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.
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/
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