Ex vivo analysis of wild-type and IRAG^Δ12/Δ12 platelets. (A) Effect of 8-pCPT-cGMP (200 μM), DEA/NO (30 μM), cBIMPS (30 μM), or prostacyclin (5 μM) on collagen-induced aggregation of wild-type (WT) and IRAG^Δ12/Δ12 (Δ12/Δ12) platelets. Representative traces of wild-type and IRAG^Δ12/Δ12 platelets are shown. The arrow indicates the addition of collagen (10 μg/mL) (CTR indicates control without addition of 8-pCPT-cGMP, DEA/NO, cBIMPS, or prostacyclin). (B) Statistical evaluation of 8-pCPT-cGMP (200 μM), DEA/NO (0.3 μM, 30 μM), or SNP (5 μM, 10 μM) on platelet aggregation induced by collagen (5 or 10 μg/mL, as indicated) or thrombin (0.1 U/mL). (C, top) Statistical evaluation of fibrinogen binding to wild-type and IRAG^Δ12/Δ12 platelets after pretreatment with 8-pCPT-cGMP (200 μM), DEA/NO (100 nM), iloprost (10 μM), or cBIMPS (30 μM) followed by induction of fibrinogen receptor activation with thrombin (0.1 U/mL). (C, bottom) Statistical evaluation of GPIIb-IIIa activation of wild-type and IRAG^Δ12/Δ12 platelets after pretreatment with 8-pCPT-cGMP (200 μM) or DEA/NO (100 μM) followed by platelet activation with thrombin (0.05 U/mL). (D) Effect of 8-pCPT-cGMP (200 μM, 10 minutes of preincubation) or DEA/NO (10 μM, 1 minute of preincubation) on thrombin-induced calcium release of Fura-2 AM (1 μM) loaded wild-type and IRAG^Δ12/Δ12 platelets. Representative traces of wild-type and IRAG^Δ12/Δ12 platelets are shown. The arrow indicates the addition of thrombin (0.4 U/mL) (CTR indicates control without addition of 8-pCPT-cGMP, DEA/NO; au, arbitrary units). (E) Statistical evaluation of thrombin-induced calcium release in wild-type and IRAG^Δ12/Δ12 platelets. Fura-2 AM (1 μM) loaded platelets were incubated with DEA/NO (10 μM, 1-5 minutes) or 8-pCPT-cGMP (100-200 μM, 10-30 minutes) and then stimulated with thrombin (0.4 U/mL).