Combined Antiplatelet Therapy Reduces the Proinflammatory Properties of Activated Platelets

Figures

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  Fig. 1. Different platelet activation pathways have no influence on chemokine release. Washed platelets (2 × 10 8 /mL) were activated with convulxin (CVX, 100 ng/mL), thrombin (IIa, 5 nM), or TRAP-6 (50 µM) for 30 minutes at 37°C. Platelets were removed and chemokines CCL5 ( A ) and CXCL4 ( B ) were determined with enzyme-linked immune sorbent assay. Control (CTRL) represents no stimulated platelets. Chemokine release was followed in time after convulxin ( C ) and thrombin ( D ) activation. Closed circles represent convulxin activation and open circles represent thrombin activation. CTRL: n = 31; CVX: n = 28–30, IIa: n = 25–29; and TRAP-6: n = 15. Median with interquartile range ( A, B ); mean ± standard deviation ( C, D ). *** p < 0.001, Kruskal–Wallis with Dunn's test.

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  Fig. 2. Effects of antiplatelet drugs against α IIb β 3 on chemokine release. Washed platelets (2 × 10 8 /mL) were incubated with indicated compounds, 5 minutes prior to platelet activation, and chemokine release was determined: CCL5 release after convulxin ( A ) and thrombin ( B ) activation and CXCL4 release after convulxin ( C ) and thrombin ( D ) activation. Closed circles represent convulxin activation and open circles represent thrombin activation. CTRL: n = 25–30; eptifibatide: n = 4–7; and tirofiban: n = 4–5. Median with interquartile range. * p < 0.05, Kruskal–Wallis with Dunn's test.

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  Fig. 3. Effect of acetylsalicylic acid (ASA) on chemokine release. Washed platelets (2 × 10 8 /mL) from healthy volunteers exposed to ASA (100 mg p.o.) were activated and CCL5 ( A ) and CXCL4 ( B ) release was determined as described. Closed circles represent convulxin activation and open circles represent thrombin activation. CTRL: n = 25–30; and ASA: n = 8. Median with interquartile range. * p < 0.05; ** p < 0.01; *** p < 0.001; Kruskal-Wallis with Dunn's test.

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  Fig. 4. Effects of P2Y 12 inhibition on chemokine release. Washed platelets (2 × 10 8 /mL) from healthy volunteers exposed to acetylsalicylic acid (ASA: 100 mg p.o.) were activated and CCL5 ( A, B ) and CXCL4 ( C, D ) release was determined as described. Prior to activation, platelets were incubated with P2Y 12 inhibitor cangrelor for 5 minutes. Closed circles represent convulxin activation and open circles represent thrombin activation. CTRL: n = 25–30; Cangrelor: n = 4; Cangr. + ASA: n = 8. Median with interquartile range. * p < 0.05; ** p < 0.01; Kruskal–Wallis with Dunn's test.

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  Fig. 5. Dual treatment with acetylsalicylic acid (ASA) + cilostazol treatment has no additional effects on chemokine release. Washed platelets (2 × 10 8 /mL) from healthy volunteers exposed to ASA (100 mg p.o.) were activated and CCL5 ( A, B ) and CXCL4 ( C, D ) release was determined as described. Platelets were incubated 10 minutes prior to activation with the PDE3 inhibitor cilostazol. Closed circles represent convulxin activation and open circles represent thrombin activation. CTRL: n = 25–29; Cilo: n = 6; ASA: n = 8; and ASA + Cilo: n = 6. Median with interquartile range. * p < 0.05; ** p < 0.01; and *** p < 0.001; analysis of variance with Sidak test.

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  Fig. 6. Combination treatment with cangrelor + cilostazol has no additional effects on chemokine release. Washed platelets (2 × 10 8 /mL) from healthy volunteers were pretreated with P2Y 12 inhibitor cangrelor, 5 minutes before activation and CCL5 ( A, B ) and CXCL4 ( C, D ) release was determined as described. Platelets were incubated 10 minutes prior to activation with the PDE3 inhibitor cilostazol. Closed circles represent convulxin activation and open circles represent thrombin activation. CTRL: n = 25–30; Cilo: n = 6–7; Cangrelor: n = 4; and Cangr. + Cilo: n = 4. Median with interquartile range. * p < 0.05 and ** p < 0.01, analysis of variance with Sidak test.

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  Fig. 7. Chemoattractant properties of activated platelets and the effects of antiplatelet drugs. Migration of monocytic cells (10 6 /mL) was induced in a 12-well chemotaxis chamber for 90 minutes at 37°C. Buffer, or supernatants of resting or activated washed platelets, was added in the bottom compartment. If applicable, platelets were activated by convulxin or thrombin without or with acetylsalicylic acid (ASA) or tirofiban ( A ), cangrelor or ASA + cangrelor (dual) ( B ), cilostazol (cilo) or ASA + cilo (dual) ( C ) , and cangrelor or cangrelor + cilo ( D ). CCL5 (0.5 µg/mL) and platelet releasates without or with Evasin-4 (Ev-4, 1 µg/mL) ( E ). n = 4–6; median with interquartile range. * p < 0.05; Kruskal–Wallis with Dunn's test.