Von Willebrand factor a2-domain structure-function studies: High fidelity detection of ADAMTS13 enzyme activity and role of calcium in regulating intra- and extra-cellular proteolysis
MetadataShow full item record
Cardiovascular diseases remain the leading cause of morbidity and mortality in industrialized nations. These diseases constitute arterial and venous thrombosis, myocardial infarction, stroke, and many more. Basic research and clinical studies have tried to determine drug targets and subsequently treat patients with suitable medications for these disorders, but the success rate is still below par. One of the main reasons for this is our incomplete understanding of the complexity of the interactions occurring in circulation that contribute to healthy hemostasis. Delving into these yet-to-be fully explored areas can greatly improve upon our current understanding of cardiovascular disorders. One such area concerns the biomolecule von Willebrand factor (VWF), the largest glycoprotein in blood, which plays a crucial role in primary hemostasis via its interaction with cell surface receptors and other blood proteins. This protein flows in blood as a series of multimers, and the distribution of multimers is regulated by a metalloprotease called ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type repeats, motif 1 type 13), which proteolytically cleaves VWF. Proper functioning of ADAMTS13 is vital to the maintenance of healthy hemostasis, and disruptions in its functioning can greatly predispose a person towards development of thrombotic disorders. This is the focus of my dissertation, which presents original research dealing with the design and results of novel methodologies to determine plasma ADAMTS13 activity and demonstrate calcium regulation of intra- and extracellular ADAMTS13-induced proteolysis. Deficiency in ADAMTS13 levels or presence of ADAMTS13 autoantibodies leads to thrombotic thrombocytopenic purpura (TTP), a disease characterized by low platelet count, hemolytic anemia and microthrombi formation. Determination of ADAMTS13 activity is essential for diagnosis of TTP, and especially to distinguish it from other disorders like hemolytic uremic syndrome (HUS) which also present with very similar clinical symptoms. Fluorescence resonance energy transfer (FRET) based assays are one of the most common type of "rapid" assays currently used for the determination of plasma ADAMTS13 activity. However, they deal with various drawbacks such as interference due to plasma autofluorescent components and bilirubin. We tried to overcome these limitations by designing a surface (flow cytometry streptavidin bead) based ADAMTS13 activity assay, and significantly succeeded in meeting our objectives. It has been established beyond doubt that ADAMTS13 cleaves VWF in blood. However, these two proteins also reside together in endothelial cells, which made us want to investigate if they interact intracellularly. Indeed, we have demonstrated for the first time that ADAMTS13 cleaves VWF intracellularly in endothelial cells via western blotting of endogenous and overexpressed VWF in HUVECs (human umbilical vein endothelial cells). Second, we demonstrate that the concentration of Ca 2+ ion is a regulator of VWF cleavage, as demonstrated in our in vitro FRET studies performed under static conditions. Higher Ca 2+ levels (representative of extracellular environment) protect VWF against proteolysis, while lower concentrations of the divalent ion (similar to intracellular milieu) readily allow cleavage. Surprisingly, this protective effect was not seen under shear conditions, suggesting that Ca 2+ regulation might only be important in intracellular static conditions. Additionally, the role of shear stress in the realization of VWF physiology is presented as a review in the first chapter of this dissertation, because shear stress is a crucial regulator of VWF structural changes and function. Its role in VWF and ADAMTS13 related blood disorders is also exhaustively covered. The chapters compiled in this dissertation contribute to both clinical and basic research by design of a novel surface-based ADAMTS13 assay and demonstration of a novel intracellular proteolysis phenomenon which is likely regulated by Ca 2+ ion. The design used in our surface-based assay could be easily adopted and redesigned to measure activity of other plasma proteases, broadening the scope of this study. Overall, we have tried to make a small but significant contribution to the understanding of VWF and ADAMTS13 physiology by our studies.