描述：（改编自研究者摘要）血小板 整合素α IIb-β 3是识别RGD的七种整合素之一 母题 当血小板被激活时，α IIb-β 3发生变化， 在构象，使这种整合素更容易接近RGD 配体。 申请人已经产生了单克隆抗体AP 7， 在其重链（H3）的第三个CDR中含有RGD序列。 这种抗体结合到静止和重组状态的 整合素，尽管与后者的亲和力高五倍。 AP7.3， 其中AP 7 H3环已被相应序列取代 从PAC 1（另一种仅与重组整联蛋白结合的抗体）， 对重组整联蛋白表现出增加的选择性， 在所有方面都相同的整体亲和力的降低 重组PAC 1 Fab片段本身的行为。 AP7.3和 PAC 1仅结合活化的细胞表面上的整合素的一个子集。 血小板 这项研究的目的是为了更好地了解 重组敏感的RGD识别的分子基础， 整合素，并建立亚群的生物学意义 整合素 为了实现这些目标，申请人将使用 作为RGD配体的AP 7的工程化衍生物和重组的，可溶的， 作为受体的人α IIb-β 3异二聚体的功能衍生物。 将追求四个具体目标：1）确定H3中的特征 AP7.3的环，允许其选择性地结合活化形式 2）确定α IIb-β 3中结合 AP7.3; 3）确定为什么只有α IIb-β 3亚群结合 AP7.3和其他活化依赖性抗体;和4）确定 AP 7/alphaIIb-beta3复合物的中间分辨率结构， 三维电子晶体学 申请人建议， α IIb-β 3和AP 7的Fab片段的分子特征 一系列的抗体，参与重组敏感的 相互作用将适用于所有含RGD的配体，包括 肽模拟物

DESCRIPTION: (Adapted from investigator's abstract) The platelet integrin alphaIIb-beta3 is one of seven integrins that recognize the RGD motif. When platelets are activated, alphaIIb-beta3 undergoes a change in conformation that renders this integrin more accessible to RGD ligands. The applicant has generated a monoclonal antibody AP7 that contains the RGD sequence in the third CDR of its heavy chain (H3). This antibody binds to both quiescent and reconformed states of the integrin, albeit with five-fold higher affinity to the latter. AP7.3, in which the AP7 H3 loop has been replaced by the corresponding sequence from PAC1 (another antibody that binds only to the reconformed integrin), exhibits an increased selectivity for reconformed integrin and a decrease in overall affinity that is identical in all respects to the behavior of recombinant PAC1 Fab fragments themselves. Both AP7.3 and PAC1 bind only a subset of integrin on the surface of activated platelets. The goal of the proposed research is to better understand the molecular basis of reconformation-sensitive RGD recognition by the integrin and to establish the biological significance of subpopulations of integrin. To address these objectives, the applicant will use engineered derivatives of AP7 as RGD ligands and recombinant, soluble, functional derivatives of human alphaIIb- beta3 heterodimers as receptor. Four specific aims will be pursued: 1) to define features in the H3 loop of AP7.3 that allow it to selectively bind to the activated form of alphaIIb-beta3; 2) to determine the sites in alphaIIb- beta3 that bind AP7.3; 3) to determine why only a subpopulation of alphaIIb- beta3 binds AP7.3 and other activation-dependent antibodies; and 4) to determine the intermediate resolution structure of AP7/alphaIIb-beta3 complexes by two- dimensional electron crystallography. The applicant suggests that molecular features in alphaIIb-beta3 and the Fab fragments of the AP7 series of antibodies that are involved in the reconformation-sensitive interaction will be applicable to all RGD-containing ligands, including peptidomimetics.