Single molecule measurements on von Willebrand factor A1 and A2 domains

描述（由申请人提供）：血管性血友病因子（VWF）是一种串联血浆糖蛋白，在止血中具有多种功能。血管性血友病（VWD）的VWF突变是遗传性出血性疾病的最常见原因。VWF的长度或每个连接体的单体数量和血管中的流量调节其止血和凝血效力。VWF可作为血流传感器，小动脉出血部位的血流升高以及与内皮下胶原蛋白的结合可激活VWF。VWF中的A1结构域与血小板上的糖蛋白Ib1结合形成止血栓。ADAMTS13是一种切割VWF A2结构域的酶，通过水动力将A2结构域展开后才进行切割，从而调节内皮细胞和血小板以超大形态分泌后VWF的大小分布。遗传性或获得性ADAMTS13缺乏可导致危及生命的血栓性血小板减少性紫癜（TTP）。在这里，我们研究了在VWF剪切传感功能中特别重要的A1和A2结构域，它们的突变分别导致VWD 2B型和2A型的定性缺陷。为了了解这些结构域在体内施加张力的生理环境中的功能，我们使用激光镊子对孤立的结构域施加张力，并使用DNA手柄将结构域连接到珠子上。A2结构域中选定的2A型突变将被用来检验A2结构域是否在较低的力下展开、更机械敏感或更慢地折叠，增加对ADAMTS13的敏感性，在患者中存在异质性的假设。对更大结构的实验将测试A2附近的结构域在抗力中的作用。A Receptor and Ligand in A Single Molecule （reality）将用于描述A1-GPIb1受体-配体结合的生理相关力依赖性，以及VWD 2B型突变如何增强这种结合。ReaLISM是一种融合蛋白，由A1结构域的N - C端、多肽连接体和GPIb1的富含亮氨酸的重复结构域组成。我们发现受体-配体复合物作为一个弯曲键，在低力下具有一个状态（状态1，弯曲），在高力下具有一个更强的状态（状态2，扩展），具有更低的koff0。健康受体-配体复合物的实验将进一步表征该键的精细结构和动力学。功能2B型和血小板型VWD突变的增益实验将验证它们稳定切换到状态2的假设。现实主义的首次使用使我们能够测量受体与配体结合的on-rate的力依赖性。我们将检验拉力减缓相遇配合物向结合配合物转化的假设。此外，我们将研究从状态2解离后，状态2的受体或配体的构象是否可以持续，并提高键形成的速率。这些结果将与我们改善VWD和血栓性血小板减少性紫癜的诊断和治疗，以及开发A1-GPIb1复合物的拮抗剂以预防血栓形成的长期目标相关。这些结果也将对理解受体-配体键的特化具有广泛的意义，受体-配体键使细胞粘附在脉管系统中以抵抗强大的流体动力。1

DESCRIPTION (provided by applicant): Von Willebrand factor (VWF) is a concatameric plasma glycoprotein that has multiple functions in hemostasis. Mutations in VWF in von Willebrand disease (VWD) are the most common cause of heritable bleeding disorders. The length, or number of monomers per concatamer, and flow in blood vessels regulate the hemostatic and thrombotic potency of VWF. VWF acts as a flow sensor, and elevated flow at sites of bleeding in arterioles, and binding to subendothelial collagen, activate VWF. The A1 domain in VWF binds to glycoprotein Ib1 on platelets to form a hemostatic plug. ADAMTS13 is an enzyme that cleaves the A2 domain of VWF, and by cleaving only after unfolding of the A2 domain by hydrodynamic force, regulates its size distribution after secretion in an ultralarge form from endothelial cells and platelets. Inherited or acquired deficiency of ADAMTS13 causes life-threatening thrombotic thrombocytopenic purpura (TTP). Here, we study the A1 and A2 domains, which are particularly important in the shear sensor function of VWF, mutations of which cause qualitative defects in VWD type 2B and 2A, respectively. To appreciate the functions of these domains in the physiologic setting in which tensile force is applied them in vivo, we apply tensile force to the isolated domains using laser tweezers and novel constructs in which the domains are linked to beads using DNA handles. Selected type 2A mutations in the A2 domain will be used to test the hypothesis that there is heterogeneity among patients in whether the A2 domain unfolds at lower force, is more mechanically sensitive, or refolds more slowly, increasing sensitivity to ADAMTS13. Experiments on larger constructs will test the role of domains that neighbor A2 in force-resistance. A Receptor and Ligand in a Single Molecule (ReaLiSM) will be used to characterize the physiologically-relevant force-dependence of the A1-GPIb1 receptor-ligand bond, and how VWD type 2B mutations enhance this bond. ReaLISM is a fusion protein that consists from N to C- terminus of the A1 domain, a polypeptide linker, and the leucine-rich repeat domain of GPIb1. We have found that the receptor-ligand complex acts as a flex-bond, with one state (state 1, flexed) at low force, and a stronger state with lower koff0 at high force (state 2, extended). Experiments on the healthy receptor-ligand complex will further characterize the fine structure and kinetics of this bond. Experiments on gain of function type 2B and platelet-type VWD mutations will test the hypothesis that they stabilize switching to state 2. The use of ReaLiSM for the first time allows us to measure the force dependence of the on-rate for receptor binding to ligand. We will test the hypothesis that tensile force slows the conversion of encounter complexes to the bound complex. Furthermore, we will examine whether after dissociation from state 2, the conformation of the receptor or ligand in state 2 can persist, and enhance the rate of bond formation. The results will be relevant to our long-term goal of improving the diagnosis and treatment of VWD and thrombotic thrombocytopenic purpura, and developing antagonists of the A1-GPIb1 complex to prevent thrombosis. The results will also be of wide significance for understanding the specializations of receptor-ligand bonds that enable cell adhesion in the vasculature to resist strong hydrodynamic forces. 1