Cofactor-Dependent Regulation of ADAMTS13 Function

ADAMTS13 功能的辅因子依赖性调节

• 批准号: 7663368
• 负责人: X. Long Zheng
• 金额: $ 36.28万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7663368
• 关键词: Address; Affect; Affinity; Atomic Force Microscopy; Binding; Biological Assay; Biology; Blood; Blood Platelets; Carrier Proteins; Cells; Chemistry; Cleaved cell; Coagulation Process; Cognition; Complex; Development; Disease; Electron Microscopy; Endothelial Cells; Endothelium; Enzymes; Factor VIII; Factor VIIIa; Functional disorder; Hemorrhage; Hemostatic function; In Vitro; Inherited; Instruction; Kinetics; Laboratories; Liquid substance; Measurement; Measures; Mediating; Megakaryocytes; Membrane; Metalloproteases; Molecular; Organelles; Paper; Peptide Hydrolases; Physiological; Plasma; Play; Predisposition; Process; Proteolysis; Proteolytic Processing; Reaction; Regulation; Research Personnel; Role; Site; Solutions; Source; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; Syndrome; Thrombotic Thrombocytopenic Purpura; base; cancer procoagulant; cofactor; in vivo; insight; meetings; prevent; programs; shear stress; tool; von Willebrand Disease; von Willebrand Factor

Program Director/PrincipalInvestigator(Last, First, Middle): Krishnaswamy, Sliram PROJECT SUMMARY (See instructions): Proteolytic cleavage of von Willebrand factor (vWF) by ADAMTS13 metalloprotease is critical for maintaining normal hemostasis. We hypothesize that factor VI11 (FVIII) may be a cofactorthat markedly accelerates this processing underflow shear stress. This hypothesis is build upon our findings in vitro and in vivo as presented in our preliminary results and in recent PNAS paper. However, there are many gaps between the observed effect and the molecular mechanisms underlying this rate enhancing effect of FVIII (and activated FVIII). Moreover, the structure-function relationship of FVIII, ADAMTS13 and vWF in this three-body problem is not fully understood. The aims of this proposal mainly focus on addressing some of these questions. Specifically, we propose: 1) To establish robust approaches to permit a quantitative description of the action of ADAMTS13 on either vWF from plasma or UL-vWF newly released from endothelial cells in the absence and presence of FVIII. This provides information about the magnitude of rate enhancing effect and differential role of FVIII in proteolytical process of these two sources of substrate. 2) To determine the mechanisms underlying the rate enhancing effect of FVIII (and its derivatives) on proteolytic cleavage of vWF in solution by ADAMTS13 under shear stress by assessing the contribution of ternary complex formation between ADAMTS13, vWF and FVIII vs. conformational change of vWF upon binding of FVIII and/or ADAMTS13 under shear stress to this process. 3) To determine the structural components of ADAMTS13 required for proteolytic cleavage of soluble vWF and membrane-bound UL-vWF on endothelial cells by focusing on the role of spacer domain and CUB domains in cognition of vWF and vWF-FVIII complexes under fluid shear stress. Along the same line, we will determine the domains of ADAMTS13 requried for proteolytic cleavage of membrane bound UL-vWF on endothelial cells in the absence and presence of shear stress. RELEVANCE (See instructions): The information obtained from the proposed study will advance our understanding of biology of vWF processing by ADAMTS13, pathophysiology of TTP and other thrombotic complications. The results will also provide more insight into the mechanisms underlying the subtypes of von Willebrand diseases that are associated with abnormal proteolysis of vWF molecules. PROJECT/

项目负责人/主要研究者（最后，第一，中间）：Krishnaswamy，Sliram 项目总结（见说明）： 血管性血友病因子（vWF）被ADAMTS 13金属蛋白酶的蛋白水解裂解对于 维持正常止血。我们假设因子VI 11（FVIII）可能是一种辅助因子， 加速了这种处理底流剪切应力。这一假设是建立在我们在体外和体内的发现之上的。 正如我们的初步结果和最近的PNAS论文所述。然而， 观察到的效应与FVIII的这种速率增强效应的分子机制之间的关系 (and激活的凝血因子VIII）。此外，还探讨了FVIII、ADAMTS 13和vWF在这一过程中的结构与功能关系。 三体问题还没有被完全理解。本提案的目的主要是解决以下一些问题： 这些问题。具体而言，我们建议： 1)建立稳健的方法，以定量描述ADAMTS 13对以下任一项的作用 来自血浆的vWF或在不存在和存在FVIII的情况下从内皮细胞新释放的UL-vWF。 这提供了关于FVIII在以下方面的速率增强效应和差异作用的程度的信息： 这两种底物来源的蛋白水解过程。 2)确定凝血因子VIII（及其衍生物）对心率增强作用的机制 在剪切应力下，通过评估ADAMTS 13对溶液中vWF的蛋白水解裂解的贡献， ADAMTS 13、vWF和FVIII之间三元复合物形成与vWF的构象变化 FVIII和/或ADAMTS 13在剪切应力下与该过程的结合。 3)确定可溶性vWF蛋白水解切割所需的ADAMTS 13结构组分 和膜结合UL-vWF对内皮细胞的作用，重点是间隔区和CUB的作用 vWF和vWF-FVIII复合物在流体剪切应力下的认知结构域。沿着同样的路线，我们将 确定ADAMTS 13的结构域，所述ADAMTS 13的结构域是膜结合UL-vWF的蛋白水解切割所需的， 内皮细胞在不存在和存在剪切应力的情况下。 相关性（参见说明）： 从这项研究中获得的信息将促进我们对vWF生物学的理解 通过ADAMTS 13处理，TTP的病理生理学和其他血栓并发症。结果也将 提供了更多的深入了解冯维勒布兰德病的亚型， 与vWF分子的异常蛋白水解有关。 项目/