Partial downregulation of platelet glycoprotein VI by low affinity antibodies confers sustained and safe antithrombotic protection

Abstract Glycoprotein (GP) VI is a platelet-specific activating receptor for collagen and fibrin(ogen), and a promising target for antithrombotic therapy. Inhibitory Fab fragments against GPVI provide robust protection in mouse models of arterial thrombosis and ischemic stroke without impairing hemostasis in mice or humans. However, their short in vivo half-life limits their suitability for long-term therapy. In contrast, GPVI-targeting IgGs (e.g., JAQ1-3) induce complete receptor depletion and a GPVI-knockout-like (GPVI KO-like ) phenotype in mice, a mechanism also observed in humans with anti-GPVI autoantibodies. While GPVI deficiency causes only moderate hemostatic defects, bleeding risk increases when combined with high-dose aspirin (100 mg/kg). Using a humanized GPVI ( hGP6 tg/tg ) mouse model, we show that high-affinity anti-human GPVI IgGs (Emf1 K D 0.490 nM, Emf2) induce a GPVI KO-like phenotype with antithrombotic efficacy. In contrast, JAQ1 IgG, which binds hGPVI with low affinity (K D : 9.6 nM), induces only partial (~50%) GPVI downregulation, generating a stable low-density GPVI (GPVI LO ) phenotype lasting up to 10 days. GPVI LO platelets showed impaired aggregation, abolished procoagulant activity, and—similar to Fab-mediated inhibition—conferred protection from arterial thrombosis and LPS-induced thrombo-inflammation without increasing bleeding, even when combined with high-dose aspirin. The low-affinity anti-mouse GPVI antibody JAQ4 (K D = 21 nM) induced a comparable GPVI LO phenotype in wild-type mice. Transfusion of human platelets in NOD/SCID mice showed that complete and partial GPVI depletion, respectively, also occurs in human platelets in vivo. These findings establish that partial, affinity-dependent GPVI downregulation can provide sustained antithrombotic protection while preserving hemostasis, offering a promising strategy for long-term platelet inhibition.