.) Platelet adhesion to sites of endothelial cell injury is critical for maintaining the integrity of the vasculature. An essential part of this process is the ability of von Willebrand factor (vWF) to promote platelet deposition by forming a """"""""bridge"""""""" between the exposed subendothelial matrix and platelet glycoprotein Ib/IX/V (GpIb/IX/V) receptor complex. Naturally occurring mutations in the A1 domain of this large complex glycoprotein have been described that perturb platelet binding but not its ability to form high molecular weight multimers (Type 2M). Much research has concentrated on the function of this domain, but progress has been hampered by 1) the inability to express and purify sufficient quantities of a functional recombinant vWF-A1 protein, and 2) lack of an in vivo mouse model to better characterize vWF-A1:GpIb/IX/V interactions in a physiologically relevant setting. The applicant has successfully employed site directed mutagenesis to define residues that are essential for GpIb/IX/V binding to recombinant human vWF-A1 (rvWF-A1) protein under flow conditions. Yet, he has found that human rvWF-A1 does not support the adhesion of mouse platelets, which precludes its use in vivo. The fundamental purpose of this application is to characterize the molecular interactions between murine vWF and GpIb/IX/V in order to improve our understanding of adhesive mechanisms involved in promoting thrombosis in vivo. The proposed studies, using murine vWF-A1 cDNA cloned by the applicant, will be directed at two specific aims.
The first aim i s to identify key structural elements with murine vWF-A1 domain that promote interactions with GpIb/IX/V. To achieve this goal, site directed mutagenesis of candidate residues, based on the crystal structure of human vWF-A1, will be performed and the function of bacterial expressed protein ascertained using an in vitro flow chamber. Homologue mutagenesis of human and mouse vWF‑A1 domains will also be done to further define the GpIb/IX/V binding site in human vWF-A1.
The second aim i s to characterize interactions mediated by vWF-A1 in a setting of acute vascular injury. Using intravital microscopy, the ability of recombinant vWF-A1 protein and antibodies to alter platelet behavior at sites of arteriolar injury will be determined. Mice will be genetically engineered to express green fluorescent protein (GFP) in platelets in order to facilitate these in vivo studies.
