# Structural basis of von Willebrand factor biology and physics

> **NIH NIH R01** · BOSTON CHILDREN'S HOSPITAL · 2022 · $673,660

## Abstract

von Willebrand factor (VWF) is a multi-domain plasma protein secreted by endothelial cells. In hemostasis,
VWF binds and crosslinks platelets to one another and the vessel wall to form the platelet plug. VWF also
binds to and stabilizes factor VIII (FVIII) in the coagulation cascade. VWF mutations cause the most common
heritable bleeding disorders called von Willebrand disease (VWD). The D1, D2, and D´D3 assemblies in VWF
are specialized domains that enable biosynthesis of VWF into ultralong concatemers that are stored as helical
tubules in Weibel-Palade bodies (WPBs). D´D3 also binds FVIII. Long length enables VWF to sense flow.
Changes in flow at sites of bleeding activate VWF by 1) elongating coiled VWF concatemers into a thread-like
conformation that exposes previously buried A1 domains and 2) activating a high-affinity state of VWF A1
domains that bind platelet glycoprotein Ibα (GPIbα) for platelet plug formation. High-resolution structures of D
assemblies and the high-affinity state of A1 are lacking. In Aim 1, we will determine the structure of the high-
affinity state of A1. Unfolding studies show that VWF A2 and A3 domains have two states, whereas A1 has
three: native, intermediate, and unfolded. Preliminary studies show that truncating the O-glycosylated linkers
N- and C-terminal destabilizes the native state of A1 and increases affinity for GPIbα. We propose that the
intermediate state corresponds to the high-affinity state of A1. We test the hypothesis that further truncation of
the linkers flanking A1, gain-of-function mutations (e.g. activating VWD mutations), and the allosteric activator
ristocetin all increase A1 affinity for GPIbα by stabilizing the intermediate state over the native state. We will
use combinations of truncations, mutations, and ristocetin to stabilize A1 in the intermediate state and to
determine the crystal structure of the putative high-affinity state of A1 and its complex with GPIbα. Aim 2 will
determine structures of D´D3 and the D´D3 dimer. Our preliminary crystal structure of the D´D3 monomer
shows how the C8, TIL, and E modules pack around the VWD module to form the D3 assembly. D´ protrudes
from the D3 assembly. The two cysteines that have been proposed to form the inter-dimer disulfide bonds are
buried. We will solve the structure of a D´D3 dimer (D´D3)2 or a D3 dimer with the protruding D´ removed to
define the structural rearrangements required for D´D3 dimerization. Proposed disulfide rearrangement that
precedes dimerization will be verified by mutation and in vitro reconstitution. As backup, we will pursue a cryo-
EM structure of VWF helical tubules to determine the structure of (D´D3)2 and how D assemblies enable
formation of highly ordered tubules. Aim 3 uses crystallography to understand how D’D3 binds FVIII, which has
the potential through protein engineering to revolutionize replacement FVIII therapy in hemophilia A. As an
alternative strategy, we will determine a cryoEM structure of a D’D3 com...

## Key facts

- **NIH application ID:** 10434710
- **Project number:** 5R01HL148755-04
- **Recipient organization:** BOSTON CHILDREN'S HOSPITAL
- **Principal Investigator:** TIMOTHY A SPRINGER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $673,660
- **Award type:** 5
- **Project period:** 2019-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10434710

## Citation

> US National Institutes of Health, RePORTER application 10434710, Structural basis of von Willebrand factor biology and physics (5R01HL148755-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10434710. Licensed CC0.

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