The Parallel Signaling Pathways Of Phosphatidylserine (PS) Exposure Downstream Of Platelet FcγRIIa

Platelet FcγRIIa is an important component of heparin-induced thrombocytopenia and other immune-mediated thrombocytopenia and thrombosis syndromes. Platelet FcγRIIa, an ITAM receptor, signals not only to cause aggregation and secretion, but also to PS exposure in the platelet procoagulant response when platelets are co-stimulated via the GCPR PARs. The molecular mechanisms downstream of FcγRIIa that lead to PS exposure are incompletely understood. Recently, we (WB; Ahmad et al., JTH 2011; 9:2077) demonstrated that downstream of PAR and GPVI/FcRγ, another ITAM receptor, there are CalDAG-GEF I (CDGI)-dependent and CDGI-independent, ADP/P2Y12-dependent parallel signaling pathways to PS exposure. In this study, we investigated the molecular signaling requirements for PS exposure downstream of FcγRIIa + PAR dual stimulation.

We studied the exposure of PS in FcγRIIa transgenic (tg) mouse platelets following dual stimulation through FcγRIIa via anti-mouse CD9 and through PAR4 via PAR4 activating peptide (PAR4AP; AYPGKF). Washed platelets from FcγRIIa-tg mice and FcγRIIa-tg/CDG1^-/- mice were stimulated with varying concentrations of anti-mouse CD9 and PAR4-AP (200uM) under static conditions and immediately measured for PS exposure by labeled Annexin-V in flow cytometry. We observed that at 0.5ug/ml of anti-mouse CD9 that the PS exposure of the FcγRIIa-tg/CDG1^-/- platelets is approximately 70% of the FcγRIIa-tg platelets. However, when the platelets are pre-incubated with P2Y12 inhibitor MesAMP at 100uM, the PS exposure of the FcγRIIa-tg platelets is decreased by approximately 50% (n=3). For the FcγRIIa-tg/CDG1^-/- platelets in the presence of MesAMP, PS exposure is completely abolished (n=3). This indicates that CDG1 contributes part of the signal that leads to PS exposure, while ADP/P2Y12 contributes the other CDGI-independent part of PS exposure downstream of FcγRIIa and GPCR dual stimulation. At a lower concentration of anti-mouse CD9 (0.25ug/ml; near threshold), the level of PS exposure of the FcγRIIa-tg/CDG1^-/- platelets is approximately 80% of the FcgRIIa-tg platelets. In addition, at 0.25 ug/ml of anti-CD9, FcγRIIa-tg/CDG1^-/- platelets pre-incubated with the P2Y12 inhibitor revealed no stimulated PS exposure. This observation indicates that ADP/P2Y12 plays a more significant role in PS exposure as the concentration of FcγRIIa stimulant nears threshold.

Eradication of procoagulant PS exposure may require targeting of both the CDGI-dependent and CDGI-independent pathways for optimum therapeutic benefit in HIT and other immune-mediated thrombocytopenia and thrombosis disorders. CDGI inhibitors useful in human platelets will allow translation of these findings.