Novel small molecule inhibitors of mitochondrial permeability transition pore (mPTP) block platelet procoagulant activity

Abstract

Abstract Subpopulations of procoagulant platelets expose phosphatidylserine (PS) on their plasma membrane, providing a catalytic surface for thrombin generation and fibrin formation. Platelet PS exposure is critical for coagulation factor assembly in hemostasis; however, dysregulated procoagulant platelet activity underlies pathological thrombus formation. In many vascular, inflammatory and thrombotic disorders, increases in platelet cytosolic calcium levels, driven by the opening of the mitochondrial permeability transition pore (mPTP), underlie robust platelet PS exposure and procoagulant activity and thrombosis. Procoagulant platelets and the platelet mPTP have long been a target of interest for antithrombotic therapies. However, traditional therapeutic mPTP inhibitors, such as cyclosporine A, which target cyclophilin D (CypD), exhibit off-target effects and cytotoxicity, limiting their clinical application. We recently characterized a series of novel, CypD-independent small molecule mPTP inhibitors through a high-throughput screen of NIH compound libraries. Our chemical biology efforts led to the development of an isoxazole-based mPTP inhibitor (MC63) and structurally related triazole analogues, including TR002, optimized for potency and stability. In this study, we evaluate the potential of MC63, TR002, and related compounds as pharmacological antithrombotic agents in preclinical models of platelet activity and thrombosis. To determine whether MC63 and TR002 target platelet mPTP, we assessed mitochondrial membrane potential by tetramethylrhodamine ethyl ester (TMRE) staining and flow cytometry in resting and procoagulant platelets prepared from anticoagulated blood from healthy human donors. Platelet glycoprotein GPVI agonist (crosslinked collagen-related peptide, CRP-XL) and thrombin stimulation caused mitochondrial depolarization, leaving only 24.4% of vehicle-treated platelets polarized, versus 97.2% in unstimulated controls. Pretreatment with MC63 or TR002 (10 µM each) preserved mitochondrial polarization (94.1% and 93.8%, respectively). In parallel, platelet PS exposure (lactadherin-FITC staining), significantly increased upon CRP-XL/thrombin stimulation (vehicle: 34.1%; resting: 0.7%). MC63 and TR002 pretreatment markedly reduced PS-positive platelets (1.9% and 4.6%; P < 0.0001). Both MC63 and TR002 also significantly reduced procoagulant platelet generation ex vivo in whole blood stimulated with GPVI and PAR4 agonists (MC63: P < 0.0001; TR002: P = 0.0114). Annexin V-Alexa Fluor 488 live-cell imaging experiments also showed significant reductions in platelet PS exposure in response to procoagulant agonists. Although MC63 or TR002 attenuated PS exposure of platelets co-stimulated with CRP-XL and thrombin, flow cytometry analysis showed no significant differences in platelet secretion (CD62P-APC) or integrin activation (PAC1-FITC) compared to controls stimulated with CRP-XL (P > 0.9999). Light transmission aggregometry of CRP-XL-stimulated platelets also indicated no significant impact of these mPTP inhibitors on platelet aggregation, which was comparable to controls (vehicle: 79.2%; MC63: 74.9%, P = 0.952; TR002: 86.1%, P = 0.995). To evaluate the antithrombotic effects of MC63 and TR002, we first used a collagen-coated Ibidi flow chamber assay. Treatment of whole blood from healthy human donors with MC63 or TR002 significantly reduced Annexin V staining and thrombus size of adherent platelets under physiologic shear (P < 0.0001). Finally, to examine the effects of mPTP inhibition on thrombosis in vivo, we assessed arteriolar thrombus formation in mouse cremaster vasculature following laser injury, quantifying thrombus growth by platelet and fibrin accumulation over time with intravital microscopy. Intravenous injection of MC63 (10 µg/g) reduced median platelet and fibrin accumulation by 59.7% (P < 0.05) and 70.8% (P < 0.0001; N = 30 injuries from 3 mice), respectively, compared with vehicle-treated controls (N = 28 injuries from 3 mice). Injury size was comparable between groups. Altogether, our findings support MC63 and TR002 as selective inhibitors of platelet mPTP, and procoagulant platelet activity, effectively limiting thrombosis without impairing essential hemostatic functions. These data highlight pharmacologic mPTP inhibition as a promising approach to safer antithrombotic therapies.