Facey Adam, Pinar Isaac, Arthur Jane F, Qiao Jianlin, Jing Jing, Mado Belden, Carberry Josie, Andrews Robert K, Gardiner Elizabeth E
Australian Centre for Blood Diseases, Monash University , Melbourne, Victoria, Australia 3004.
Department of Mechanical and Aerospace Engineering, Monash University , Clayton, Victoria, Australia 3168.
Biochemistry. 2016 Mar 1;55(8):1187-94. doi: 10.1021/acs.biochem.5b01102. Epub 2016 Feb 16.
The primary platelet collagen receptor, glycoprotein VI (GPVI), plays an important role in platelet activation and thrombosis. The ectodomain of human GPVI (sGPVI) is proteolytically shed from human platelets by a-disintegrin-and-metalloproteinase 10 (ADAM10). In this study, we used a novel ADAM10-sensitive fluorescence resonance energy transfer sensor to analyze ADAM10-mediated shedding of GPVI from human platelets in response to the exposure of GPVI ligands collagen-related peptide (10 μg/mL), collagen (10 μg/mL), and convulxin (0.1 μg/mL) to shear stress (1000-10000 s(-1), 5 min), or a generic activator of metalloproteinases, N-ethylmaleimide (NEM, 5 mM). Elevated shear, NEM, or ligand engagement of GPVI all induced shedding of GPVI, as detected by release of sGPVI; however, only shear or NEM significantly increased ADAM10 enzyme activity. ADAM10 activity was also detectable on the surface of thrombi formed on a collagen-coated surface under flow conditions. Our findings indicate different mechanisms regulate shear- and ligand-induced shedding and shear forces found within the vasculature can regulate ADAM10 activity.
主要的血小板胶原受体糖蛋白VI（GPVI）在血小板活化和血栓形成中起重要作用。人GPVI的胞外域（sGPVI）通过a-解整合素和金属蛋白酶10（ADAM10）从人血小板中蛋白水解性脱落。在本研究中，我们使用一种新型的对ADAM10敏感的荧光共振能量转移传感器，来分析ADAM10介导的GPVI从人血小板中的脱落情况，这是在GPVI配体胶原相关肽（10μg/mL）、胶原（10μg/mL）和convulxin（0.1μg/mL）暴露于剪切应力（1000 - 10000 s(-1)，5分钟）或金属蛋白酶的通用激活剂N-乙基马来酰亚胺（NEM，5 mM）的情况下发生的。如通过sGPVI的释放所检测到的，升高的剪切力、NEM或GPVI的配体结合均诱导了GPVI的脱落；然而，只有剪切力或NEM显著增加了ADAM10酶活性。在流动条件下，在胶原包被表面形成的血栓表面也可检测到ADAM10活性。我们的研究结果表明，不同的机制调节剪切力和配体诱导的脱落，并且脉管系统内发现的剪切力可调节ADAM10活性。
