Single molecule measurements on von Willebrand factor A1 and A2 domains

描述（由申请方提供）：血管性血友病因子（VWF）是一种多联体血浆糖蛋白，具有多种止血功能。血管性血友病（VWD）中VWF的突变是遗传性出血性疾病的最常见原因。每个多联体的单体长度或数量以及血管中的流动调节VWF的止血和血栓形成效力。VWF充当流量传感器，并且在小动脉中出血部位的流量升高，并且与内皮下胶原蛋白结合，激活VWF。VWF中的A1结构域与血小板上的糖蛋白Ib 1结合形成止血栓。ADAMTS 13是切割VWF的A2结构域的酶，并且仅在A2结构域通过流体动力解折叠后才切割，从而在从内皮细胞和血小板以超大形式分泌后调节其尺寸分布。ADAMTS 13的遗传性或获得性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，我们研究的A1和A2域，这是特别重要的剪切传感器功能的VWF，突变导致定性缺陷的VWD型2B和2A，分别。为了理解这些结构域在生理环境中的功能，其中张力在体内施加给它们，我们使用激光镊子和新的构建体将张力施加到分离的结构域，其中结构域使用DNA手柄连接到珠。A2结构域中选定的2A型突变将用于检验以下假设：患者之间存在异质性，即A2结构域是否在较低的力下展开，机械敏感性更高，或再折叠更慢，从而增加对ADAMTS 13的敏感性。对较大结构的实验将测试邻近A2的结构域在力抵抗中的作用。单分子中的受体和配体（ReaLiSM）将用于表征A1-GPIb 1受体-配体键的生理相关力依赖性，以及VWD 2B型突变如何增强该键。ReaLISM是一种融合蛋白，由A1结构域的N至C末端、多肽接头和GPIb 1的富亮氨酸重复结构域组成。我们已经发现，受体-配体复合物作为柔性键，在低力下具有一个状态（状态1，弯曲），并且在高力下具有较低的koff 0的较强状态（状态2，延伸）。对健康受体-配体复合物的实验将进一步表征这种键的精细结构和动力学。关于功能类型2B和血小板型VWD突变的获得的实验将测试它们稳定切换到状态2的假设。首次使用ReaLiSM使我们能够测量受体与配体结合速率的力依赖性。我们将检验张力减缓相遇复合物向结合复合物转化的假设。此外，我们将研究从状态2解离后，受体或配体在状态2中的构象是否可以持续，并提高键形成的速率。这些结果将与我们的长期目标有关，即改善VWD和血栓性血小板减少性紫癜的诊断和治疗，并开发A1-GPIb 1复合物的拮抗剂以预防血栓形成。这些结果对于理解受体-配体键的特化也具有广泛的意义，这些键使血管系统中的细胞粘附能够抵抗强的流体动力。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