P2x1 Receptors Amplify Fc Gamma Riia-Induced Ca2+ Increases And Functional Responses In Human Platelets

Platelets express key receptors of the innate immune system such as Fc gamma RIIa and Toll-like receptors (TLR). P2X1 cation channels amplify the platelet responses to several major platelet stimuli, particularly glycoprotein (GP) VI and TLR2/1, whereas their contribution to Src tyrosine kinase-dependent Fc gamma RIIa receptors remains unknown. We investigated the role of P2X1 receptors during activation of Fc gamma RIIa in human platelets, following stimulation by cross-linking of an anti-Fc gamma RIIa monoclonal antibody (mAb) IV. 3, or bacterial stimulation with Streptococcus sanguinis. Activation was assessed in washed platelet suspensions via measurement of intracellular Ca2+ ([Ca2+](i)) increases, ATP release and aggregation. P2X1 activity was abolished by pre-addition of a, beta-meATP, exclusion of apyrase or the antagonist NF449. Fc gamma RIIa activation evoked a robust increase in [Ca2+](i) (441 +/- 33 nM at 30 mu g/mL mAb), which was reduced to a similar extent (to 66-70% of control) by NF449, pre-exposure to a, beta-meATP or apyrase omission, demonstrating a significant P2X1 receptor contribution. Fc gamma RIIa activation-dependent P2X1 responses were partially resistant to nitric oxide (NO), but abrogated by 500 nM prostacyclin (PGI(2)). Aggregation responses to bacteria and Fc gamma RIIa activation were also inhibited by P2X1 receptor desensitization (to 66 and 42% of control, respectively). However, Fc gamma RIIa-mediated tyrosine phosphorylation and ATP release were not significantly altered by the loss of P2X1 activity. In conclusion, we show that P2X1 receptors enhance platelet Fc gamma RIIa receptor-evoked aggregation through an increase in [Ca2+] i downstream of the initial tyrosine phosphorylation events and early dense granule release. This represents a further route whereby ATP-gated cation channels can contribute to platelet-dependent immune responses in vivo.