剪切诱导的全长血管性血友病因子多聚体的展开与酶切
Shear-Induced Unfolding and Enzymatic Cleavage of Full-Length VWF Multimers.
作者信息
Lippok Svenja, Radtke Matthias, Obser Tobias, Kleemeier Lars, Schneppenheim Reinhard, Budde Ulrich, Netz Roland R, Rädler Joachim O
机构信息
Faculty of Physics and Center for NanoScience, Ludwig Maximilian University, Munich, Germany.
Department of Physics, Freie Universität Berlin, Berlin, Germany.
出版信息
Biophys J. 2016 Feb 2;110(3):545-554. doi: 10.1016/j.bpj.2015.12.023.
Proteolysis of the multimeric blood coagulation protein von Willebrand Factor (VWF) by ADAMTS13 is crucial for prevention of microvascular thrombosis. ADAMTS13 cleaves VWF within the mechanosensitive A2 domain, which is believed to open under shear flow. In this study, we combine fluorescence correlation spectroscopy (FCS) and a microfluidic shear cell to monitor real-time kinetics of full-length VWF proteolysis as a function of shear stress. For comparison, we also measure the Michaelis-Menten kinetics of ADAMTS13 cleavage of wild-type VWF in the absence of shear but partially denaturing conditions. Under shear, ADAMTS13 activity on full-length VWF arises without denaturing agent as evidenced by FCS and gel-based multimer analysis. In agreement with Brownian hydrodynamics simulations, we find a sigmoidal increase of the enzymatic rate as a function of shear at a threshold shear rate γ˙1/2 = 5522/s. The same flow-rate dependence of ADAMTS13 activity we also observe in blood plasma, which is relevant to predict hemostatic dysfunction.
多聚体血液凝固蛋白血管性血友病因子（VWF）被ADAMTS13进行蛋白水解对于预防微血管血栓形成至关重要。ADAMTS13在机械敏感的A2结构域内切割VWF，据信该结构域在剪切流作用下会打开。在本研究中，我们结合荧光相关光谱法（FCS）和微流控剪切池来监测全长VWF蛋白水解的实时动力学，该动力学是剪切应力的函数。为作比较，我们还在无剪切但部分变性的条件下测量了ADAMTS13切割野生型VWF的米氏动力学。在剪切作用下，FCS和基于凝胶的多聚体分析表明，ADAMTS13在无变性剂的情况下对全长VWF具有活性。与布朗流体动力学模拟结果一致，我们发现在阈值剪切速率γ˙1/2 = 5522/s时，酶促反应速率随剪切呈S形增加。我们在血浆中也观察到ADAMTS13活性具有相同的流速依赖性，这对于预测止血功能障碍具有重要意义。
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