Background Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have now examined the role of connexins in platelets, blood cells that circulate in isolation, but upon tissue injury adhere to each other and the vessel wall to prevent blood loss and facilitate wound repair. Methods and Results We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses prior to platelet-platelet contact, and reduced laser induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion and clot retraction indicating an important role for Cx37 hemichannels and gap junctions in platelet thrombus function. Conclusions Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of haemostasis and thrombosis and represent potential therapeutic targets.

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.