Platelet Ca2+ responses coupled to glycoprotein VI and Toll-like receptors persist in the presence of endothelial-derived inhibitors: roles for secondary activation of P2X1 receptors and release from intracellular Ca2+ stores by C. Y. Eleanor Fung, Sarah Jones, Adwoa Ntrakwah, Khalid M. Naseem, Richard W. Farndale, and Martyn P. Mahaut-Smith Blood Volume 119(15):3613-3621 April 12, 2012 ©2012 by American Society of Hematology

The potent endogenous inhibitor PGI2 abolishes platelet Ca2+ responses to G protein-coupled receptors but not those after activation of GPVI, Toll-like receptors, or P2X1 cation channels. The potent endogenous inhibitor PGI2 abolishes platelet Ca2+ responses to G protein-coupled receptors but not those after activation of GPVI, Toll-like receptors, or P2X1 cation channels. Effect of a supramaximal concentration of PGI2 (60μM; 90 seconds) on [Ca2+]i increases mediated via a range of receptors. Representative responses from more than 4 donors are shown in panels A through E, and the average as a percentage of a paired control is shown in panel F. The G protein–coupled receptor agonists U46619 (A,F; U4) and thrombin (B,F; Thr) were used at near-EC50 concentrations (0.5μM U46619 and 0.03 U mL−1 thrombin). GPVI and TLR2/1 receptor Ca2+ responses were stimulated with a low or intermediate concentration of collagen (0.5 and 2 μg mL−1; C,F; Coll) or Pam3CSK4 (1 and 10 μg mL−1, D,F; Pam), respectively. P2X1 receptors were maximally activated with 10μM α,βmeATP (E-F). The asterisks indicate significance levels compared with samples in the absence of PGI2; the hash symbols indicate significance level compared with thrombin in the presence of PGI2. U4 indicates U46619; Pam, Pam3CSK4; Thr, thrombin; and Coll, collagen. C. Y. Eleanor Fung et al. Blood 2012;119:3613-3621 ©2012 by American Society of Hematology

PGI2-resistant Ca2+ responses to GPVI and Toll-like receptors are mediated through both P2X1 receptor-dependent entry and release of intracellular stores. PGI2-resistant Ca2+ responses to GPVI and Toll-like receptors are mediated through both P2X1 receptor-dependent entry and release of intracellular stores. (A-D) Sample [Ca2+]i responses to collagen and Pam3CSK4 in the presence of 60μM PGI2 with and without functional P2X1 receptors. P2X1 receptors were desensitized by addition of 0.6μM α,βmeATP 60 seconds before addition of external Ca2+ and 90 seconds before agonist addition. PGI2-resistant Ca2+ responses at a low concentration of agonist (A, 0.5 μg mL−1 collagen and B, 1 μg mL−1 Pam3CSK4), were mainly because of P2X1 receptors. At a higher agonist concentration (C, 2 μg mL−1 collagen and D, 10 μg mL−1 Pam3CSK4), a slower, P2X1-independent component was revealed. (E-F) Average peak [Ca2+]i responses to 2 μg mL−1 collagen (coll; E) and 10 μg mL−1 Pam3CSK4 (Pam; F) under various conditions (60μM PGI2, desensitization of P2X1 receptors with 0.6μM α,βmeATP, 1mM EGTA (0 Ca2+) medium, 10μM U73122, and 100nM wortmannin. Asterisks indicate the significance level compared with control samples in the absence of PGI2, and hash symbols indicate the significance level compared with samples in the presence of PGI2 after desensitization of P2X1. C. Y. Eleanor Fung et al. Blood 2012;119:3613-3621 ©2012 by American Society of Hematology

Dense granule secretion stimulated by collagen and Pam3CSK4 is partially resistant to PGI2. Dense granule secretion stimulated by collagen and Pam3CSK4 is partially resistant to PGI2. Representative (A-D) and average peak (E) extracellular ATP levels monitored by luciferin:luciferase showing the effect of a supramaximal concentration of PGI2 (60μM; 90 seconds) on dense granule secretion evoked by U46619, thrombin, collagen, and Pam3CSK4. Agonist concentrations match those used in Ca2+ responses (Figures 1–2), with the exception that no detectable ATP release was observed with the lower concentration of Pam3CSK4 (1 μg mL−1). Asterisks indicate the significance level compared with control samples in the absence of PGI2, and hash symbols indicate the significance level compared with thrombin in the presence of PGI2. C. Y. Eleanor Fung et al. Blood 2012;119:3613-3621 ©2012 by American Society of Hematology

