OC 09.2 - Blocking thrombin-mediated cleavage of platelet glycoprotein V to treat hemorrhagic complications.

Background: Treatment of genetic or drug-related hemorrhagic complications is an unmet clinical need. We have shown that antibody-mediated blockade of the thrombin cleavage site of platelet glycoprotein (GP)V enhances fibrin formation in a spatio-temporal manner. Therefore, we hypothesized that promoting the pro-hemostatic functionality of platelet GPV is a promising approach for potential clinical applications.

Aims: Evaluation of the pro-hemostatic potential of anti-GPV antibodies, which block thrombin-mediated GPV cleavage, in the treatment of hemorrhagic complications.

Methods: We used our newly generated humanized GPV knock-in mice (hGP5KI) to investigate the effects of in-house generated anti-human GPV (hGPV) monoclonal antibodies (mAbs, termed LUMs) on platelet function and coagulation. We assessed clotting time, fibrin formation, thromboelastography (TEG), calibrated automated thrombography (CAT), thrombin generation, and tail bleeding time.

Results: In our previous study, inhibition of thrombin-mediated cleavage of mouse GPV (mGPV) with anti-mGPV mAb increased fibrin formation and hemostatic platelet function without affecting PAR-mediated platelet activation. We also developed a panel of anti-hGPV antibodies (LUMs) and identified LUM/B interfering with thrombin-mediated hGPV cleavage. Remarkably, fibrin formation was accelerated and enhanced in the presence of LUM/B but not in the presence of other mAbs that bind to different GPV epitopes. Thrombin-induced platelet activation was unaltered in hGP5KI platelets and unaffected in the presence of LUM/B. The parameters of clotting time, TEG, and thrombin generation stayed unaltered in hGP5KI mice independent of LUM/B. Most importantly, hemostatic function improved during thrombocytopenia-related and clopidogrel-induced bleeding in the hGP5KI mice treated with LUM/B compared to the control.

Conclusion(s): Drug-induced defects in platelet hemostatic function are efficiently attenuated by blocking thrombin-mediated cleavage of human GPV. The data presented indicate a high clinical potential of anti-GPV treatment to improve hemostasis, promote fibrin formation, and thereby treat genetic or drug-related hemorrhagic complications.