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.
