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

Abstract

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.

Piezo1 channel activates ADAM10 sheddase to regulate Notch1 and gene expression

“…An important observation of ours was that shear stress activates ADAM10 via Piezo1 (Figure 2h). It is consistent with previous work describing activation of ADAM10 by shear stress in human platelets 31 , which also express Piezo1 32 . The finding has implications for Notch1 signalling but also more widely because ADAM10 is a sheddase that also targets E-and VE-cadherin, CD44 and cellular prion protein 17,[33][34][35] .…”

Matrix Mechanosensation in the Erythroid and Megakaryocytic Lineages

“…Yoda-1 stimulation of platelets increased Ca 2+ influx by over 170% [96]. In vitro thrombus formation of whole blood was reduced by GsMTx-4; however, platelet aggregation was unaffected [96]. As noted above, the role of Piezo1 function has been well-characterized in RBC physiology; Piezo1 activation by Yoda-1 alters erythroid differentiation kinetics [86,90,94].…”

“…Piezo1 was activated in platelets and Meg-01 by fluid shear stress, and inhibition of Piezo1 with GsMTx-4 abrogated the Ca 2+ influx [96]. Yoda-1 stimulation of platelets increased Ca 2+ influx by over 170% [96]. In vitro thrombus formation of whole blood was reduced by GsMTx-4; however, platelet aggregation was unaffected [96].…”

“…As discussed above, both the erythroid and megakaryocytic lineages detect and react to mechanical forces through integrins as well as mechanosensitive ion channels. Extracellular Ca 2+ plays an important role in MK ability to sense the ECM, as MKs and/or MK cell lines (Meg-01) have been observed to express the mechanosensitive ion channels [30,95,96]. The mechanosensitive receptor TRPC6 has been linked to Ca 2+ influx and platelet function [97][98][99].…”

“…Piezo1 expression was found on platelets and the MK cell line, Meg-01 [96]. Piezo1 was activated in platelets and Meg-01 by fluid shear stress, and inhibition of Piezo1 with GsMTx-4 abrogated the Ca 2+ influx [96]. Yoda-1 stimulation of platelets increased Ca 2+ influx by over 170% [96].…”