Page 202 - Haematologica May 2022

Letters to the Editor
nor CCCP had any impact on thrombus growth in the presence of ticagrelor, an ADP receptor antagonist (Figure 2B, G-I). Furthermore, adhesion and spreading of platelets pretreated with tirofiban, an aIIbβ3 integrin blocker, on collagen matrix under shear were unaffected in the presence of antimycin, oligomycin or CCCP (Figure
2C, J-L). Thus, ATP sourced from mitochondria was dis- pensable for platelet aggregation, adhesion and procoag- ulant activity but was essential for granule secretion, platelet-neutrophil interactions, as well as thrombus growth.
We asked whether differential modulation of platelet
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Figure 1. Effects of pre-treatment with mitochondrial inhibitors (antimycin, oligomycin) or an uncoupler (CCCP) on human platelet function in vitro. Washed human platelets were pre-treated with vehicle (control), antimycin (2 μg/mL), oligomycin (10 μg/mL) or CCCP (100 μM) for 15 min at room temperature, followed by addition of human thrombin (0.5 U/mL) for 30 min, or as indicated. (A) Representative tracings showing thrombin (0.2 U/mL)-induced aggregation (1-4) or ATP release (1’-4’) of platelets pre-treated with vehicle (1,1’), antimycin (2,2’), oligomycin (3,3’) or CCCP (4,4’). (B, C) Bar diagram quantifying mean platelet aggregation and platelet dense granule secretion in different samples, respectively. (D-F) Representative histogram overlay plots showing binding of FITC-PAC1 (BD Biosciences #340507), PE-annexin V (Biolegend #640908), and PE-anti-CD62P (BD Biosciences #550561), respectively. (G-I) Corresponding bar diagrams quantifying aIIbβ3 integrin activation, phosphatidylserine exposure and P-selectin expression, respectively. Each dot represents an independent observation. Data are presented as mean ± standard error of mean. *P<0.05 with respect to vehicle-treated thrombin-stimulated platelets. #P<0.05 with respect to vehicle- treated unstimulated platelets. Significance in difference of means was tested by repeated measures analysis of variance and the Dunnett multiple comparison test. PS: phosphatidylserine; Thr: thrombin.
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