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CME Evaluation

Oral Communication Session

Coagulation Factors and Co-Factors

Session: Mechanical forces and thrombus dynamics

OC 59.3 - A Novel Mechanism Mediated by Allosteric Disulfide Bonds Fine-Tunes Thrombus Formation

Tuesday, June 25, 2024
10:00 – 10:15 ICT
Room: 208 A-D

    Presenting Author(s)

  • Keyu Lv, phD

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

    • Co-Author(s)

  • SC

    Shuai Chen

    PhD student
    Department of Pharmacology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology
    wuhan, Hebei, 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)

  • JC

    Joyce Chiu

    Lecturer
    The Centenary Institute, University of Sydney
    Sydney, New South Wales, Australia

  • HW

    Haoqing J. Wang

    PhD student
    School of Biomedical Engineering, Faculty of Engineering, The University of Sydney
    Darlington, New South Wales, Australia

  • YH

    Yunyun Han

    Professor
    Department of Neurobiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology
    wuhan, Hebei, China (People's Republic)

  • XY

    Xiaodan Yang

    PhD student
    Department of Neurobiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology
    wuhan, Hebei, China (People's Republic)

  • SB

    Sheryl R Bowley

    Postdoctoral researcher
    Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School
    Boston, Massachusetts, United States

  • Zhaoming Tang, MD

    Director of the Clinical Hemostasis and Thrombosis Laboratory
    Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology
    Wuhan, Hubei, China (People's Republic)

  • Ning Tang, MPH

    Associate Professor
    Tongji hospital, Tongji Medical College, Huazhong University of Science and Technology
    Wuhan, Hubei, China (People's Republic)

  • Aizhen Yang, PhD

    Associate researcher
    The Cyrus Tang Hematology Center, Soochow University
    SuZhou, Jiangsu, China (People's Republic)

  • YW

    Yi Wu, MD, PhD

    Professor
    Soochow University
    Suzhou, Jiangsu, China (People's Republic)

  • Lining A. Ju, PhD

    University of Sydney, Australia

  • Alvin H. Schmaier

    Professor
    Department of Medicine, University Hospitals Cleveland Medical Center and Case Western Reserve University
    Cleveland, Ohio, United States

  • Philip Hogg, BSc(Hon), PhD

    University of Technology Sydney and Centenary Institute

  • 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) plays a vital role during thrombosis by catalyzing disulfide rearrangement. Histidine-rich glycoprotein (HRG) was identified to be a substrate of PDI by mechanism-based kinetic trapping.

Aims: This study aims to determine the effects of PDI-mediated disulfide cleavage on HRG functions and thrombosis.

Methods: Mass spectrometry was performed to identify the disulfide bonds targeted by PDI. ELISA, biomembrane force probe and thrombin generation assay were used to determine the in vitro effects of PDI on HRG-regulated coagulation. The murine model of laser-induced thrombosis was performed to examine the in vivo effects of PDI on HRG-mediated thrombus formation.

Results: PDI formed disulfide-linked complexes with HRG and cleaved three disulfide bonds in the histidine-rich region that is important for HRG’s interaction with heparan sulfate (HS) and factor XII (FXIIa). Compared to inert-PDI, wt-PDI enhanced the procoagulant and anticoagulant activities of HRG by neutralization of endothelial HS and inhibition of FXIIa activity, respectively. Following laser-induced injury of cremaster arterioles, HRG accumulated rapidly at the interface between platelet thrombi and endothelium depending on platelet-derived Zn2+. Treatment with Rutin, a PDI inhibitor, inhibited HRG accumulation but increased antithrombin deposition in the growing thrombus. HRG deficiency (Hrg-/-) led to delayed onset but enhanced formation of thrombus compared to WT. However, there was further thrombosis reduction in HRG and FXII double-knockout mice (DKO) compared to FXII deficiency alone (F12-/-), confirming HRG’s procoagulant activity independent of FXIIa. Mutation of target disulfides of PDI led to a gain-of-function HRG that promoted its activities during coagulation.

Conclusion(s): PDI cleaves allosteric disulfide bonds on HRG to fine-tune thrombosis. At the early stage, PDI reduces HRG to promote its neutralization of endothelial HS allowing for rapid activation of the clotting system. At the late stage, PDI reduces HRG to enhance its inhibition of FXIIa preventing excessive clot formation.