PB0873 - Small-molecule cyclophilin inhibitors potently reduce platelet procoagulant activity

Monday, June 26, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Jens Van Bael

PhD student
Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium

Co-Author(s)

AV

Aline Vandenbulcke

Lab technician
Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
AA

Abdelhakim Ahmed-Belkacem

Dr.
INSERM U955, Team Viruses, Hepatology Cancer, Créteil, France
Créteil, Ile-de-France, France
JG

Jean-François Guichou

Prof. Dr.
Centre de Biologie Structurale (CBS), INSERM U1054, CNRS UMR5048, Université de Montpellier, Montpellier, France
Montpellier, Languedoc-Roussillon, France
JP

Jean-Michel Pawlotsky

Prof. Dr.
INSERM U955, Team Viruses, Hepatology Cancer, Créteil, France
Créteil, Ile-de-France, France
JS

Jelle Samyn

Student
Laboratory for Thrombosis Research, KU Leuven Campus Kulak, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
AB

Arjan D. Barendrecht

Lab technician
Department of CDL Research, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands
Utrecht, Utrecht, Netherlands
Coen Maas, PhD

Associate Professor
CDL Research, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands
Utrecht, Utrecht, Netherlands
SD

Simon F. De Meyer

Prof., Dr., Eng.
Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
Karen Vanhoorelbeke, PhD

Prof., Dr.
KU Leuven
Kortrijk, West-Vlaanderen, Belgium
CT

Claudia Tersteeg

Prof., Dr.
Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium

Background: Cyclophilins (CyP) are involved in various pathways regulating platelet function. CyPD mediates opening of the mitochondrial permeability transition pore resulting in procoagulant platelet formation. Procoagulant platelets acts as a scaffold for the prothrombinase complex, catalyzing fibrin formation. Inhibition of platelet CyPD has therefore been suggested as a therapeutic strategy to limit thrombosis. Indeed, Cyclosporin A (CsA) inhibits procoagulant platelet formation via CypD. However, CsA treated patients have increased thrombotic complications due to CsA enhanced platelet aggregation. Therefore, potent CyP inhibitors unrelated to CsA are needed.

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

Methods: Prior to the experiment, human platelets were incubated with CsA or SMCypIs. Platelet aggregation was measured using light transmission aggregometry. Procoagulant platelet formation and platelet activation was measured using flow cytometry. Procoagulant platelet function was evaluated using a modified activated Partial Thromboplastin Time assay to measure the procoagulant platelet-dependent clotting time. Fibrin formation under flow was visualized by perfusing recalcified whole blood in the presence of tPA-Alexa488 over collagen coated surfaces.

Results: While CsA increased both ADP- and collagen-induced platelet aggregation, aggregation in the presence of the SMCypIs was unaffected. No effect was observed on platelet activation. Importantly, SMCypIs were able to significantly decrease procoagulant platelet formation upon dual-agonist stimulation, as shown by a decreased phosphatidylserine (PS) exposure as well as a reduction in the loss of mitochondrial membrane potential. Additionally, the SMCypIs as well as CsA potently reduced procoagulant platelet-dependent clotting time as well as fibrin formation under flow.

Conclusion(s): We demonstrate that the CsA-induced platelet aggregation is not observed upon specific cyclophilin inhibition, while a clear reduction in procoagulant platelets is observed. Reducing platelet procoagulant activity by specific cyclophilin inhibition could be a promising strategy to limit thrombosis.