Immunocytochemical Study on the Redistribution of Signal Molecules in Human Platelet Adherent to Extracellular Matrices

利用免疫荧光显微镜，我们研究了粘附在胶原蛋白上的人血小板的细胞骨架模式。肌动蛋白被检测为对齐的补丁，并位于沿着肌动蛋白丝。虽然在肌动蛋白丝的末端没有检测到塔林，但它集中在血小板与相邻血小板紧密接触的血小板周围。检测到PP 125 μ g<FAK>为小斑点，所有这些斑点作为一个整体在粘附血小板的中心区域形成一个环。这些结果表明，粘附的血小板不能形成典型的局灶性接触，在激动剂诱导的血小板聚集或在基质上铺展时，局灶性粘附激酶（FAK）在酪氨酸上被磷酸化。本研究采用免疫细胞化学方法，观察凝血酶诱导血小板活化和聚集过程中FAK的分布。我们发现FAK在聚集和酪氨酸磷酸化之前从细胞质转位到质膜下的亚膜区。接下来，我们用渥曼青霉素和LY 294002检测了磷酸肌醇3-激酶（P13-K）在血小板粘附和聚集中的作用，两种不相关的P13抑制剂-用抑制剂预处理血小板可防止血小板在胶原蛋白上的铺展和凝血酶受体激活剂诱导的聚集肽。这些结果表明，P13-K是激活α IIb β3整合素和维持整合素活性状态所必需的。

Using immunofluorescence microscopy, we have examined the cytoskeletal patterns in human platelets adherent to collagen. Vinculin was detected as aligned patches, and located along actin filaments. Although talin was not detected at the ends of actin filaments, it was concentrated at the periphery of the platelets where the platelets were in close contact with adjacent platelets. pp125^<FAK> was detected as small spots, all of which, as a whole, formed a ring at the central areas of adherent platelets. These results suggest that the adherent platelets fail to form typical focal contacts.Focal adhesion kinase (FAK) is phosphorylated on tyrosine during agonist-induced platelet aggregation or spreading on matrices. In this study, using a immunocytochemical method, we focused on (re)distribution of FAK during platelet activation and aggregation induced by thrombin. We found that FAK translocates to submembrane zone just beneath plasma membrane from cytoplasm prior to both aggregation and tyrosine phosphorylation of the kinase.Next, we have examined the role of phosphoinositide 3-kinase (P13-K) in platelet adhesion and aggregation using wortmannin and LY294002, two unrelated inhibitors of P13-K.Pretreatment of the platelets with the inhibitors prevented platelet spreading on collagen and aggregation induced by thrombin receptor activator peptide. These results suggest that P13-K is necessary for the activation of αllbβ3 integrin and maintenance of the integrin in its active state.