Evidence for shear-mediated Ca²⁺ entry through mechanosensitive cation channels in human platelets and a...

The role of mechanosensitive (MS) Ca²⁺-permeable ion channels in platelets is unclear, despite the importance of shear stress in platelet function and life-threatening thrombus formation. We therefore sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca²⁺ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to glass coverslips within flow chambers that allowed applications of physiological and pathological shear stress. Arterial shear (1002.6 s^-1) induced a sustained increase in [Ca²⁺] _i in Meg-01 cells and enhanced the frequency of repetitive Ca²⁺ transients by 80% in platelets. These Ca²⁺ increases were abrogated by the MS channel inhibitor Grammostola spatulata mechanotoxin 4 (GsMTx-4) or by chelation of extracellular Ca²⁺ Thrombus formation was studied on collagen-coated surfaces using DiOC₆-stained platelets. In addition, [Ca²⁺] _i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4, independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation or on Ca²⁺ influx via TRPC6 or Orai1 channels and caused only a minor inhibition of P2X1-dependent Ca²⁺ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca²⁺ transients by 170%. Piezo1 mRNA transcripts and protein were detected with quantitative RT-PCR and Western blotting, respectively, in both platelets and Meg-01 cells. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca²⁺ entry and thrombus formation under arterial shear.