Friday, April 26, 2024
4/26/2024
PROJECT SUMMARY/ABSTRACT Von Willebrand Disease (VWD), the most common bleeding disorder worldwide, is caused by mutations in von Willebrand Factor (VWF), a large multidomain protein. In the blood, VWF circulates as a long multimer of head- to-head disulfide linked dimers of mature VWF. These long multimers are critical for VWF function as they give circulating VWF polyvalency for activating and binding platelets at sites of endothelial injury, forming a hemostatic plug to staunch bleeding. Additionally, long VWF multimers stabilize coagulation factor VIII (FVIII) in the blood. To form these long multimers, crucial for normal hemostasis, VWF forms helical tubules in the low pH of the late- Golgi and Weibel-Palade bodies (WPB). The helical tubule templates the disulfide bond formation needed to form long multimers by positioning D3 domains in close proximity. At the same time, VWF’s prodomain is cleaved, generating the mature VWF that binds FVIII in the blood. Aberrancy in these maturation steps due to VWF mutations causes several VWD subtypes. Despite the importance of the helical tubule for VWF multimerization, the high-resolution structure of the helical tubule is not known. This fellowship proposal aims to determine structures of VWF helical tubules at three stages of maturation, test Type 2A VWD mutations for causing short tubules, and interrogate the implications of prodomain cleavage for FVIII-VWF tubule association. In Aim 1, using a C-terminally truncated VWF construct, a high-resolution structure of the VWF tubule before and after head-to-head disulfide bonds form will be determined using cryo-electron microscopy (cryo-EM) and helical reconstruction. Using cryo-electron tomography (cryo-ET) and subtomogram averaging, a three- dimensional reconstruction of the in situ VWF tubule will be determined to test if the close packing of VWF helical tubules inside the native WPB environment has consequences for the molecular structure of VWF in the tubule. Guided by this structural insight, a subset of VWD mutations will be tested for their effect on robust tubule formation and normal VWF multimer length. Aim 2 will determine the structural rearrangements in VWF upon prodomain cleavage and test if the cleaved tubule can bind FVIII. This research will elucidate the molecular mechanism of VWF head-to-head disulfide bond formation, necessary for VWF multimerization and normal hemostasis. Structural characterization of the VWF tubule will lead to a molecular understanding of VWD caused by inefficient multimerization. Identification of FVIII-VWF tubule binding will provide a novel context to understand their association and inform therapeutic efforts to modulate FVIII-VWF binding before secretion into the blood. A preliminary VWF tubule reconstruction indicates that additional data collection will allow atomic model building. This research will be carried out under the sponsorship of Dr. Timothy Springer, experienced in structural characterization of VWF, and Dr. Alan Brown, an expert in cryo-EM, creating a strong training environment for predoctoral physician-scientist training.
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