Investigation of antibody-induced Fc-gamma-RIIA-mediated procoagulant platelet formation in real-time

Heparin-induced thrombocytopenia is a prothrombotic disease characterized by a rapid drop in platelet (PLT) count and high risk for the onset of thromboembolic events. Anti-platelet factor 4/heparin immunoglobulin G (IgG) immune complexes that activate PLTs and different immune cells are the causing factor for HIT. Although PLTs are well characterized for being pivotal in the complex pathophysiology of HIT, a deeper knowledge regarding the kinetics of antibody (Ab)-induced Fc-gamma-RIIA-mediated PLT alterations is missing. Utilizing a flow cytometry (FC)-based kinetic protocol and a simplified in vitro model of HIT, we aimed to investigate Ab-induced PLT alterations in real time.

PLTs from healthy individuals were loaded with the calcium-indicator Fluo-5N which was followed by dual agonist stimulation with thrombin (THR; [0.125 U/mL]) and convulxin (CVX, agonist of collagen GPVI receptor; [500 ng/mL]) or with monoclonal antibody (moAb) anti-CD9 or isotype control (Iso; [both 5 µg/mL]) for 15 min under continuous measurement in FC. Changes in PLTs phenotype were assessed by co-staining with PE-labelled moAb PAC-1 and Annexin-V (AnxV)-Cy5, detecting the activated fibrinogen receptor and procoagulant PLT phosphatidylserine (PS), respectively.

Compared to control, stimulation of PLTs with anti-CD9 resulted in the formation of procoagulant PLTs that were characterized by a high F5N signal, increased binding of AnxV and low binding of PAC-1. Although dual agonist stimulation with THR+CVX resulted in significant higher formation of F5N positive PLTs (mean %±SEM: 33.6±2.8 vs. 15.2±1.2), higher binding of AnxV was detected following stimulation with anti-CD9 (mean fold increase of AnxV binding±SEM: 25.23±1.4 vs. 3.7±0.5, p<0.0001). Most importantly, while the presence of the direct thrombin inhibitor hirudin did not show the potential to prevent anti-CD9-induced procoagulant PLT formation, specific FcγRIIA blockade using moAb IV.3 resulted in a nearly complete abrogation of Ab-induced procoagulant PLT formation (mean %±SEM: 15.2±1.2 vs. 0.80±0.3).

First findings from our kinetic studies indicate that anti-CD9 induces procoagulant PLTs in a FcγRIIA-dependent manner. Future studies with this time resolved model of Ab-induced PLT activation will allow the identification of the critical events that are essential for the generation of Ab-induced procoagulant PLTs in HIT.

The authors have no conflicts of interest to disclose.