PB0344 - The impact of spleen tyrosine kinase-inhibition on 5B9 HIT antibody-induced procoagulant platelet formation

Background: Heparin-induced thrombocytopenia (HIT) is a prothrombotic disease caused by antibodies (Abs) that recognize complexes of platelet (PLT) factor 4 and heparin. HIT immune complexes can engage PLT immune receptor Fc-gamma-RIIA (FcγRIIA) which results in an immunoreceptor tyrosine-based activation motif (ITAM)-mediated activating signal downstream with a pivotal role of spleen tyrosine kinase (Syk). 5B9 is a chimeric monoclonal HIT antibody that resembles the pathogenesis of HIT.

Aims: This study aims to characterize 5B9 HIT Ab-induced PLT subpopulations and to investigate the therapeutic potential of different Syk inhibitors on Ab-induced PLT alterations.

Methods: PLTs from healthy individuals were incubated with 5B9 HIT Ab in the presence of different heparin concentrations. Afterwards, PLTs were analyzed for changes in the expression level of the PLT surface activation marker P-selectin (CD62p) and for procoagulant phosphatidylserine (PS) externalization in flow cytometry (FC).

Results: 5B9 induced a significant formation of procoagulant PLTs (CD62p/PS double positive) in the presence of low- (0.2 IU/mL) dose heparin whereas these changes were absent under buffer conditions or in the presence of high-dose heparin (p=0.0009 and 0.0011, respectively). Formation of Ab-induced procoagulant PLTs seem to be mainly mediated by PLT FcγRIIA as the inhibition of PLT protease activated receptor-1 (PAR-1) with a specific PAR-1 inhibitor did not affect procoagulant PLT formation. Additionally, experiments with the Syk inhibitors Fostamatinib (R406), PRT-060318 and Lanraplenib revealed that procoagulant PLT formation seems to be dependent on Syk-mediated signaling mechanisms as the pretreatment with these inhibitors resulted in a marked reduction of Ab-induced procoagulant PLTs (IC50:14.35±1.27μM; 0.14±1.1μM; and 0.22±1.29μM, respectively).

Conclusion(s): Findings of our study suggest that 5B9 HIT Ab induces procoagulant PLTs via the engagement of PLT FcγRIIA and subsequent Syk-mediated signaling events. This in vitro model could have the potential to investigate the therapeutic potential of different Syk inhibitors for the prevention of thromboembolic events in HIT