Subventions et des contributions :

Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023)

The coagulation system consists of plasma proteins and circulating platelets that coordinate to form a blood clot at sites of blood vessel damage. Von Willebrand Factor (VWF) is a large multimeric plasma protein that initiates clot formation by capturing circulating platelets to the injured vessel wall. ADAMTS13 is a metalloprotease that cleaves VWF to regulate its platelet binding activity. Too much ADAMTS13 activity leads to smaller VWF multimers with insufficient platelet binding activity, which can cause bleeding. Too little ADAMTS13 activity causes VWF to have excessive platelet binding activity, which can cause life-threatening thrombosis. Therefore, regulation of ADAMTS13 activity is critical to maintaining proper balance in the blood clotting system. ADAMTS13 is constitutively secreted into the circulation as an active protease and is not inhibited by any known natural protease inhibitors. Despite this seemingly unregulated activity, VWF is currently the only known substrate for ADAMTS13. For these reasons, the regulation of ADAMTS13 activity is poorly understood. This proposed research program seeks to understand the regulation of ADAMTS13 protease activity in an effort to better define its role in biology. Currently, my lab has 3 specific research goals that will contribute to our understanding of ADAMTS13 specificity and regulation. Project 1 will utilize substrate phage display to establish the rules that govern ADAMTS13 specificity. By incorporating high throughput DNA sequencing into our phage display methodology, we can simultaneously measure enrichment and depletion for all 64 million peptides in the library in a single reaction. The resulting substrate recognition motif is highly refined, and will aid in the identification of novel ADAMTS13 substrates. Project 2 seeks to understand the molecular mechanism for ADAMTS13 resistance to natural protease inhibitors. Because ADAMTS13 is the only member of the ADAMTS protease family that is resistant to all natural protease inhibitors, we will engineer (a) deletion constructs of ADAMTS13 and (b) chimeras with other ADAMTS proteases, to define the features of ADAMTS13 that promote resistance to protease inhibitors. Project 3 will utilize a new method called Bio-ID to identify novel physiologic substrates or ligands of ADAMTS13. This project will make a substantial impact on the field by uncovering potential new roles for ADAMTS13 in the cardiovascular system. Overall, our research will address important questions regarding ADAMTS13 regulation as well as break exciting new ground on our understanding of the role of ADAMTS13 in biology. Many of the methods that we are developing to investigate ADAMTS13 are widely applicable, and will make a broad impact to protease research in the natural sciences.