Cofactor-Dependent Regulation of ADAMTS13 Function

项目主任/首席调查员(最后、第一、中间)：Krishnaswamy，Sliram 项目总结(见说明)： ADAMTS13金属蛋白酶对von Willebrand因子(VWF)的蛋白水解性切割 维持正常止血。我们假设因子VI11(FVIII)可能是一个显著 加速了这一加工过程中的底流剪应力。这一假说建立在我们在体外和体内的发现之上。 活体，如我们的初步结果和最近的PNAS论文中所述。然而，还有许多差距。 FVIII促心率作用的分子机制与观察效应之间的关系 (并激活了FVIII)。此外，还研究了FVIII、ADAMTS13和vWF的结构与功能关系 三体问题还没有完全被理解。这项提案的目标主要集中在解决一些 这些问题。具体来说，我们建议： 1)建立可靠的方法，以便能够定量描述ADAMTS13对任何一种情况的行动 在FVIII不存在的情况下，来自血浆的VWF或从内皮细胞新释放的UL-vWF。 这提供了有关FVIII的速率增强效应的大小和区别作用的信息。 这两种底物的蛋白质降解过程。 2)确定FVIII(及其衍生物)的促速作用机制。 剪切力作用下ADAMTS13对vWF的切割作用 ADAMTS13、vWF和FVIII三元络合物的形成及vWF的构象变化 在剪应力下FVIII和/或ADAMTS13与这一过程的结合。 3)确定可溶性vWF蛋白水解性切割所需的ADAMTS13的结构成分 和膜结合的UL-vWF通过关注间隔区和CUB的作用对内皮细胞的影响 流体剪应力作用下vWF和vWF-FVIII复合体的认知结构域。沿着同样的路线，我们将 确定膜结合UL-vWF蛋白水解性切割所需的ADAMTS13结构域 内皮细胞在没有和存在剪切力的情况下。 相关性(请参阅说明)： 从拟议的研究中获得的信息将促进我们对vWF生物学的了解 经ADAMTS13处理，TTP的病理生理学及其他血栓并发症。结果也将是 提供更多关于von Willebrand疾病亚型潜在机制的见解 与vWF分子的异常蛋白分解有关。 项目/

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/