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

Abstract

Platelets can respond to multiple antagonists and agonists, implying that their activation state is a consequence of past exposure to these substances. While platelets are often considered as one-time responsive cells, they likely can respond to sequential application of inhibitors and stimuli. We hypothesized that the ability of platelets to sequentially respond depends on the time and type of repeated agonist application. The present proof-of-concept data show that iloprost (cAMP elevation), tirofiban (integrin αIIbβ3 blocker) and Syk kinase inhibition subacutely modulated platelet aggregation, i.e. halted this process even when applied after agonist. In comparison to thrombin-activated receptor (PAR) stimulation, glycoprotein VI (GPVI) stimulation was less sensitive to time-dependent blockage of aggregation, with Syk inhibition as an exception. Furthermore, cytosolic Ca2+ measurements indicated that, when compared to PAR, prior GPVI stimulation induced a more persistent, priming activation state of platelets that influenced the response to a next agent. Overall, these data point to an unexpected priming memory of activated platelets in subacutely responding to another inhibitor or stimulus, with a higher versatility and faster offset after PAR stimulation than after GPVI stimulation.

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.

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.

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.

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.

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.

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.