Journal: The Journal of Biological Chemistry

Article Title: Evidence for shear-mediated Ca 2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

doi: 10.1074/jbc.M116.766196

Figure Lengend Snippet: Effect of GsMTx-4 on Ca 2+ entry via TRPC6, P2X1, and store-operated channels in platelets. A–C , representative [Ca 2+ ] i recordings ( left panels ) and average peak [Ca 2+ ] i responses ( right panels ) for store-operated ( n = 4) ( A ), TRPC6 ( n = 4) ( B ), and P2X1 cation channels ( n = 3) ( C ) in suspensions of platelets in the presence and absence of GsMTx-4. Store-operated Ca 2+ entry was assessed by addition of 1.26 m m CaCl 2 15 min after treatment with the SERCA inhibitor thapsigargin. TRPC6 was activated using the diacylglycerol analogue OAG. P2X1 was activated with the non-hydrolyzable ATP analogue α,β-meATP (10 μ m ). **, p < 0.01; ns , not significant.

Article Snippet: The antibody concentrations used were: FAM38A (Piezo1) rabbit anti-human polyclonal antibody (15939-1-AP; ProteinTech, Manchester, UK), 1:2000; P2X1 rabbit anti-human polyclonal antibody (APR-001; Alomone Labs, Israel), 1:1000; and FAM38B (Piezo2) rabbit anti-human polyclonal antibody (G-20 Santa Cruz Biotechnology, Heidelberg, Germany), 1:200.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Evidence for shear-mediated Ca 2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

doi: 10.1074/jbc.M116.766196

Figure Lengend Snippet: Assessment of P2X1 activity in Fura-2-loaded platelet and Meg-01 cell suspensions in the presence and absence of extracellular apyrase. A , selective activation of platelet P2X1 channels using α,β-meATP in the presence and absence of 0.32 unit/ml apyrase. B , representative [Ca 2+ ] i recordings ( panels i and ii ) and average Ca 2+ increases ( panel iii ) demonstrating that α,β-meATP does not induce [Ca 2+ ] i elevations in Meg-01 cells similar to vehicle control, indicating no P2X1 activity in these cells. As a positive control, the addition of a supramaximal concentration of ADP is shown to cause sharp [Ca 2+ ] i elevations indicating intact P2Y responses. ****, p < 0.0001. ns , not significant.

Article Snippet: The antibody concentrations used were: FAM38A (Piezo1) rabbit anti-human polyclonal antibody (15939-1-AP; ProteinTech, Manchester, UK), 1:2000; P2X1 rabbit anti-human polyclonal antibody (APR-001; Alomone Labs, Israel), 1:1000; and FAM38B (Piezo2) rabbit anti-human polyclonal antibody (G-20 Santa Cruz Biotechnology, Heidelberg, Germany), 1:200.

Techniques: Activity Assay, Activation Assay, Positive Control, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Evidence for shear-mediated Ca 2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

doi: 10.1074/jbc.M116.766196

Figure Lengend Snippet: GsMTx-4 inhibits thrombus formation independently of P2X1 receptors. A , P2X1-dependent Ca 2+ entry (α,β-meATP, 10 μ m ) in platelet suspensions is completely inhibited by 1 μ m NF449. B , effect of 2.5 μ m GsMTx-4 and 1 μ m NF449, individually and combined, on thrombi formed on a collagen surface. The average values are shown ( n = 7) for thrombus volume ( panel i ), surface coverage ( panel ii ), and thrombus height ( panel iii ). *, p < 0.05. ns , not significant.

Article Snippet: The antibody concentrations used were: FAM38A (Piezo1) rabbit anti-human polyclonal antibody (15939-1-AP; ProteinTech, Manchester, UK), 1:2000; P2X1 rabbit anti-human polyclonal antibody (APR-001; Alomone Labs, Israel), 1:1000; and FAM38B (Piezo2) rabbit anti-human polyclonal antibody (G-20 Santa Cruz Biotechnology, Heidelberg, Germany), 1:200.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Evidence for shear-mediated Ca 2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

doi: 10.1074/jbc.M116.766196

Figure Lengend Snippet: Mechanosensitive ion channel expression in human platelets and the Meg-01 cell line. A , relative expression of mRNA transcripts for three MS cation channels (Piezo1, Piezo2, and TRPC6) in human platelets and the Meg-01 cell line, relative to GAPDH. n.d. , not detected. The values are shown only for detectable levels of expression. B , Western blots for Piezo1 (233 kDa) and P2X1 receptors (55 kDa) in Meg-01 and human platelet lysates, compared with α-tubulin housekeeping control (60 kDa). The sizes (in kDa) and positions of protein standards are indicated with arrowheads . Meg-01 samples were from three different culture passages ( lanes P:2 , P:4 , and P:6 ), and platelet samples were from three different donors ( lanes 1 , 2 , and 3 ). The blank lane lacked protein lysate.

Article Snippet: The antibody concentrations used were: FAM38A (Piezo1) rabbit anti-human polyclonal antibody (15939-1-AP; ProteinTech, Manchester, UK), 1:2000; P2X1 rabbit anti-human polyclonal antibody (APR-001; Alomone Labs, Israel), 1:1000; and FAM38B (Piezo2) rabbit anti-human polyclonal antibody (G-20 Santa Cruz Biotechnology, Heidelberg, Germany), 1:200.

Techniques: Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Evidence for shear-mediated Ca 2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

doi: 10.1074/jbc.M116.766196

Figure Lengend Snippet: The Piezo1 agonist Yoda1 induced increases in [Ca 2+ ] i in platelets and Meg-01 cells. A–C , [Ca 2+ ] i responses to Yoda1 (25 μ m ) assessed in stirred Fura-2-loaded washed suspensions of platelets ( top panels ) and Meg-01 cells ( bottom panels ). A and B show representative recordings, and C shows the average peak [Ca 2+ ] i increases ( n = 4) for Yoda1 in the presence of extracellular Ca 2+ compared with its vehicle control (DMSO) and following removal of external Ca 2+ (EGTA). D , representative intracellular Ca 2+ recording (Fluo-3 F / F 0 fluorescence) from a single platelet attached to a PECAM-1-coated glass coverslip in the presence of Yoda1 and exposed to two cycles of no flow ( white regions ) and arterial shear ( gray regions ). See C , ( panel i ) for the control trace. E , average Ca 2+ increases above baseline ( F / F 0 ·4 min) in the presence and absence of Yoda1 under conditions of no flow and normal arterial shear ( n = 20, 35, 20, and 35 cells in HBSS no flow, Yoda1 no flow, HBSS normal arterial, and Yoda1 normal arterial conditions, respectively). **, p < 0.01; †, p < 0.01 compared with no flow, in the presence of HBSS; ‡, p < 0.001 compared with no flow in presence of Yoda1. Apyrase was omitted from the extracellular buffer to avoid P2X1 receptor responses (see ).

Article Snippet: The antibody concentrations used were: FAM38A (Piezo1) rabbit anti-human polyclonal antibody (15939-1-AP; ProteinTech, Manchester, UK), 1:2000; P2X1 rabbit anti-human polyclonal antibody (APR-001; Alomone Labs, Israel), 1:1000; and FAM38B (Piezo2) rabbit anti-human polyclonal antibody (G-20 Santa Cruz Biotechnology, Heidelberg, Germany), 1:200.

Techniques: Fluorescence