PB0756 - A Novel Potent Inhibitor of Protein Disulfide Isomerase Suppresses Platelet Aggregation and Thrombus Formation

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Lili Zhang

PhD Student
Huazhong University of Science and Technology
wuhan, Hubei, China (People's Republic)

Co-Author(s)

Keyu Lv, phD

PhD student
Huazhong University of Science and Technology
Wuhan, Hebei, China (People's Republic)
HW

Hao Wang

PhD Student
Huazhong University of Science and Technology
Wuhan, Hubei, China (People's Republic)
GZ

Guijuan Zheng

Postdoctoral researcher
Huazhong University of Science and Technology
Wuhan, Hubei, China (People's Republic)
XX

Xulin Xu

Associate Professor
Department of Pharmacology, School of Basic Medicine, Tongji Medical College and State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Huazhong University of Science and Technology
Wuhan, Hubei, China (People's Republic)
MH

Mingdong Huang

Professor
College of Chemistry, Fuzhou University
Fuzhou, Fujian, China (People's Republic)
GY

Guangmin Yao

Professor
Huazhong University of Science and Technology
Wuhan, Hubei, China (People's Republic)
Chao Fang, PhD

Professor of Pharmacology
Department of Pharmacology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology
Wuhan, Hubei, China (People's Republic)

Background: Protein disulfide isomerase (PDI) regulates the functions of coagulation-related proteins by catalyzing disulfide rearrangements. Inhibition of PDI is emerging as a new antithrombotic approach. A class of novel inhibitors of PDI were identified from Pieris japonica, a medicinal plant used for the treatment of clotting diseases.

Aims: This study aims to evaluate the antithrombotic effects of compound 8-2, the isolated constituent with the highest potency in the inhibition of PDI.

Methods: Insulin reduction assay was used to measure the reductase activity of PDI. Molecular docking, tryptophan fluorescence assay and rescue experiments using PDI fragments were performed to determine the mode of actions of 8-2. Platelet aggregation, adhesion, and spreading were used to evaluate in vitro platelet functions. The murine model of laser-induced thrombosis was utilized to evaluate the in vivo effects of 8-2. The cytotoxicity was examined by measuring lactate dehydrogenase. The bleeding risks were evaluated using murine models of tail bleeding and saphenous vein bleeding.

Results: 8-2 inhibited PDI reductase activity (IC50 at 0.23 μM, lower than that of Rutin 3.93 μM) by directly interacting with the b′x region of PDI, and without affecting the activities of ERp5, ERp57 and ERp72. 8-2 significantly suppressed α-thrombin-induced platelet aggregation (IC50 at 2.23 μM, lower than that of isoquercetin 21.61 μM) as well as platelet adhesion and spreading on collagen. 8-2 inhibited laser-induced thrombus formation in the cremaster arterioles in a dose-dependent manner with a higher efficacy than isoquercetin. The recombinant b′x fragment of PDI rescued both platelet functions in vitro and thrombus formation in vivo in the presence of 8-2. 8-2 didn’t not result in significant cytotoxicity or prolong the bleeding time.

Conclusion(s): The novel PDI inhibitor 8-2 inhibits platelet activation and thrombus formation by binding to b′x, the substrate binding domain of PDI, without causing significant cytotoxicity or affecting physiological hemostasis.