# FRET Visualization of High Mechanosensation of von Willebrand Factor to Hydrodynamic Force

## TL;DR

This study uses a FRET biosensor to show how von Willebrand factor responds to shear force in blood vessels, revealing two stages of unfolding that help in clot formation.

## Contribution

A novel FRET biosensor was developed to visualize vWF's mechanosensation at low shear forces, revealing two-level unfolding dynamics.

## Key findings

- A 2.8 dyn/cm2 shear force caused a ~60% FRET change in vWF within 30 minutes.
- Low shear forces as low as 0.14 dyn/cm2 induced a ~16% FRET change.
- Higher shear forces (2.8 to 28 dyn/cm2) led to decreasing FRET changes, indicating second-level A2 domain unfolding.

## Abstract

von Willebrand factor (vWF) is a large glycoprotein in the circulation system, which senses hydrodynamic force at vascular injuries and then recruits platelets in assembling clots. How vWF mechanosenses shear flow for molecular unfolding is an important topic. Here, a Förster resonance energy transfer (FRET) biosensor was developed to monitor vWF conformation change to hydrodynamic force. The vWF-based biosensor is anchored on the cell surface, in which the A2 domain is flanked with a FRET pair. With 293T cells seeded into microfluidic channels, 2.8 dyn/cm2 of shear force (i.e., 28 μN/cm2, or 264.1/s in shear rate) induced a remarkable FRET change (~60%) in 30 min. A gradient micro-shear below 2.8 dyn/cm2 demonstrated FRET responses positively related to flow magnitudes, with 0.14 dyn/cm2 (1.4 μN/cm2) inducing an obvious change (~16%). The FRET increases indicate closer positioning of A2’s two terminals in vWF or the addition of a more parallel orientation of the FRET pair, supported with the high FRET of the A2-only-based biosensor, which probably resulted from flow-induced A2 dissociation from vWF intramolecular binding such as that in A1/A3 domains. Interestingly, gradient flow increases from 2.8 to 28 dyn/cm2 led to decreasing FRET changes, suggesting the second-level unfolding in the A2 domain. The LOCK-vWF biosensor with bridged A2 two terminals or an A2-only biosensor could not sense the shear, indicating a structure-flexible A2 and large vWF molecules that are important in the mechanosensation. In conclusion, the developed vWF-based biosensor demonstrated the high mechanosensation of vWF with two-level unfolding to shear force: the dissociation of the A2 domain from vWF intramolecular binding under a micro-shear, and then the unfolding of A2 in vWF under a higher shear; the FRET response to shear force at a very low scale may support the observed clot formation at microvascular wounds. This study provides new insights into the vWF’s mechanosensitive feature for its physiological functions and implicated disorders.