Tucker Katherine L, Kaiser William J, Bergeron Angela L, Hu Hongbo, Dong Jing-fei, Tan Tse-Hua, Gibbins Jonathan M
Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, The University of Reading, Whiteknights, Reading, UK.
Proteomics. 2009 Sep;9(18):4340-54. doi: 10.1002/pmic.200900024.
The platelet surface is a dynamic interface that changes rapidly in response to stimuli to co-ordinate the formation of thrombi at sites of vascular injury. Tight control is essential as loss of organisation may result in the inappropriate formation of thrombi (thrombosis) or excessive bleeding. In this paper we describe the comparative analysis of resting and thrombin-stimulated platelet membrane proteomes and associated proteins to identify proteins important to platelet function. Surface proteins were labelled using a biotin tag and isolated by NeurtrAvidin affinity chromatography. Liquid phase IEF and SDS-PAGE were used to separate proteins, and bands of increased intensity in the stimulated platelet fractions were digested and identified by FT-ICR mass spectrometry. Novel proteins were identified along with proteins known to be translocated to the platelet surface. Furthermore, many platelet proteins revealed changes in location associated with function, including G6B and Hip-55. HIP-55 is an SH3-binding protein important in T-cell receptor signalling. Further analysis of HIP-55 revealed that this adaptor protein becomes increasingly associated with both Syk and integrin beta3 upon platelet activation. Analysis of HIP-55 deficient platelets revealed reduced fibrinogen binding upon thrombin stimulation, suggesting HIP-55 to be an important regulator of platelet function.
血小板表面是一个动态界面，它会根据刺激迅速变化，以协调血管损伤部位血栓的形成。严格的控制至关重要，因为失去组织性可能导致血栓形成不当（血栓形成）或出血过多。在本文中，我们描述了对静息和凝血酶刺激的血小板膜蛋白质组及相关蛋白质的比较分析，以鉴定对血小板功能重要的蛋白质。使用生物素标签标记表面蛋白质，并通过抗生物素蛋白亲和色谱法进行分离。液相IEF和SDS-PAGE用于分离蛋白质，对刺激的血小板组分中强度增加的条带进行消化，并通过傅里叶变换离子回旋共振质谱法进行鉴定。鉴定出了新的蛋白质以及已知易位至血小板表面的蛋白质。此外，许多血小板蛋白质显示出与功能相关的位置变化，包括G6B和Hip-55。HIP-55是一种在T细胞受体信号传导中起重要作用的SH3结合蛋白。对HIP-55的进一步分析表明，这种衔接蛋白在血小板活化后与Syk和整合素β3的结合越来越多。对缺乏HIP-55的血小板的分析表明，凝血酶刺激后纤维蛋白原结合减少，这表明HIP-55是血小板功能的重要调节因子。
