Background: Cyclophilins (CyP) are involved in various pathways regulating platelet function. CyPA is a Ca2+ regulator and is involved in integrin αIIbβ3 bidirectional signaling, while CyPD mediates opening of the mitochondrial permeability transition pore resulting in procoagulant platelet formation. Platelet CyP has therefore been suggested as a therapeutic target to limit thrombosis. Many studies utilize the immunosuppressant Cyclosporin A (CsA) or derivatives. However, these have several disadvantages including side-effects unrelated to CyP inhibition. Among others, CsA was shown to enhance platelet aggregation, which was suggested to be responsible for the thrombotic complications observed in patients treated with CsA. Therefore, potent CyP inhibitors unrelated to CsA are needed.

Aims: Test the effect of F759, a novel non-peptidic small-molecule CyP inhibitor (SMCypI), on platelet activation, aggregation and procoagulant platelet formation, in comparison with CsA.

Methods: Washed human platelets were incubated with 4 µM CsA or 125 µM F759 prior to activation. Procoagulant platelet formation was measured using flow cytometry, by analyzing AnnexinV binding and P-selectin expression, as well as tetramethylrhodamine methyl ester staining demonstrating an intact mitochondrial membrane potential. P-selectin expression and activated integrin αIIbβ3 were measured using flow cytometry. Platelet aggregation was measured using light transmission aggregometry.

Results: Addition of F759 resulted in a decrease in AnnexinV+/P-selectin+ platelets upon dual-agonist activation, comparable to the inhibition observed with CsA. Similarly, F759 prevented the dual-agonist induced loss of mitochondrial membrane potential analogous to CsA. No effect was observed of F759 and CsA on P-selectin expression and integrin αIIβ3 activation after thrombin, collagen related peptide or ADP activation. F759 induced a moderate inhibition of sub-optimal concentrations of thrombin-, collagen- and ADP-induced platelet aggregation, while a significant increased aggregation was observed with CsA.

Conclusion(s): A novel non-peptidic SMCypI reduced procoagulant platelet formation and moderately reduced platelet aggregation. Further experiments are needed to demonstrate its potential therapeutic application to limit thrombosis.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/small-molecule-cyclophilin-inhibitors-as-a-potential-therapeutic-application-to-limit-thrombosis/