Single molecule measurements on von Willebrand factor A1 and A2 domains

冯维勒布兰德因子 A1 和 A2 域的单分子测量

• 批准号: 8434898
• 负责人: TIMOTHY A SPRINGER
• 金额: $ 44.41万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-06 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8434898
• 关键词: Affect; Binding; Biophysics; Blood Clot; Blood Platelets; Blood Vessels; Blood coagulation; Cell Adhesion; Chimeric Proteins; Cleaved cell; Collagen; Complex; Cysteine; DNA; Defect; Dependence; Development; Diagnosis; Disease; Dissociation; Endothelial Cells; Enzymes; Equation; Fluorescence; Glycoproteins; Goals; Hemorrhage; Hemostatic Agents; Hemostatic function; Heterogeneity; Inherited; Kinetics; Lasers; Length; Leucine-Rich Repeat; Life; Ligands; Link; Measurement; Measures; Memory; Modeling; Modification; Molecular Conformation; Mutation; Patients; Peptides; Phase; Physiological; Plasma; Polymers; Proteins; Pseudo von Willebrand disease; Relaxation; Resistance; Role; Site; Structure; Testing; Thrombosis; Thrombotic Thrombocytopenic Purpura; Time; arteriole; base; crosslink; gain of function; gain of function mutation; improved; in vivo; insight; laser tweezer; molecular dynamics; monomer; novel; novel therapeutics; polypeptide; prevent; public health relevance; receptor; receptor binding; research study; sensor; single molecule; therapeutic target; tryptophyl-proline; von Willebrand Disease; von Willebrand Factor

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

描述（由申请人提供）：von Willebrand因子（VWF）是一种伴侣血浆糖蛋白，在止血中具有多个功能。 Von Willebrand疾病（VWD）中VWF的突变是遗传出血疾病的最常见原因。每个辅导员的长度或单体数量以及血管中的流动调节VWF的止血和血栓性效力。 VWF充当流动传感器，并在小动脉出血部位升高流动，并与下皮胶原蛋白结合，激活VWF。 VWF中的A1域与血小板上的糖蛋白IB1结合，形成止血塞。 ADAMTS13是一种裂解VWF的A2结构域的酶，并且仅在通过流体动力展开A2结构域后才切割，可以从内皮细胞和Platelets中以超大性形式的分泌后调节其大小分布。 ADAMTS13的遗传或获得的缺乏会导致威胁生命的血栓性血小板减少紫癜（TTP）。在这里，我们研究了A1和A2结构域，这些结构域在VWF的剪切传感器功能中尤为重要，其突变分别导致VWD 2B型和2A型的定性缺陷。为了欣赏这些结构域在体内施加拉伸力的生理环境中的功能，我们使用激光镊子和新型结构将拉伸力应用于孤立的结构域，并使用DNA柄将域与珠子链接到珠子。 A2结构域中选定的2A型突变将用于检验以下假设：患者在A2域在较低力下展开，更敏感或更缓慢地重折，从而提高对ADAMTSS13的敏感性。对较大构建体的实验将测试邻近A2在力抵抗中的域的作用。单个分子（现实主义）中的受体和配体将用于表征A1-GPIB1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。现实主义是一种融合蛋白，由A1结构域的N到C末端组成，多肽接头和GPIB1的富含亮氨酸重复结构域。我们已经发现，受体配体复合物充当柔性键，一个状态（状态1，弯曲）在低力下，高力量较低（状态2，扩展）。关于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。关于2B型功能增长和血小板型VWD突变的实验将检验以下假设，即它们稳定转换为状态2。第一次使用现实主义使我们能够测量速率对受体与配体结合的受体依赖性。我们将测试以下假设，即拉伸力减慢了相遇复合物向结合复合物的转化。此外，我们将检查与状态2分离后，状态2中受体或配体的构象是否可以持续存在，并提高键形成速率。结果将与我们的长期目标有关，即改善VWD和血栓性血小板细胞减少紫红色的诊断和治疗，以及发展A1-GPIB1复合物的拮抗剂以防止血栓形成。对于理解能够在脉管系统中的细胞粘附以抵抗强大的流体动力的受体粘附的专业知识，结果也将具有广泛的意义。 1