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.