Collagen-evoked ATP release requires PLC activation but not P2X1 receptors and involves costimulation of Ca2+ mobilization and PKC. Sample (A,C,D) and average (B,E) ATP secretion responses to collagen (0.5 μg mL−1), monitored by luciferin:luciferase. Collagen-evoked ATP release requires PLC activation but not P2X1 receptors and involves costimulation of Ca2+ mobilization and PKC. Sample (A,C,D) and average (B,E) ATP secretion responses to collagen (0.5 μg mL−1), monitored by luciferin:luciferase. (A-B) Effect of desensitization of P2X1 receptors using 0.6μM α,βmeATP before addition of external Ca2+. (C-E) Effect of the PLC inhibitor U73122 (10μM; 10 minutes), cytosolic loading of the Ca2+ chelator BAPTA (15μM BAPTA-AM), the PKC inhibitor GF109203X (20μM; 10 minutes; GF), or combined BAPTA loading and GF109203X. C. Y. Eleanor Fung et al. Blood 2012;119:3613-3621 ©2012 by American Society of Hematology

Toll-like receptor–evoked ATP release requires activation of PLC, PKC, and an increase in intracellular Ca2+. Toll-like receptor–evoked ATP release requires activation of PLC, PKC, and an increase in intracellular Ca2+. Sample (A-C) and average (D) ATP release stimulated by 10 μg mL−1 Pam3CSK4, showing the effect of the PLC inhibitor U73122 (10μM; 10 minutes), intracellular loading with the Ca2+ chelator BAPTA (15μM BAPTA-AM) or the PKC inhibitor GF109203X (20μM; 10 minutes; GF). C. Y. Eleanor Fung et al. Blood 2012;119:3613-3621 ©2012 by American Society of Hematology

Secondary P2X1 receptor activation is resistant to nitric oxide but is substantially attenuated by increased ectonucleotidase activity. [Ca2+]i responses (A-E) and ATP secretion (F) induced by a range of agonists (U46619 [U4], thrombin [Thr], collagen [Coll... Secondary P2X1 receptor activation is resistant to nitric oxide but is substantially attenuated by increased ectonucleotidase activity. [Ca2+]i responses (A-E) and ATP secretion (F) induced by a range of agonists (U46619 [U4], thrombin [Thr], collagen [Coll], Pam3CSK4 [Pam], and α,βmeATP) were studied under control conditions and after exposure to either spermine NONOate (sNO; 100μM) or increased apyrase (3.2 U mL−1). In addition, the contribution of P2X1 receptors was assessed by desensitization before addition of external Ca2+ using 0.6μM α,βmeATP. Asterisks indicate the significance level compared with control samples in the absence of sNO, and hash symbols indicate the significance level compared with samples in the presence of sNO (E) or compared with thrombin in the presence of sNO (F). C. Y. Eleanor Fung et al. Blood 2012;119:3613-3621 ©2012 by American Society of Hematology

Amplification of collagen-evoked Ca2+ responses by P2X1 receptors is independent of platelet density. Amplification of collagen-evoked Ca2+ responses by P2X1 receptors is independent of platelet density. Representative (A,C) and average (B,D,E) ATP secretion (A-B) and [Ca2+]i responses (C-E) evoked by collagen (0.5 μg mL−1) at platelet densities of 10% to 100% (where 100% is equivalent to that in plasma). The contribution of P2X1 receptors was assessed by desensitization before addition of external Ca2+ using 0.6μM α,βmeATP (desensitized P2X1). (D) Absolute peak Ca2+ response under control conditions (dark bars) and after desensitization of P2X1 receptors with 0.6μM α,βmeATP (light bars). (E) Peak Ca2+ response as a percentage of control responses after P2X1 receptor desensitization with 0.6μM α,βmeATP; there was no significant difference between responses in panel E across the range of platelet densities. C. Y. Eleanor Fung et al. Blood 2012;119:3613-3621 ©2012 by American Society of Hematology