Fig 1: Small-molecule cyclophilin inhibitors inhibit procoagulant platelet formation. Washed platelets were incubated with 125 µM F759 or F83236, or 4 µM CsA. Platelets were then activated with dual agonists CRP-XL (5 µg/mL) and thrombin (2 U/mL) in the presence of 2.5 mM CaCl2. (A) During activation, platelets were labeled with anti-CD42b, anti-CD62P and AnnexinV. Using flow cytometry, CD42b+ platelets were gated, and the percentage of CD62P+/AnnexinV+ platelets was analyzed. (B) Platelets were incubated with TMRM before incubation with Cyp inhibitors. During activation, platelets were labeled with anti-CD42b. CD42b+/TMRMlow platelets were analyzed using flow cytometry. (C) SEM images of washed platelets. Platelets were allowed to adhere to fibrinogen-coated coverslips after incubation with Cyp inhibitors. Spread platelets are seen, as well as remnants of procoagulant platelets, which are indicated by arrows. (D) Washed platelets were incubated with 125 µM F759 or F83236, or 4 µM CsA. Platelets were then incubated without (NA) or with dual agonists PAR-4 (62.5 µM) and CRP-XL in the presence of 2.5 mM CaCl2. Next, platelets were incubated with PPP and ellagic acid before the addition of 25 mM CaCl2 to initiate coagulation. Data are represented as mean ± SD of 8 (panel A), 4 (panel B) or 5 (panel C) separate experiments. * p < 0.05, ** p < 0.005 compared to vehicle control by One-Way ANOVA. SMCypIs, small-molecule cyclophilin inhibitors; CRP-XL, collagen-related peptide; TMRM, tetramethylrhodamine methyl ester; Cyp, cyclophilin; CsA, Cyclosporin A; NA, no agonists; PPP, platelet-poor plasma.