Structure and Function of the VWF Helical Tubule Required for Hemostasis

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.

Grant Number: 5F30HL162128-04
NIH Institute/Center: NIH
Principal Investigator: Jacob Anderson