PB0353 - Novel GPIb-independent platelet aggregation induced by botrocetin: Implications for diagnosis and antithrombotic therapy

Sunday, June 23, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

CS

Chuanbin Shen

Professor
Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China
Qingdao, Shandong, China (People's Republic)

Co-Author(s)

DM

Daniel Mackeigan

Master students
University of Toronto
Toronto, Ontario, Canada
AS

Aron Shoara

Post-doc fellow
St. Michael's Hospital
Toronto, Ontario, Canada
PB

Preeti Bhoria

Post Doc
University of Toronto
Toronto, Ontario, Canada
GZ

Guangheng Zhu

Research staff
St. Michael's Hospital
Toronto, Ontario, Canada
DK

Danielle Karakas

PhD students
University of Toronto
Toronto, Ontario, Canada
WM

Wenjing Ma

Post Doc
University of Toronto
Toronto, Ontario, Canada
RX

Runjia Xu

Research assistant
University of Toronto
Toronto, Ontario, Canada
ZC

ZiYan Chen

Master student
University of Toronto
Toronto, Ontario, Canada
SS

Sladjana Slavkovic

Post-doc fellow
St. Michael's Hospital
Torotno, Ontario, Canada
DZ

Dachuan Zhang

Master student
University of Toronto
Toronto, Ontario, Canada
VP

Viktor Prifti

Master student
University of Toronto
Toronto, Ontario, Canada
ZL

Zhenze Liu

Post Doc
University of Toronto
Toronto, Ontario, Canada
PC

Pingguo Chen

Research scientist
St Michael's Hospital
Toronto, Ontario, Canada
Heyu Ni, MD, PhD

Professor
University of Toronto / Canadian Blood Services
Toronto, Ontario, Canada

Background: Snake venom botrocetin facilitates von Willebrand factor (VWF) binding to platelet GPIbα and has been widely used for the diagnosis of von Willebrand diseases and GPIb-related disorders. Botrocetin is also commonly employed for the development/characterization of antithrombotic drugs targeting the GPIb-VWF axis.

Aims: To explore the alternative receptor(s)/mechanisms participate in botrocetin-mediated platelet aggregation and inhibition.

Methods: The effects of botrocetin on platelet aggregation were examined using platelets from wild-type, VWF and fibrinogen-deficient, GPIbα-deficient, IL4Rα/GPIbα-transgenic and αIIbβ3-deficient mice, and human. Platelet-fibrinogen and platelet-VWF A1 interaction were measured using flow cytometry. GPIbα-VWF A1 interaction was evaluated by ELISA. Botrocetin-αIIbβ3 and botrocetin-GPIbα interaction was measured using ELISA and fluorescence spectrophotometer. Heparinized whole blood from healthy donors was used to test thrombus formation and growth in perfusion chamber.

Results: Botrocetin could induce aggregation of platelets from a Bernard-Soulier syndrome patient and GPIbα-deficient mice as well as platelets lacking the N-terminal extracellular domain of GPIbα. Botrocetin could interact with αIIbβ3 and facilitated αIIbβ3-VWF interaction independent of GPIb. Botrocetin competitively bound to the ligand-binding domain of activated rather than resting αIIbβ3. Although botrocetin-induced platelet aggregation requires VWF, strikingly, in the absence of VWF, botrocetin blocked fibrinogen and other ligand binding to αIIbβ3, and inhibited platelet aggregation and thrombus formation. Consistently, recombinant botrocetin defective in VWF binding inhibited αIIbβ3 and GPIb-mediated platelet aggregation, spreading and thrombus formation.

Conclusion(s): Our study provides insights into avoiding the misdiagnosis of GPIb-related disorders and developing botrocetin mutants as potential new anti-thrombotics that may simultaneously target both αIIbβ3 and GPIbα.