Antibody-induced procoagulant platelets are important mediators of neutrophil extracellular trap formation

Neutrophil activation and subsequent formation of neutrophil extracellular traps (NETs) are increasingly identified to play an important role in the context of thromboinflammation and immunothrombosis. Activated platelets (PLTs) are reported to amplify neutrophil-mediated effector mechanisms. However, the relevance of other PLT subpopulations remains elusive. In this study, we aimed to dissect the role of antibody (Ab)-induced procoagulant PLTs in neutrophil activation and NET formation in vitro.

PLTs from healthy individuals were stimulated with monoclonal antibody (Ab) anti-CD9 to induce the formation of P-selectin (CD62p) and phosphatidylserine (PS) double positive procoagulant PLTs. Afterwards anti-CD9 or control Ab treated PLTs were co-incubated with purified neutrophils which was followed by the assessment of NET formation in an immunefluorescence microscopy-based NET detection assay.

Coincubation of neutrophils with anti-CD9-induced procoagulant PLTs resulted in a dramatic increase of NET formation compared to control (mean percentage [%] of NETs±SEM: 60.2±6.8 vs. 6.5±2.7, p<0.001). Interestingly, specific blockade of PLT Fc-gamma-RIIA (FcγRIIA) using IV.3 monoclonal blocking Ab resulted in a near complete abrogation of procoagulant PLT-mediated NET formation in vitro (mean% NETs±SEM: 65.4±4.3 vs. 6.4±2.8, p=0.02). Additionally, pretreatment of PLTs with a selective inhibitor of PLT spleen tyrosine kinase (SYK), a key regulatory enzyme in the PLT activating FcγRIIA-mediated signaling cascade, prior to incubation with anti-CD9, resulted in a significant reduction of Ab-induced procoagulant PLT-mediated NET formation in vitro (mean %±SEM: 65.5±3.1 vs. 9.5±2.3, p<0.0001).

Our findings indicate that Ab-induced procoagulant PLTs mediate increased NET formation in a PLT FcγRIIA-dependent manner. Selective inhibition of PLT SYK could be a potential therapeutic approach to prevent from procoagulant PLT-mediated NET formation and subsequent associated prothrombotic effects in different Ab-induced PLT FcγRIIA -mediated diseases.

All authors have no potential conflicts to disclose.