Abstract
Platelets support coagulation by externalizing phosphatidylserine (PS), creating a catalytic surface for thrombin generation and fibrin formation. Although platelet PS exposure is required for hemostasis, excessive procoagulant platelet activity drives thrombosis. Sustained calcium influx after strong agonist stimulation promotes mitochondrial permeability transition pore (mPTP) opening, mitochondrial depolarization and calcium amplification that commit platelets to PS-high procoagulant states. Existing approaches to suppress this pathway are limited by the hemostatic requirement for platelet PS exposure and pleiotropic effects of cyclophilin-targeting agents. Here, we use a series of newly developed cyclophilin D-independent small molecule mPTP inhibitors to define how mPTP opening regulates procoagulant platelet activity. Under GPVI/PAR-driven procoagulant conditions, MC63, TR001 and TR002 preserved mitochondrial membrane potential, prevented intracellular calcium overload and reduced PS/P-selectin-positive platelets, microparticle release and membrane blebbing, while preserving agonist-induced calcium mobilization, secretion, integrin activation, aggregation, F-actin assembly and clot retraction. Mechanistically, mPTP inhibition limited calcium-dependent phosphatase, protease and mitochondrial activities associated with procoagulant platelet conversion. MC63, TR001 and TR002 reduced platelet-dependent thrombin generation and fibrin formation without affecting platelet-independent thrombin activity. In whole blood perfused over collagen, mPTP inhibitors reduced PS-positive platelets within thrombi without impairing platelet accumulation. In vivo, mPTP inhibitors decreased platelet and fibrin accumulation after laser-induced arteriolar injury but did not prolong tail bleeding alone or in combination with acetylsalicylic acid. Together, these findings identify mPTP opening as a druggable checkpoint for platelet procoagulant commitment and support mPTP inhibition as an antithrombotic strategy that may preserve hemostasis.