Morphological and Biochemical Analyses in Human Platelets Activated Through Integrin a6b1

• Main Authors: Jui-Chin Chang, 張瑞瑾
• Other Authors: Szecheng J. Lo
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/89776874586058754147

博士 === 國立陽明大學 === 微生物及免疫學研究所 === 94 === Platelets play an essential role of hemastasis in prevention of bleeding from damaged blood vessels. Integrins, a superfamily of adhesion receptors, are involved in platelet activation and aggregation by interaction with exposed subendothelial matrix and secreted extracellular matrix, respectively. The platelet morphological changes and activations through integrin aIIbb3 and a2b1 are well characterized, but that via integrin a6b1 is less understood. To examine the activation of platelets through integrin a6b1, anti-integrin a6 mAb, GoH3, were used as substrates. GST-rhodostomin, a recombinant snake disintegrin which activates integin aIIbb3, was parallel used as a comparison. Results of scanning electronic microscopy (SEM) and differential interference contrast (DIC) microscopy showed that platelets formed filopodia and lamellipodia but never fully spread on plates coated with laminin or GoH3; in contrast, platelets fully spread on GST-rhodostomin substrate within 15 minutes. By using fluorescence microscopy and whole-mount TEM to analyze the actin cytoskeleton distribution, we found that actin formed bundles in the peripheral and central portion of platelets when they adhered on GST-rhodostomin while actin were radiated out from the central region of platelets when they adhered on GoH3 mAb. Platelets treated with Src kinase inhibitor (PP1) and PI3 kinase inhibitors (Wortmannin and LY294002) showed that the morphology and actin patterns were disturbed both on GoH3 mAb- and GST-rhodostomin-coated plates. Results of immunoprecipitation and immunoblotting showed that Src and Syk were activated when platelets adhered on both of GST-rhodostomin and GoH3 mAb substrates. And a higher phosphotyrosine signal of PI3K catalytic subunit p110 and a higher PI3K activity were detected when platelets adhered on GoH3 mAb than they spread on GST-rhodostomin substrates. To further investigate the small GTPase Rho family participated in inducing different morphology of platelets through integrin aIIbb3 and a6b1 activation, small GTPase activity assay was performed. A higher amount of activated Cdc42 was detected when platelets adhered on GoH3 mAb than they spread on GST-rhodostomin substrate. The activity of Cdc42 and Rac was increased in platelet pretreated with PI3K inhibitor, wortmannin. Taken together, the results suggested that PI3K is a modulator to regulate the level of activated Rac/Cdc42 in filopodia formation of platelets which induced by a6b1 integrin.