Small-Molecule Cyclophilin Inhibitors Potently Reduce Platelet Procoagulant Activity

Figures

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  Figure 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…

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  Figure 2. Small-molecule cyclophilin inhibitors do not affect platelet activation. Washed platelets were incubated with 125 µM F759 or F83236, or 4 µM CsA. Platelets were then activated with either ADP (1 or 10 µM), CRP-XL (0.1 or 1 µg/mL) or thrombin (0.2 or 0.8 U/mL). During activation, platelets were labeled with anti-CD42b, anti-CD62P and anti-activated αIIbβ3 (PAC-1 clone). CD42b+/CD62P+/activated αIIbβ3+ platelets were analyzed using flow cytometry. Data are represented as mean ± SD of 4 separate experiments. SMCypIs, small-molecule cyclophilin inhibitors; CsA, Cyclosporin A; ADP, adenosine 5′-diphosphate; CRP-XL, collagen-related peptide.

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  Figure 3. Cyclosporin A enhances platelet aggregation, in contrast to small-molecule cyclophilin inhibitors. Platelet Rich Plasma (PRP) or washed platelets were incubated with 125 µM SMCypls (F759 or F83236) or with 4 µM CsA. (A) Aggregation in PRP was induced with 0.9 µM ADP (A), while aggregation of washed platelets was induced by (B) 2.5 µg/mL Horm collagen or (C) 0.1 U/mL thrombin. Left: Aggregation curves from representative experiments upon incubation with vehicle (black) or CsA (red). Right: Maximal aggregation values are shown. Data are represented as mean ± SD of 4 separate experiments. * p < 0.05 compared to the vehicle by One-Way ANOVA. SMCypIs, small-molecule cyclophilin inhibitors; CsA, Cyclosporin A; ADP, adenosine 5′-diphosphate.

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  Figure 4. Small-molecule cyclophilin inhibitors inhibit coagulation induced by procoagulant platelets. (A) Whole blood was incubated with 125 µM F759 or F83236, 4 µM CsA before perfusion over a Horm collagen-coated surface in the presence of 6.6 mM CaCl2 and 3.08 mM MgCl2. Shown are representative images after 9 min of top row: DIC images of platelet aggregates; middle row: anti-GpIb-VhH Alexa647 indicating platelets and bottom row: tPA-Alexa488 binding to fibrin. (B) Quantification of Δ MFI anti-GpIb-VhH Alexa647 signal during 9 min. (C) MFI tPA-Alexa488 over time. (D) Quantification of Δ MFI tPA-Alexa488 signal during 9 min. Data represented as mean ± SD of 5–7 separate experiments. * p < 0.05 compared to vehicle by One-Way ANOVA. SMCypIs, small-molecule cyclophilin inhibitors; CsA, Cyclosporin A; tPA, tissue plasminogen activator.