Mechanosensitive and FcγRIIa-mediated Platelet Calcium Entry Mechanisms

Elevation of intracellular Ca2+ ([Ca2+]i) is essential for platelet function. Despite the established role of shear stress in haemostasis and thrombosis, the possible contribution of mechanosensitive (MS) Ca2+-permeable ion channels to platelet activation remains unknown. One well-established Ca2+-permeable ion channel which enhances platelet responses at high shear is the ATP-gated P2X1 channel. The relative contribution of this channel to platelet function was studied following the activation of the FcγRIIa immune receptor. qRT-PCR and Western blotting revealed that human platelets and a megakaryocytic cell line, Meg-01, express the MS cation channel Piezo1. To investigate Ca2+-permeable MS channel activity, single platelets and Meg-01 cells were loaded with the Ca2+ indicator Fluo-3, and exposed to arterial shear in flow chambers which induced increases in [Ca2+]i in both cell types in physiological salines. The MS channel blocker GsMTx-4 inhibited these responses and reduced thrombus formation over collagen, whereas Piezo1 channel agonist Yoda1 potentiated platelet shear-induced Ca2+ transients. In Fura-2-loaded platelet suspensions, the GsMTx-4-sensitive shear-evoked responses were shown to be independent of P2X1, Orai1 and TRPC6. FcγRIIa-mediated [Ca2+]i elevations and aggregation were monitored using ratiometric [Ca2+]i measurements and light transmission, respectively. The contribution of P2X1 channels was assessed following inhibition by NF449, and inactivation by pre-addition of α,β-meATP or apyrase exclusion. These treatments significantly reduced antibody- or bacteria-induced FcγRIIa-mediated responses, indicating a significant P2X1 channel contribution. Phosphorylation assays indicated that P2X1 amplifies FcγRIIa-mediated responses via direct Ca2+ influx, rather than via a feedforward effect on early tyrosine phosphorylation. In conclusion, this thesis provides evidence that platelets express functional MS Piezo1 channels which can provide a direct route for Ca2+ entry under normal and pathological arterial shear, and contribute to thrombus formation. Additionally, P2X1 channels were shown to amplify FcγRIIa-mediated platelet Ca2+ signalling and aggregation, which can contribute to platelet activation under shear in infective endocarditis