血管性血友病因子的单受体-单配体研究

Mutations in Von Willebrand Factor (VWF) are the most common causes of heritable bleeding disorders and life-threatening thrombosis, whereas the molecular mechanisms remain unknown. Our collaborators at Harvard Medical School have found that VWF A1 domain and GPIb on platelet interacts as a flex-bond instead of a catch-bond. We have expressed and purifed multiple mutants of A1 and GPIb complex, as well as different peptide linkers. Our preliminary rupture force data from magnetic and laser tweezers indicates that mutations from patients shift off-rates to extended states. Single receptor-ligand molecule enables us for the first time to solve the haunting issue of force-dependence of on-rate. Preliminary on-rate force-clamp measurements demonstrate that tensile force decreases the molecular encounter rate and the speed of bond formation. We will continue the rupture force and force-clamp experiments and carry out the re-binding force measurements. We will study after bond dissociation whether the conformation of receptor or ligand can maintain and can accelerate re-binding. We will also establish a theoretical model for the relation of force and on-rate, and bridge the gap between the one dimensional and the three dimensional on-rates. The results will lead to our long-term goal of improving the diagnosis and treatment of von willebrand disease and thrombotic thrombocytopenic purpura, and developing antagonists of the A1-GPIb complex to prevent thrombosis.

血管性血友病因子（VWF）的异常是先天性白血病的主要原因，也能引起恶性血栓的形成，但机制不详。我们在哈佛的合作者发现VWF A1域能与血小板上的GPIb形成一种从未观测到的柔性键，与逆锁键有争议。在本项目中，我们重组出多种A1域和GPIb疾病类型的单受体-单配体分子，并改变连接肽段的长度。以这些分子在磁镊光镊上做单分子断裂力预实验，发现疾病类型的变异体使负反应速率向延伸态漂移。单受体-单配体分子的出现，也使我们能解决一个几十年来悬而未决的课题：正反应速率与力的关系。力钳预实验发现拉力会降低反应体系中分子相遇的概率、并减慢键形成的速度。除了断裂力和力钳实验，我们还将做结合力实验。我们将研究从延伸态解离后，受体或配体的构象能否维持并加快键的形成。我们将建立力与正反应速率的理论模型，并把一维速率推导到三维。结果将有助于实现长期目标：提高诊治血管性血友病和血小板减少性紫癜的效率，开发拮抗剂以抗血栓。

血管性血友病因子在血流异常时被激活，并能与血小板结合形成栓子。VWF及其配体分子的遗传变异导致2B类型和血小板类型的血友病。受体-配体的相互作用如何受到血流剪应力等的调控，如何导致凝血异常，是有关药物研发的关键问题，亟待研究。在本项目中，我们重组出多种疾病类型的VWF A1域和GPIba的单受体-单配体分子。以这些分子在光镊和磁镊上做单分子断裂力实验，发现在10 pN以上拉力作用时，疾病类型变异体的负反应速率小于野生型。我们采用0-20 pN的力程和精细的面元，发现负反应速率随力呈现滑移-逆锁-滑移键的交替变化。分析单受体-单配体分子的数据，建立了正反应速率与力的重要关系。力钳实验发现拉力会降低反应体系中分子相遇的概率、并减慢键形成的速度。以上结果揭示了VWF和GPIba的作用机理，有助于诊治血管性血友病和开发抗血栓药剂。