PB0779 - Anticoagulant effects of a new inhibitor of PDI isomerases

Background: Protein disulfide isomerases (PDIs) support thrombus formation via the regulation of platelet activation and coagulation. C-3399, recently developed a new inhibitor of PDIA1, PDIA3 and PDIA6 has antiplatelet effect, but its influence on thrombin generation remains unknown.

Aims: To investigate procoagulant phenotype of platelets in mice with atherosclerosis as compare with healthy mice, and to study the effects of newly in-house synthesized inhibitor C-3399 on thrombin generation in mouse plasma in vitro.

Methods: Murine platelet-poor plasma (PPP) and platelet-rich plasma (PRP) of wild-type (C57BL/6) and aged-matched ApoE LDLR -/- mice were incubated with C-3399, bepristat 2a, acetylsalicylic acid or cangrelor, then thrombin generation was triggered with convulxin, thrombin or tissue factor and recalcification. Thrombin generation was measured using Calibrated Automated Thrombography. Additionally, the effect of C-3399 on thromboxane A2 production by platelets was evaluated by measuring the concentration of TXB2 (a stable metabolite of TXA2) by ELISA kit.

Results: PRP of ApoE LDLR -/- mice exhibited elevated thrombin generation compared to PRP of C57BL/6 as expressed by higher endogenous thrombin potential (ETP) and thrombin peak. C-3399 did not affect thrombin generation in convulxin-stimulated PRP, while bepristat decrease ETP and peak, and prolonged lag time and time to peak. Interestingly, acetylsalicylic acid had no effect on convulxin-induced thrombin generation in PRP, however cangrelor reduced ETP and peak and prolonged time to peak and lag time. Surprisingly, PDI inhibitors, acetylsalicylic acid and cangrelor did not influence TG parameters in thrombin stimulated samples. Additionally, C-3399 and acetylsalicylic acid blocked tromboxane A2 synthesis in platelets stimulated with convulxin.

Conclusion(s): Platelets of ApoE/LDLR -/- mice exhibited procoagulant phenotype compared to wild-type age-matched mice. C-3399 blocked GPVI-mediated TXA2 synthesis but in contrast to bepristat had no inhibitory effects on GPVI-mediated thrombin generation in PRP.

This work was supported by the National Science Centre, Poland, grant SONATA-17 no. UMO 2021/43/D/NZ7/03366.