Structural studies of mechanisms of shear stress on VWF-GPIbalpha interactions and mechanism of cleavage of VWF by ADAMTS-13

• Antragsteller: Dr. Subhanjan Mondal
• Fachliche Zuordnung: Biochemie
• Förderung: Förderung von 2008 bis 2010
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 106187983

von Willebrand factor (VWF), the largest adhesion ligand in the blood, is critical for bleeding arrest at the site of vessel injury. Its hemostatic function depends on the unique composition of multimers: large multimeric forms are crucial for sheardependent platelet aggregation, whereas small multimers, such as dimers and tetramers, are unable to support platelet aggregation. The multimer distribution of VWF in plasma is regulated in part by ADAMTS-13, a metalloprotease that cleaves the ultra-large form of VWF (ULVWF) secreted by activated endothelial cells and platelets into smaller less thrombogenic multimers. We will investigate the hypothesis that plasma VWF is derived from ultralarge VWF and that shear stress induces conformational changes that lead to binding to GP1bα and cleavage by ADAMTS13. For this we will use a structural biology approach and (1.) crystallize VWF domain D’D3A1 as a dimer which will reveal if, as a dimer, the A1 domain is masked by D’- D3, (2.) co-crystallize an A2 polypeptide containing the cleavage siteTyr1605-Met1606 bond and the metalloprotease domain of ADAMTS13 to understand molecular details of ADAMTS13 cleavage, (3.) determine structures of ends of VWF multimers present in the plasma after ADAMTS13 cleavage. For this we will determine structures of A2 fragments, one from the beginning of the A1 domain and ending in 1605 at the cleavage site and the other beginning at 1606 and going to the end of the A3 domain. We will also generate monoclonal antibodies to these fragments and screen for antibodies that bind only to the neoepitopes made available upon ADAMTS13 cleavage and not to native A2. These antibodies will be useful to assay ADAMTS13 activity. This research will contribute to delineating mechanisms of shear stress and VWF-GPIbα interactions and the mechanism of cleavage by ADAMTS-13.

• DFG-Verfahren: Forschungsstipendien
• Internationaler Bezug: USA
• Gastgeber: Professor Timothy A. Springer