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

• DOI: 10.20944/preprints202502.0915.v1
• Publication Date: 2025-02-14T05:07:59Z
• Authors: Mingxing Ouyang; Yao Gao; Binqian Zhou; Jia Guo; Lei Lei; Yingxiao Wang; Linhong Deng

von Willebrand factor (vWF) is a large glycoprotein in 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, Förster resonance energy transfer (FRET) biosensor was developed to monitor vWF conformation change to hydrodynamic force. The full-length vWF-based biosensor is anchored on cell surface, in which A2 domain is flanked with FRET pair. With 293T cells seeded into microfluidic channels, 2.8 dyn/cm2 shear force induced remarkable FRET change (~60%) in 30 min. Gradient micro-shear below 2.8 dyn/cm2 demonstrated FRET responses positively related to flow magnitudes with 0.14 dyn/cm2 inducing obvious change (~16%). The FRET increases indicate closer positioning of A2’s two termini in vWF, supported with high FRET of A2 only-based biosensor, which probably resulted from flow-induced A2 dissociation from vWF intramolecular binding. Interestingly, gradual increase of flow from 2.8 to 28 dyn/cm2 led to decreasing FRET changes, suggesting the second-level unfolding in A2 domain. LOCK-vWF biosensor with bridged A2 two termini or A2 only biosensor couldn’t sense the shear, indicating structure-flexible A2 and large vWF molecules important in the mechanosensation. In conclusion, the developed vWF-based biosensor demonstrated high mechanosensation of vWF with two-level unfolding to shear force: the dissociation of A2 domain from vWF intramolecular binding under micro shear, and then unfolding of A2 in vWF under higher shear. This study provides new insights on vWF mechanosensitive feature for its physiological functions and implicated disorders.