Inter-domain regulation of Von Willebrand factor's function: Platelet adhesion, activation and aggregation in blood
Madabhushi, Sri Ranganayaki
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Cardiovascular diseases are the primary cause of deaths worldwide and according to the World Health Organization; it is projected to cause the death of about 25 million people by 2030. Multiple factors cause the abnormalities of heart and blood vessels that lead to these deadly cardiovascular diseases. One such factor involves the abnormal functioning of the blood protein called von Willebrand factor (VWF) that is involved in hemostasis and thrombosis. VWF is a critical protein involved in pathogenesis of myocardial infarction, platelet adhesion and activation at sites of vascular injury especially at high shear regimes found in arterial stenosis as well as regions of atherosclerotic plaque rupture. The level of this protein is also an indicator of acute coronary heart diseases and adverse cardiac disorders. Additionally, VWF plays a role in the pathogenesis of von Willebrand disease (VWD), which is the most predominant form of hereditary bleeding disorders. Therefore, it is vital to understand the role of VWF in blood and mechanisms involved in regulation of its function. The regulatory processes include factors such as the inter-domain organization of VWF, size of the protein, presence of inhibitory molecules in blood, glycosylation, and shear stress. This dissertation focuses on understanding the mechanism of inter-domain regulation of VWF function, with focus on its platelet adhesion, activation and aggregation properties. Using surface plasmon resonance and ELISA based assays, the presence of interaction between the von Willebrand factor propeptide (VWFpp) and VWF in circulation has been established and a model of their interaction over physiological pH and calcium conditions is proposed. Monoclonal antibodies against the D’D3 domain (DD3.1) of VWF were developed that completely block the interaction between VWFpp and VWF reinforcing the fact that D’D3 is the only domain of VWF that participates in this association. Functional studies revealed that VWFpp (and DD3.1), through its binding to the D’D3 domain, inhibits the VWF-A1 domain mediated platelet adhesion, activation and aggregation. Structural insights into the inter-domain regulation of VWF function has been obtained using mass spectrometry of cross-linked VWF. A set of C++ programs, called XPA, have been developed to analyze the large set of MS data and identify inter-domain cross-linked peptides. Results from this study, for the first time, identify the peptides in D’D3 and A1 domains that come in close proximity in the solution conformation of VWF. One of the A1 peptides in the D’D3-A1 crosslink forms the interaction interface with the platelet receptor GpIbα. Static and shear based assays using ΔD’D3-VWF (VWF with deletion of D’D3 domain) and full-length VWF molecules showed significantly higher platelet binding affinity and platelet translocation velocity in case of the ΔD’D3-VWF constructs. Thus, this study provides novel insights into the structural basis for the regulation of VWF A1 domain function by the neighboring D’D3 domain. Additionally, characterization of monoclonal antibodies against the D’D3 domain provide evidence that the conformational changes in VWF either in the presence of urea or shear stress involve changes in the D’D3 domain. Lastly, while the role of VWF in cardiovascular diseases has been well established, its role in the thrombogenecity of cardiovascular devices is understated. An effort has been made in this direction to highlight the role of VWF in causing the issues with cardiovascular devices. In summary, this dissertation advances the knowledge in the area of inter-domain organization of VWF and the structural regulation of VWF function. The results provide interesting insights on various questions in this field.