DYNAMIC REGULATION OF ADHESIVE FORCES MEDIATED BY AIBB3

整合素AlphaIIbbeta3在细胞凋亡过程中起着关键作用。 血小板血栓的形成，但调节 其对静息和活化血小板的不同作用尚不清楚。 完全理解。这项提议的长期目标是 对粘附力的动态调节的洞察 AlphaIIbbeta3在规定的功能条件下。第一 实验模型将评估受体与表面结合的相互作用 不含剪切力的快速(旋转式)粘合试验中的配基。 另一种方法将直接测量两种材料之间的粘合强度 表达重组αIIbbeta3和表面结合配体的细胞 使用单细胞微吸管系统。最后，它的影响 AlphaIIbbeta3功能状态上的不同剪切力将是 采用平行板层流模型系统进行评估。 αIIbbeta3基因在中国仓鼠卵巢细胞中的重组表达 (A5细胞)将允许详细研究结构-功能 通过评估特定靶向突变的影响来建立相互关系 在受体的相关区域。此外，GP Ib-IX复合体具有 与AlphaIIbbeta3在同一细胞中成功共表达，从而 使评估这两者的协同作用成为可能 高剪应力下支持黏附的受体。两个外部 激动剂，激活的单抗(AP5或抗Libs-6)和 二价离子，将被用来调节不同的功能状态 感受器。洞察AlphaIIbbeta3的进展 通过它的激活途径，有以下特定的目的 建议：1)定义每个状态的功能属性 重组AlphaIIbbeta3；2)检测配体结合 该受体在固定化和可溶性方面的特异性 配体和离散的配位域；3)功能的表征 流动条件下受体的性质；4)建立 受体不同结构域的作用，如阳离子结合部位， 配体结合位点和细胞质尾部序列，在维持 重组AlphaIIbbeta3的不同功能状态。结果是 这些研究结果将有助于阐明这一机制。 AlphaIIbbeta3的激活；这反过来将阐明一个重要的 血栓形成过程中的血小板功能。

The integrin alphaIIbbeta3 is of key importance in the process of platelet thrombus formation, but the sequence of events that modulate its distinct functions on resting and activated platelets is not yet fully understood. The long term objective of this proposal is to gain insight into the dynamic regulation of the adhesive forces mediated by alphaIIbbeta3 under defined functional conditions. The first experimental model will evaluate receptor interaction with surface-bound ligand in a rapid (spin-on) adhesion assay not involving shear forces. Another method will measure directly the adhesive strength between a cell expressing recombinant alphaIIbbeta3 and a surface bound ligand using the single cell micropipette system. Lastly, the effects of varying shear forces on the functional state of alphaIIbbeta3 will be assessed using the parallel plate laminar flow model system. Recombinant expression of alphaIIbbeta3 on Chinese hamster ovary cells (A5 cells) will allow the detailed study of structure-function relationships by evaluating the effects of specific targeted mutations in relevant domains of the receptor. Moreover, the GP Ib-IX complex has been successfully coexpressed in the same cell with alphaIIbbeta3, thus making it possible to evaluate the synergistic role of these two receptors in supporting adhesion under high shear stress. Two external agonists, an activating monoclonal antibody (AP5 or anti-LIBS-6) and divalent ions, will be used to modulate different functional states of the receptor. To gain insight into the progression of alphaIIbbeta3 through its pathway of activation, the following specific aims are proposed: 1) To define the functional properties of each state of recombinant alphaIIbbeta3; 2) to examine the ligand binding specificities of this receptor with respect to immobilized and soluble ligands and discrete ligand domains; 3) to characterize the functional properties of the receptor under conditions of flow; 4) to establish the role of distinct domains of the receptor, such as cation binding sites, ligand binding sites and cytoplasmic tail sequences, in maintaining the distinct functional states of recombinant alphaIIbbeta3. The results obtained from these studies will contribute to elucidating the mechanism of alphaIIbbeta3 activation; this, in turn, will clarify an important aspect of platelet function during thrombogenesis.