Long-term platelet priming after glycoprotein VI stimulation in comparison to Protease-Activating Receptor (PAR) stimulation

Figures

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  Fig 1. Post-hoc inhibitory effect of iloprost on TRAP6- and CRP-XL-induced platelet aggregation. Platelets in suspension were stimulated with 10 μM TRAP6 (A-C) or 5 μg/mL CRP-XL (D-F), and aggregation was recorded by light transmission aggregometry. Iloprost (10 nM) was added before (-2, -1 or -0.5 min), simultaneously with (0 min) or after (+0.5, +1, +2 min) the indicated agonist. Representative traces are shown); arrows indicate addition of agonist, arrowheads addition of iloprost (A, D). Graphs of aggregation slope as %T/min (B, E) and aggregation-area-under-the-curve (AUC, 10 min) (C, F), as fractions of control without iloprost. For maximal %T values, see S1 Data. Means ± SEM (n = 3). *P<0.05, **P<0.001 vs. control traces, paired Student t-test.

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  Fig 2. Post-hoc inhibitory effect of integrin antagonist on TRAP6- and CRP-XL-induced platelet aggregation. Platelets in suspension were stimulated with 10 μM TRAP6 (A-C) or 5 μg/mL CRP-XL (D-F), and aggregation was recorded by light transmission aggregometry. Tirofiban (1 μg/mL) was added before (-2, -1 or -0.5 min), simultaneously with (0 min) or after (+0.5, +1, +2 min) the indicated agonist. Representative traces are shown; arrows indicate addition of agonist, arrowheads addition of tirofiban (A, D). Bar graphs indicate the aggregation slope as %T/min (B, E) and aggregation-area-under-the-curve (AUC, 10 min) (C, F), as fractions of control without tirofiban. Means ± SEM (n = 3). *P<0.05, **P<0.001 vs. control traces, paired Student t-test.

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  Fig 3. Post-hoc inhibitory effect of Syk blocker on CRP-XL-induced platelet aggregation. Platelets in suspension were stimulated with 10 μM TRAP6 (A-C) or 5 μg/mL CRP-XL (D-F), and aggregation was recorded by light transmission aggregometry. Syk-IN (5 μM) was added before (-2, -1 or -0.5 min), simultaneously with (0 min) or after (+0.5, +1, +2 min) the indicated agonist. Representative traces; arrows indicate addition of agonist, arrowheads addition of Syk-IN (A, D). Bar graphs of aggregation slope as%T/min (B, E) and aggregation-area-under-the-curve (AUC, 10 min) (C, F), expressed as fractions of control without Syk-IN. Means ± SEM (n = 3). *P<0.05, **P<0.001 vs. control traces, paired Student t-test.

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  Fig 4. Post-hoc inhibitory effect of iloprost plus tirofiban on agonist-induced platelet aggregation. Platelets in suspension were stimulated with 10 μM TRAP6 (A-C) or 5 μg/mL CRP-XL (D-F), and aggregation was recorded by light transmission aggregometry. Iloprost (10 nM) in combination with tirofiban (1 μg/mL) was added before (-2, -1 or -0.5 min), simultaneously with (0 min) or after (+0.5, +1, +2, +4, +6 min) the indicated agonist. See Fig 1. Means ± SEM (n = 3). *P<0.05, **P<0.001 vs. control traces, paired Student t-test.

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  Fig 5. Recurrent Ca2+ signal generation induced by consecutive agonists. Calibrated [Ca2+]i rises were recorded during 25 min of Fura-2-loaded platelets in 96-wells plates. Platelets were stimulated at indicated time point (arrow) as second agonist with 10 μM TRAP6 (A, C, E) or 5 μg/mL CRP-XL (B, D, F). (A, B) Preaddition of vehicle (control), TRAP6 (10 μM) or CRP-XL (5 μg/mL) at 10 min before second agonist. (C, D) Preaddition of vehicle control or iloprost (10 nM) with/without TRAP6 (10 μM) at 10 min before second agonist. (E, F) Preaddition of iloprost (10 nM) with/without CRP-XL (5 μg/mL) at 10 min before second agonist. Means ± SEM (n = 3 experiments). *P<0.05, **P<0.001 vs. controls at t = 1000 s, paired Student t-test.

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  Fig 6. Effect of agonist stimulation on Ca2+ signalling induced by a second agonist. Calibrated [Ca2+]i rises were recorded during 25 min of Fura-2-loaded platelets in 96-wells plates. Platelets were stimulated at indicated time point (arrow) as second agonist with 10 μM TRAP6 (A, C), 5 μg/mL CRP-XL (B, D), 1 nM thrombin (E) or 1 μM Me-S-ADP (F). (A, B) Preaddition of vehicle (control) or thrombin (1 nM) at 10 min before second agonist. (C, D) Preaddition of vehicle (control) or Me-S-ADP (1 μM) at 10 min before second agonist. (E, F) Preaddition of vehicle (control), TRAP6 (10 μM) or CRP-XL (5 μg/mL) at 10 min before second agonist. Means ± SEM (n = 3 experiments). *P<0.05, **P<0.001 vs. controls at t = 1000 s, paired Student t-test.