OC 33.1 - G6b-B bi-specific cis-acting platelet receptor inhibitors (CAPRIs)-a novel therapy in thrombosis

Background: G6b-B is a tyrosine-based inhibition motif (ITIM)-containing platelet inhibitory receptor which controls platelet reactivity. Two distinct bispecific single-chain variable fragment (bi-scFv) CAPRIs have been developed to human G6b-B – either the tyrosine-based activation motif (ITAM)-containing receptor human FcRIIA (CD32A) or GPVI – bringing G6b-B-bound Shp1/Shp2 within the molecular reach of the ITAM-proximal signaling complex that they dephosphorylate and inactive.

Aims: Our aim was to investigate the efficacy and mechanistic basis of these bivalent CAPRIs in blocking prothrombotic disorders like HIT (heparin-induced thrombocytopenia), VITT (vaccine-induced immune thrombotic thrombocytopenia) and APS (antiphospholipid syndrome) in a human endothelial cell-coated microfluidic system.

Methods: Microfluidics studies were performed in human umbilical vein endothelial cell (HUVEC)-lined channels followed by photochemical injury. Whole human blood from heathy donors was flowed into these channels after the blood had been pre-incubated with patient-derived HIT, VITT, APS IgGs (100 µg/ml) ± PF4 (25 µg/ml) ± CAPRIs (30-50 µg/ml) for 15 minutes. In vitro thrombus formation followed by platelet adhesion and complement activation were investigated.

Results: G6B-CD32A-bi-scFv inhibited platelet adhesion in three thrombosis models as compared to G6B-GPVI-bi-scFv and even more potently when compared to the individual scFvs (Figure 1). Confocal images from these same channels showed significantly lower complement C3c and C5-b9 activation by G6B-CD32A-bi-scFv for HIT (Figure 2) and VITT and APS (not shown).

Conclusion(s): These human endothelial-lined microfluidic studies support that a CAPRI strategy targeting G6b-B through FcRIIAis highly effective at preventing platelet accumulation – at least in part – by blocking complement activation in several strongly prothrombotic settings. Further studies to confirm efficacy in murine models of these three disorders and examine efficacy in the presence of standard anticoagulant as well as the bleeding risks of the CAPRIs alone or combination with anticoagulant need now be done. Nonetheless, these studies support CAPRIs as a novel anti-platelet, antithrombotic therapy.