REGULATION OF PLATELET FUNCTION BY GPIIB-IIIA

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。健康的志愿者将被用于研究。抽血，分离血小板。评估钙蛋白酶、幽灵蛋白和骨骼蛋白作用的实验将涉及抑制血小板内的特定途径和相互作用，并评估其功能后果。途径将被特定的药理学抑制剂和棕榈酰化肽的引入所抑制。后一种肽将被设计用于消除感兴趣的相互作用，例如谱蛋白SH3结构域与已识别的结合伙伴之间的相互作用，骨骼蛋白与整合素之间的相互作用，或钙蛋白酶与已识别的结合伙伴之间的相互作用。消融特定信号分子或相互作用的后果将通过测量整合素aIIbb3的激活能力、血小板粘附能力或血小板扩散能力来评估。抑制的详细效果将通过免疫荧光研究来评估，确定整合素、肌动蛋白细丝、谱蛋白、钙蛋白酶和骨骼蛋白在扩散细胞中的重新分布。在其他研究中，血小板将用于检测特定的信号分子，如钙蛋白酶、Rac、cdc42。从血小板中分离纯化的蛋白将用于体外结合试验，提高抗体，并用于体外鉴定钙蛋白酶裂解产物的试验。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Healthy volunteers will be used for the studies. Blood will be drawn and platelets isolated. Experiments to assess the role of calpain, spectrin, and skelemin will involve inhibiting specific pathways and interactions within platelets and assessing the functional conseqences. Pathways will be inhibited with specific pharmacological inhibitors and by the introduction of palmitylated peptides. The latter peptides will be designed to ablate interactions of interest, such as those between spectrin SH3 domain and identified binding partners, that between skelemin and integrin, or that between calpain and identified binding partners. Consequence of ablating specific signaling molecules or interactions will be assessed by meausuring the ability of the integrin aIIbb3 to be activated, the ability of platelets to adhere or the ability of platelets to spread. Detailed effects of inhibition will be assessed by immnofluorescent studies identifying the redistribution of integrin, actin filaments, spectrin, calpain and skelemin in spreading cells. In other studies, platelets will be used for assays of specific signaling molecules such as calpain, Rac, cdc42. Isolation of purified proteins from platelets will be performed for in vitro binding assays, raising antibodies, and for in vitro assays identifying calpain cleavage products.