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

Introduction: Heparin-induced thrombocytopenia (HIT) is a prothrombotic disease characterized by a rapid drop in platelet (PLT) count and high risk for the onset of thromboembolic events. The interaction of PLTs with anti-platelet factor 4 (PF4)/heparin IgG immune complexes via PLT Fc-gamma-RIIA (FcγRIIA) is well characterized to contribute to the prothrombotic coagulopathy in HIT. However, a deeper knowledge regarding the kinetics of antibody (Ab)-induced FcγRIIA-mediated PLT alterations is missing. Using a flow cytometer (FC)-based kinetic protocol, we investigated real-time PLT alterations induced by thrombin (THR) plus convulxin (CVX), a collagen GPVI receptor agonist associated with the Fc receptor γ chain (FcRγ), and a monoclonal antibody (moAb) targeting the tetraspanin CD9, which activates PLTs via FcγRIIA.

Method: PLTs from healthy individuals were loaded with the calcium-indicator Fluo-5N followed by dual agonist stimulation with THR (0.125 U/mL) and CVX (500 ng/mL), or with moAb anti-CD9 and its isotype control (both 5 µg/mL) for 15 min under continuous measurement in FC. Changes in PLT 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).

Results: Stimulation with THR+CVX resulted in the formation of procoagulant PLTs that were characterized by a high F5N signal and high binding of AnxV to PS. Although activation with anti-CD9 resulted in a lower percentage of F5N-positive PLTs compared to THR+CVX (mean%±SEM:15.2±1.2 vs. 33.6±2.8, p=0.001) a higher binding of AnxV was detected (mean FI of AnxV binding±SEM: 25.2±1.4 vs. 3.7±0.5, p<0.0001). In addition, while the presence of the direct thrombin inhibitor hirudin inhibited the formation of F5N-positive PLTs dual agonist stimulation (mean%±SEM: 34.2%±1.2% vs. 12.5%±1.7%, p=0.0014), it had no effect on anti-CD9-induced procoagulant PLT formation (F5N mean%±SEM:15.2±1.2 vs. 13.7±1.3, ns). Specific FcγRIIA blockade using moAb IV.3 had no effect on the activation with THR+CVX (F5N mean%±SEM: 33.6±2.8 vs. 32.8±1.7, ns) but resulted in a near complete reduction of Ab-induced procoagulant PLT formation (F5N mean%±SEM: 15.21±1.17% vs. 0.8%±0.3%, p=0.0079). Additionally, formation of F5N positive PLTs and high AnxV-binding following dual agonist as well as anti-CD9 stimulation was significantly reduced in the presence of spleen tyrosine kinase (SYK) inhibitor Lanraplenib (F5N mean%±SEM: 33.6±2.8 vs. 0.8±0.2 and 15.2%±1.2% vs. 0.9±0.2).

Conclusion: First findings of this study indicate that binding of anti-CD9 induces the formation of a procoagulant PLT subpopulation in a FcγRIIA-dependent SYK-mediated pathway that is not affected when thrombin is not present. Our model of Ab-induced PLT activation allows the dissection of critical events that are essential for the generation of procoagulant PLTs and can be used as a simplified in vitro model of HIT and other PLT Fc-gamma-RIIA-mediated prothrombotic diseases.

Publikationsverlauf

Artikel online veröffentlicht:
13. Februar 2025