Abstract Body: Arterial thrombosis is a leading cause of death and disability worldwide. A key feature of arterial thrombosis is biomechanical platelet aggregation, i.e., large-scale platelet aggregation driven by high shear stress. It is well known that biomechanical platelet aggregation is initiated by the interaction of platelet surface GPIbα with plasma protein von Willebrand factor at its A1 domain (VWFA1). However, blocking this interaction compromises hemostasis and causes hemorrhage.
Physiologically, VWF exists in both soluble (in blood) and immobilized (on sub-endothelial collagen or inflamed endothelium) forms, mediating platelet aggregation and adhesion, respectively. We find that NMC4, an antibody targeting positively charged residues in VWFA1 α4/α5 helices (Fig. 1A), preferentially inhibits biomechanical platelet aggregation over adhesion (Fig. 1B). Consistently, in mice, NMC4 preferentially inhibits arterial thrombosis over hemostasis: at 0.625 μg per 25 g body weight, it prevents FeCl₃-induced artery occlusion without prolonging tail bleeding time (Fig. 1C).
These findings prompted us to test whether the positively charged residues in VWFA1 helices α4/α5 form a regulatory hotspot that differentially regulates soluble and immobilized VWF. We engineered VWFA1 mutants M3, M9, M13, M10 that replace 1, 2, 3 and 5 positive residues in α4/α5 helices with alanine, respectively. The mutations are found to progressively suppress soluble VWF–mediated platelet aggregation while enhancing immobilized VWF–mediated adhesion (Fig. 2A,B,E,F). The magnitude of inhibition/enhancement correlates with the number of positive charges removed (Fig. 2D,H). Biophysical analyses reveal that removing positive charges from helices α4/α5 weakens VWFA1–GPIbα long-range electrostatic attraction, thereby inhibiting VWFA1–GPIbα 3-dimensional (3D) binding required for platelet aggregation (Fig. 2C). Conversely, these mutations strengthen VWFA1–GPIbα 2D binding by easing VWFA1–GPIbα “lock-and-key” docking and relieving VWFA1 auto-inhibition, thus promoting platelet adhesion (Fig. 2G).
Our findings delineate the intricate functions of VWF in vascular health. They also suggest a new therapeutic principle: selectively inhibiting soluble, but not immobilized, VWF to prevent thrombosis with minimal bleeding risk. The identified VWFA1 hotspot represents a promising target for next-generation biophysically tuned antithrombotic agents.