胶原诱导的VWF激活的力学调控机制和分子结构基础

Regulated by vascular wall shear stress, the collage-mediated VWF activation is a first key event for triggering the hemostatic function of platelet. This force-dependent and ligand-induced process is not yet well clear, and become a focus in cellular and molecular biomechanics research. In the present proposal, the force-induced transition from closed to opened conformation of VWF-A1A2A3 triplet domain is selected as starting point of this research, based on the supposition that both collagen and type 2B mutations will reduce the mechanical stability of the closed A1A2A3 structure by weakening the force-dependent interaction between internal functional subdomains of A1A2A3. Through combining with biomechanical modeling, molecular dynamics simulation, residue mutation, electron microscope, molecular imaging, AFM and flow chamber techniques, we will measure the force-dependent kinetics between monomer A1, A2 and A3 of VWF, reveal the topology and affinity (to GPIb) of A1A2A3 bound with collagen under different shear stresses, and attempt to uncover the mechanical mechanism and molecular basis of the type 2B mutation- and collagen-induced VWF activation. This research should be useful in better understanding of physiological and pathological processes of the hemostasis and thrombosis, in providing and novel strategy for clinical treatment and rehabilitation, in discovery of new drug targets, and in development of new antibody drugs, for cardiovascular and cerebrovascular disease.

胶原介导的VWF的激活是启动血小板发挥凝止血功能的关键事件，受血管壁面剪应力和化学信号的双重调控，这一尚不清晰的力化学耦合诱导过程，是当前细胞分子生物力学中的前沿热点课题。本项目拟基于胶原和2B型突变将通过减弱VWF-A1A2A3内部功能结构域间的相互作用来降低A1A2A3封闭结构的力学稳定性这一猜想，以力诱导的A1A2A3三联体构象的改变为研究切入点，结合生物力学建模与分子动力学模拟、残基突变、显微电镜、分子影像、AFM和流动腔等实验技术，测定A1、A2和A3单体间的反应动力学常数及力依赖特性，探明不同剪切力条件下结合在胶原上的A1A2A3的拓扑结构及其与GPIba的结合亲和力，力图揭示力和配体或2B型突变协同介导的VWF活化的生物力学调控机制和分子结构基础，旨在加深理解凝止血生理及病理过程，为心脑血管疾病患者的临床治疗和康复、相关分子药物靶点的发现和抗体药物的设计提供新思路。

血管性血友病因子是连接血小板与受损血管处胞外基质的桥梁，在介导血小板的初始黏附中扮演关键角色。但由于复杂的流动环境和分子内相互作用，VWF的激活机制和构象演变突至今未明。这里，我们通过实验、理论建模和数值模拟计算相结合，系统深入地研究了突变与力共同调控的VWF-A1A2A3构象与功能变化及相应分子结构基础，初步探索了冠状动脉血管分型几何特征、血流动力学与动脉粥样硬化斑块之间的相关性。原子力显微镜和流动腔实验结果表明：外力通过逆锁键机制调节A1与A2、A3结构域之间的相互作用，诱导A1A2A3三联体构象随着剪应力的增加呈现先压缩后伸展趋势，并最终双相调控A1A2A3/GPIbα的结合亲和力；而A1的突变则通过增强其与相邻结构域A2、A3的相互作用使A结构域三联体难以伸展，而功能增强型突变则反之。在胶原诱导的VWF的激活过程中，恒速度分子动力学拉伸模拟结果显示W1745C-A3突变削弱了A3与胶原复合物的力学稳定性，却强化了与A1的相互作用，因此通过A3/collagen传递的力不足以克服A1A2A3分子内部的相互作用以暴露A1上结合血小板GPIbα的位点。力可诱导A2上金属蛋白酶ADAMTS13酶切位点的暴露，而钙离子则通过增强A2疏水核心的稳定性和维持ADAMTS13的闭合构象，降低ADAMTS13酶切A2的效率。在流场中，流体剪应力开启、增强和加快A1/GPIb介导的血小板的钙响应和P-selectin的表达。此外，我们还发展了一种新的基于分子动力学模拟的计算策略，并成功预测整合素活化过程中胞内信号通路的力学稳定性、力诱导的构象变化及关键残基。最后，收集了南方人群冠状动脉影像资料，通过影像的三维重建、理论建模和数值计算，发现冠心病患者冠状动脉的几何分型偏离Murray定律，在血管分叉处产生更加复杂的流体动力学环境，有利于血小板的黏附。上述的研究进展将加深对血小板凝止血失调病理过程的理解，为心血管疾病的快速诊断和抗血栓单克隆药物的研发提供有益启发。