Calpain调节血小板GPIbα膜外功能区酶切的机制研究

The ectodomain of platelet glycoprotein Ibα contains the binding site for von Willebrand factor (VWF). The binding of GPIbα to VWF on the injured vessel wall is the initial and critical step for thrombus formation. However, under physiological and pathological conditions, the ectodomain of GPIbα can be shed by ADAM17 and thus the platelets lose the function in thrombosis and haemostasis. To date, the mechanism of GPIbα shedding is still unclear. We found that calpain was involved in the ADAM17-mediated GPIbα ectodomain shedding. This study will focus on the mechanisms through the regulation of ADAM17 activity by calpain and the regulation of the sensitivity of GPIbα to ADAM17 by calpain, and also explore the relationship between the increase of platelet cytoplasm calcium concentration and GPIbα shedding and the critical role of calpain in it. This study aims to explain the regulatory mechanism of calpain in GPIbα ectodomain shedding, which may help to understand the regulation of platelet function under physiological and pathological states and suggest a novel strategy to develop new drugs for thrombotic and haemostatic diseases.

血小板膜糖蛋白(GP)Ibα膜外功能区含有血管性血友病因子（VWF）的结合位点。GPIbα与损伤血管内皮表面VWF的结合是血栓形成的起始和关键步骤。然而，在生理和病理情况下，GPIbα的膜外功能区均可被ADAM17酶切，使得血小板失去参与血栓与止血的功能。到目前为止，酶切发生的机制尚未阐明。申请人研究发现，calpain对ADAM17介导的GPIbα膜外功能区酶切起重要的正调控作用。本研究将分别从calpain调节ADAM17酶活性和calpain调节GPIbα对ADAM17的敏感性两个方面阐明其机制，并研究血小板胞内钙离子浓度升高与GPIbα被酶切之间的关系以及calpain在其中的关键作用。本研究有望阐明calpain调控GPIbα膜外功能区酶切的机制，对认识病理、生理情况血小板功能的调控机制，以及研制新型抗血栓、出血药物提供新思路。

血小板膜糖蛋白(GP)Ibα膜外功能区含有血管性血友病因子（VWF）结合位点。GPIbα与损伤血管内皮表面VWF的结合是血栓形成的起始和关键步骤。然而，在生理和病理情况下，GPIbα的膜外功能区均可被ADAM17酶切，使得血小板失去参与血栓与止血的功能。到目前为止，酶切发生的机制尚未阐明。申请人之前的研究发现，calpain对ADAM17介导的GPIbα膜外功能区酶切起重要的正调控作用。 本研究结果表明激活calpain可诱导显著的ROS释放，同时清除ROS可抑制calpain活化诱导的GPIbα胞外端被酶切，证明calpain通过调控活性氧簇（ active oxygen species , ROS）的释放调控ADAM17的活性。本研究还比较了野生型与截短型及突变型GPIbα和GPIbβ细胞株中GPIbα被酶切的情况，结果表明钙离子载体诱导野生型及截短型和突变型GPIbα的被酶切程度没有差别，提示calpain不改变底物GPIbα对ADAM17的敏感性。本研究通过比较激动剂处理后的血小板钙离子浓度变化和GPIbα被酶切的程度，证实血小板胞内钙离子浓度升高可通过激活calpain诱导GPIbα被酶切，但与GPIbα被酶切并非直接相关。本研究阐明了calpain调控GPIbα膜外功能区酶切的机制，找到了calpain－X－GPIbα通路中的X因子－ROS，对认识病理、生理情况血小板功能的调控机制，以及研制新型抗血栓、出血药物提供新思路。