DYNAMIC REGULATION OF ADHESIVE FORCES MEDIATED BY AIBB3

AIBB3 介导的粘附力的动态调节

• 批准号: 6056569
• 负责人: INGRID STUIVER
• 金额: $ 9.25万
• 依托单位: MAXIA PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6056569
• 关键词: CHO cells; blood coagulation; calcium ion; cell adhesion; confocal scanning microscopy; divalent cations; fibronectins; flow cytometry; hemodynamics; human subject; integrins; molecular site; monoclonal antibody; phlebotomy; platelet activation; platelets; protein structure; protein structure function; receptor binding; receptor expression; recombinant proteins; von Willebrand factor

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.

整联蛋白α IIb β 3在细胞凋亡过程中具有关键的重要性。 血小板血栓形成，但调节血小板血栓形成的事件序列 其对静息血小板和活化血小板的不同功能尚不清楚， 完全理解 本提案的长期目标是获得 洞察力的动态调节的粘合力介导的 α IIb β 3在确定的功能条件下。 第一 实验模型将评估受体与表面结合的 配体在不涉及剪切力的快速（旋涂）粘附测定中的应用。 另一种方法将直接测量粘合剂之间的粘合强度。 表达重组α IIb β 3和表面结合配体的细胞 使用单细胞微量移液管系统。 最后，影响 改变α IIb β 3功能状态上的剪切力， 使用平行板层流模型系统评估。 α IIb β 3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， 在受体的相关区域。 此外，GP Ib-IX复合物具有 在同一细胞中与alphaIIb β 3成功共表达，因此 从而可以评估这两种方法的协同作用， 受体在高剪切应力下支持粘附。 两个外部 激动剂、活化单克隆抗体（AP 5或抗LIBS-6）和 二价离子，将用于调节不同的功能状态， 受体。 为了深入了解alphaIIbbeta 3 通过其激活途径， 建议：1）定义每个状态的功能属性， 重组α IIb β 3; 2）检查配体结合 该受体相对于固定的和可溶的 配体和离散的配体结构域; 3）表征功能性的 在流动条件下受体的性质; 4）建立 受体的不同结构域，如阳离子结合位点， 配体结合位点和胞质尾序列，在维持 重组α IIb β 3的不同功能状态。 结果 从这些研究中获得的信息将有助于阐明该机制 alphaIIbbeta 3激活;这反过来，将澄清一个重要的 血栓形成过程中血小板功能的一个方面。