Abstract Body: Background: Excessive platelet procoagulant activity, marked by sustained and high-magnitude phosphatidylserine (PS) externalization, promotes pathological thrombosis. While sustained cytosolic Ca²⁺ elevation is required for this phenotype, the mitochondrial mechanisms that govern this process remain incompletely defined. The mitochondrial permeability transition pore (mPTP) has been implicated as a potential regulator of Ca²⁺ overload and mitochondrial dysfunction in platelets; however, its causal role in regulating highly procoagulant platelet phenotypes has been difficult to establish, in part due to the lack of selective pharmacological tools. Existing approaches, such as cyclophilin D (CypD) inhibition with cyclosporine A, are confounded by off-target effects, limiting mechanistic interpretation.
Aim: To determine whether mPTP opening functions as a critical regulatory target in the generation of highly procoagulant platelet phenotypes, using a newly developed series of cyclophilin D–independent small-molecule mPTP inhibitors (MC63, TR001, and TR002) as mechanistic tools.
Results: Dual stimulation of washed human platelets with a GPVI agonist and thrombin induced mitochondrial depolarization, cytosolic Ca²⁺ overload, and robust phosphatidylserine (PS) exposure. Pretreatment with MC63, TR001, or TR002 (10 µM) preserved mitochondrial membrane potential (~90% polarized vs. 16.8% in vehicle-treated platelets), attenuated Ca²⁺ elevation, and markedly reduced PS exposure, as assessed by lactadherin binding (<10% vs. 47.5% in vehicle). These effects were confirmed by live-cell imaging of Annexin V–Alexa Fluor 488–stained platelets. In ex vivo whole blood under physiological shear (300 s^-1), mPTP inhibition significantly suppressed procoagulant platelet formation while preserving granule secretion, integrin activation, and aggregation. To assess in vivo efficacy, we utilized a mouse cremaster arteriole laser injury model with intravital microscopy. Intravenous administration of MC63 (10 µg/g) resulted in a significant reduction in thrombus burden, decreasing median platelet accumulation by 59.7% and fibrin deposition by 70.8% compared to vehicle controls. Injury size was comparable between groups.
Conclusion: These findings identify mPTP opening as a critical and druggable target controlling platelet commitment to highly procoagulant phenotypes and supporting mPTP inhibition as an antithrombotic strategy that may spare hemostatic platelet functions.