Fig 2: Fluid shear stress induces Ca2+ transients in single platelets that are inhibited by GsMTx-4 and chelation of extracellular Ca2+. A, a cartoon representation of single platelet attachment to PECAM-1 antibody-coated biochip surface via the Ig domains 1 and 2 of the platelet PECAM-1. B, representative Fluo-3 fluorescence and bright field (B.F.) images of individual Fluo-3-loaded attached platelets before and during exposure to arterial shear. Scale bars, 20 µm. The magnified rectangular sections have been enlarged 3-fold. C, representative F/F0 Fluo-3 recordings in single platelets during no applied shear stress (white regions) and normal arterial shear (gray regions). Two successive cycles of 4 min without shear followed by 4 min of arterial shear were applied, in which the second cycle was used to compare the control conditions (i.e. HBSS + Ca2+ only) (panel i), with the effect of GsMTx-4 (panel ii), or removal of extracellular Ca2+ (panel iii). D–G, average Ca2+ increases, calculated as the 4-min F/F0 integral of all [Ca2+]i transients. D, responses in the absence of shear and during arterial shear, in the presence of extracellular Ca2+ with and without GsMTx-4 and in the absence of extracellular Ca2+ (n = 46, 46, 23, and 14 cells in no shear, HBSS, GsMTx-4, and EGTA, respectively). *, p < 0.05, compared with HBSS-only control under shear. E, comparison of Ca2+ responses during cycles 1 and 2 of normal arterial flow with Ca2+-containing HBSS only (n = 46 and 13 cells, respectively). No significant difference was found between F/F0 integrals of the calcium transients from cycles 1 and 2. F, Ca2+ responses in Ca2+-containing HBSS in the absence of shear (no applied flow) and during arterial stenotic shear with and without GsMTx-4 (n = 36, 36, 38, 13, and 13 cells in no applied flow, HBSS normal arterial, HBSS stenotic arterial, GsMTx-4 normal arterial, and GsMTx-4 stenotic arterial flow conditions, respectively). *, p < 0.05, compared with HBSS-only control under stenotic shear. G, no significant difference was found between F/F0 integrals of the calcium transients from cycles 1 and 2 of stenotic arterial flow, using Ca2+-containing HBSS only (n = 38 and 20 cells, respectively). ****, p < 0.0001; ***, p < 0.001; **, p < 0.01. ns, not significant.