Background: KKO is a mouse monoclonal antibody that mimics the sentinel biological features of pathogenic antibodies that cause HIT, including thrombocytopenia and a prothrombotic state, when it is injected into “HIT mice” lacking mouse PF4 and transgenic for human (h) PF4 and FcγRIIA. We have shown that like HIT antibodies, KKO activates platelets, neutrophils and monocytes through Fcγ receptor-mediated mechanisms, primarily FcγRIIA, and induces complement-dependent cytotoxicity after binding to complexes of hPF4 and polyanions.

Aims: We tested whether deglycosylation of the Fc portion of KKO (DGKKO) would: a) compete with HIT antibodies, b) eliminate FcγR and complement activation, and c) be protective in HIT.

Methods: ​N-linked glycans in the Fc-region of KKO were specifically hydrolyzed by endoglycosidase from Streptococcus pyogenes EndoS (Genovis). Binding of DGKKO and KKO to PF4-heparin complexes was studied by ELISA and dynamic light scattering. FcγRIIA-mediated platelet activation and complement fixation was evaluated by flow cytometry. In vivo thrombocytopenia and thrombosis in the HIT mice were done following passive immunization with the antibody or antibodies of interest.

Results: Deglycosylation did not affect KKO binding to multimolecular PF4-polyanion complexes, but eliminated its interaction with FcgRIIA, abrogated platelet activation (Figure 1, left), and decreased complement activation (Figure 1, right). Treatment of HIT mice with DGKKO did not induce thrombocytopenia (not shown) or thrombosis in a laser injury model in cremaster arterioles (Figure 2, left). Infusion of DGKKO post KKO reversed thrombocytopenia induced by KKO both when infused 1 or 6 hours after the KKO (Figure 2, right).

Deglycosylation of KKO abrogates platelet activation through FcγRIIA and decreases complement fixation. Whole blood was incubated with PF4 (10 µg/ml) and KKO or DGKKO at the indicated concentrations. Left: Platelet activation was determined by expression of P-selectin. Right: Complement fixation by measuring the deposition of C3c on platelets by flow cytometry.

 

DGKKO studies in HIT mice. Left: Laser injury of cremaster muscle arterioles was induced in HIT mice. The mice were infused with the indicated antibody (1 µg/g, i.v.) 5 minutes after laser injury. Representative images are shown of thrombi immediately pre-antibody infusion (top) and 15 minutes after antibody infusion (bottom). Green = platelet accumulation. Red = fibrin deposition. Blue = antibody deposition. Right: HIT mice were injected with KKO (200 µg i.p) either alone (red line) or subsequently given DGKKO (20 µg i.v.) 1 hour post KKO (blue line) or 6 hours post KKO (green line) as indicated by vertical arrows. N=4 animals per arm.

Conclusions: DGKKO, which retains the binding capacity of parental KKO but lacks Fc- and complement-mediated activities, blocked the induction of thrombocytopenia and thrombosis in a murine model of HIT. Studies of the effect of DGKKO on polyspecific HIT patient IgG are underway.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/prevention-of-thrombocytopenia-and-thrombosis-in-heparin-induced-thrombocytopenia-hit-using-deglycosylated-kko-a-novel-therapeutic-for-hit/