Department of Pediatrics, University of Manitoba, Winnipeg, Manitoba, Canada.
Thromb Res. 2010 Feb;125(2):152-8. doi: 10.1016/j.thromres.2009.07.005. Epub 2009 Jul 29.
CD63 and CD9 are members of the tetraspanin superfamily of integral membrane proteins that function as organizers of multi-molecular signaling complexes involved in cell morphology, motility and proliferation. Tetraspanin complexes cluster dynamically in unique cholesterol-rich tetraspanin-enriched microdomains (TEMs). In resting platelets, CD63 is located in the membranes of lysosomes and dense granules. Following platelet activation and granule exocytosis, CD63 is expressed on the plasma membrane, co-localizes with the alphaIIbbeta3-CD9 complex and is incorporated into the Triton-insoluble actin cytoskeleton, dependent on fibrinogen binding to alphaIIbbeta3. In nucleated cell lines, the assembly and maintenance of TEMs depends on the palmitoylation of both tetraspanins and some partner proteins. This study investigated the role of palmitoylation in platelet TEM assembly and maintenance. [(3)H]-palmitate-labeled, washed human platelets were studied at rest, or following activation with thrombin (0.1 U/ml). CD63 and CD9 were separated by density gradient centrifugation, isolated by immunoprecipitation, and [(3)H]-palmitate was measured in each fraction. Palmitate levels increased in all fractions following thrombin activation. However, the relative inter-fraction distribution of the tetraspanins did not change. 2-bromopalmitate (2-BP), an inhibitor of protein palmitoylation as demonstrated by decreased [(3)H]-palmitate labeling of platelet proteins, blocked both thrombin-induced platelet aggregation and platelet spreading on immobilized fibrinogen in a dose-dependent manner. 2-BP also inhibited the activation-dependent association of CD63 with CD9, and the incorporation of CD63 into the Triton-insoluble actin cytoskeleton. In contrast, 2-BP had no effect on the incorporation of alphaIIbbeta3 into the activated platelet cytoskeleton. These results demonstrate that palmitoylation is required for platelet tetraspanin-tetraspanin and tetraspanin-integrin interaction and for complete platelet spreading on a fibrinogen substrate.
CD63 和 CD9 是四跨膜蛋白超家族的成员，作为涉及细胞形态、运动和增殖的多分子信号复合物的组织者发挥作用。四跨膜蛋白复合物在富含胆固醇的独特四跨膜蛋白富集微域（TEM）中动态聚集。在静息血小板中，CD63 位于溶酶体和致密颗粒的膜上。血小板激活和颗粒胞吐后，CD63 表达于质膜上，与 alphaIIbbeta3-CD9 复合物共定位，并依赖于纤维蛋白原与 alphaIIbbeta3 的结合而纳入 Triton 不溶性肌动蛋白细胞骨架。在有核细胞系中，TEM 的组装和维持依赖于两种四跨膜蛋白和一些伴侣蛋白的棕榈酰化。本研究探讨了棕榈酰化在血小板 TEM 组装和维持中的作用。用 [(3)H]-棕榈酸标记、洗涤的人血小板在静息时或用凝血酶（0.1 U/ml）激活后进行研究。通过密度梯度离心分离 CD63 和 CD9，通过免疫沉淀分离，并用 [(3)H]-棕榈酸测量每个级分中的棕榈酸。凝血酶激活后，所有级分中的棕榈酸水平均增加。然而，四跨膜蛋白的相对级分分布没有改变。2-溴棕榈酸（2-BP），如血小板蛋白的 [(3)H]-棕榈酸标记减少所示，是一种蛋白质棕榈酰化的抑制剂，以剂量依赖性方式阻断凝血酶诱导的血小板聚集和血小板在固定化纤维蛋白原上的铺展。2-BP 还抑制 CD63 与 CD9 的激活依赖性结合，以及 CD63 纳入 Triton 不溶性肌动蛋白细胞骨架。相比之下，2-BP 对 alphaIIbbeta3 纳入激活的血小板细胞骨架没有影响。这些结果表明，棕榈酰化是血小板四跨膜蛋白-四跨膜蛋白和四跨膜蛋白-整合素相互作用以及血小板在纤维蛋白原底物上完全铺展所必需的。
