PB0812 - Partial GPVI depletion protects against arterial thrombosis in vivo by inhibiting platelet procoagulant activity without worsening the aspirin-associated bleeding risk.

Background: Glycoprotein VI (GPVI) is the major platelet receptor for collagen/fibrin(ogen) and an emerging antithrombotic target. GPVI can be depleted in circulating platelets by anti-GPVI IgGs, resulting in a knockout-like phenotype and long-term antithrombotic protection. Here, we explore the implications of human GPVI depletion and determine the therapeutic potential of scaled GPVI depletion in vivo.

Aims: Investigation of the effects of partial GPVI depletion using low affinity antibodies regarding thrombosis and hemostasis.

Methods: The efficacy of GPVI downregulation was investigated in WT (for mGPVI) and humanized (hGP6tg/tg for hGPVI) mice using low (mGPVI: JAQ4 – huGPVI: JAQ1) and high affinity (mGPVI: JAQ1 – huGPVI: Emf1) anti-GPVI IgGs. Functional consequences were determined in flow-cytometry, aggregometry and flow-adhesion assays. The in vivo effects were investigated using the tail bleeding time assay (hemostasis) and mechanical injury of the abdominal aorta (thrombosis).

Results: High affinity anti-GPVI IgGs resulted in knockout-like platelets, while low-affinity antibodies generated low-density GPVI (GPVILO) platelets in hGP6tg/tg as well as WT mice by causing partial (~50%) GPVI depletion. Remarkably, partial GPVI deficiency was sufficient to protect mice from arterial thrombosis in vivo. Whereas GPVILO platelets showed impaired aggregation to collagen, this was overcome at increased collagen concentrations. Interestingly, GPVILO platelets showed a reduced ability to become procoagulant, when exposed to a collagen surface under arterial shear rate. Repeated injections of low-affinity antibodies did not result in thrombocytopenia or a further decrease in receptor abundance, while this prolonged the time of GPVI depletion as with the high-affinity antibodies. Finally, the tail bleeding revealed that aspirin (1 mg/kg) treatment of mice with low-affinity IgGs and aspirin (1mg/kg) did not enhance the bleeding tendency.

Conclusion(s): The partial depletion of GPVI with low affinity antibodies is a robust anti-thrombotic strategy with a better safety profile than a complete GPVI immunodepletion.