OC 41.3 - Histidine-rich glycoprotein binds GPIIb/IIIa and attenuates platelet aggregation

Monday, July 11, 2022

3:15 PM – 3:30 PM

Location: ExCel Center, S11A

Presentation Format: In-Person

Presenting Author(s)

Rida Malik, HBSc

PhD Candidate
McMaster University, Hamilton, ON, Canada | Thrombosis and Atherosclerosis Research Institute (TaARI), Hamilton, ON, Canada
Hamilton, Ontario, Canada

Co-Author(s)

MN

Miguel Neves

Postdoctoral Fellow
Department of Laboratory Medicine and Pathobiology, University of Toronto | Keenan Research Center for Biomedical Science, St. Michael’s Hospital, Toronto, ON, Canada | Canadian Blood Services Centre for Innovation, Toronto, ON, Canada
Toronto, Ontario, Canada
JZ

Ji Zhou

Research Scientist
McMaster University, Hamilton, ON, Canada | Thrombosis and Atherosclerosis Research Institute (TaARI), Hamilton, ON, Canada
Hamilton, Ontario, Canada
RH

Rawaa Hussain

Research Technician
McMaster University, Hamilton, ON, Canada | Thrombosis and Atherosclerosis Research Institute (TaARI), Hamilton, ON, Canada
Hamilton, Ontario, Canada
JF

James Fredenburgh

Laboratory Manager
McMaster University, Hamilton, ON, Canada | Thrombosis and Atherosclerosis Research Institute (TaARI), Hamilton, ON, Canada
Kitchener, Ontario, Canada
HN

Heyu Ni

Principal Investigator
St. Michael's Hospital, Keenan Research Centre
Toronto, Ontario, Canada
JW

Jeffrey I. Weitz, MD

Principal Investigator
McMaster University | Thrombosis and Atherosclerosis Research Institute (TaARI)
Hamilton, Ontario, Canada

Background: Histidine-rich glycoprotein (HRG) circulates in plasma at a concentration of 1-3 µM and is released from activated platelets. HRG downregulates the contact system by inhibiting (F) XIIa. In murine models, HRG attenuates FeCl3- and polyphosphate-induced thrombosis and colocalizes with platelets and fibrin in thrombi. Therefore, we hypothesized that HRG binds to platelets and modulates their function.

Aims: Determine whether HRG binds to platelets and if so, identify the mechanism and consequences of this interaction.

Methods: The binding of HRG to glycoprotein (GP) Ibα, thrombin-binding GPIbα peptide, and active GPIIb/IIIa was examined using surface plasmon resonance (SPR), ELISA, and receptor-coated silica beads. HRG binding to washed human and murine platelets was assessed by flow cytometry. The effect of HRG on platelet aggregation in whole blood and platelet-rich plasma was examined using multiple electrode and light transmission aggregometry, respectively.

Results: Using SPR, HRG binds to immobilized GPIbα and thrombin-binding GPIbα peptide with Kd values of 1.0 nM and 0.2 nM, respectively. Using an ELISA, HRG binds immobilized GPIIb/IIIa with a Kd value of 247 nM. HRG binds to GPIIb/IIIa-coated beads with a 4-fold higher affinity than to GPIbα-coated beads. HRG binding increases 3-fold when resting platelets are activated with thrombin. HRG does not bind to resting platelets from GPIbα-deficient mice or activated platelets from Beta3-integrin-deficient mice, suggesting that binding to resting and activated platelets is mediated by GPIbα and GPIIb/IIIa, respectively. HRG competes with fibrinogen for binding to GPIIb/IIIa on thrombin-activated human or mouse platelets and inhibits aggregation in mouse whole blood or human platelet-rich plasma. Finally, whole blood platelet aggregation is enhanced in HRG-deficient mice compared with wild-type mice.

Conclusion(s): HRG binds to GPIbα on resting platelets and GPIIb/IIIa on activated platelets where it competes with fibrinogen, thereby attenuating platelet aggregation. Therefore, HRG may modulate platelet aggregation as well as coagulation.