PB0808 - The L33P polymorphism of integrin β3 PSI domain displays enhanced thiol isomerase activity and upregulates the coagulation cascade

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Co-Author(s)

BH

Brock K. Hoard

MSc Student
University of Toronto
Oshawa, Ontario, Canada

Co-Author(s)

EC

Eric Cerenzia

MSc
University of Toronto
Toronto, Ontario, Canada
PB

Preeti Bhoria

Researcher
St. Michaels Hospital
Toronto, Ontario, Canada
SS

Sladjana Slavkovic

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

Pingguo Chen

Research scientist
St Michael's Hospital
Toronto, Ontario, Canada
XL

Xi Lei

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

Guangheng Zhu

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

Andras Kapus

Professor
University of Toronto
Toronto, Ontario, Canada
KC

Kim Connelly

Professor
St. Michael's Hospital
Toronto, Ontario, Canada

Presenting Author(s)

Heyu Ni, MD, PhD

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

Background: Integrin αIIbβ3 is the essential platelet receptor for hemostasis and thrombosis via intercellular and extracellular matrix interactions. We recently discovered that the plexin-semaphorin-integrin (PSI) domain of the β3 subunit possess endogenous thiol-isomerase activity. Inhibiting this activity reduced platelet aggregation in-vitro and attenuated thrombus formation in-vivo (Blood, 2017). Interestingly, the L33P polymorphism within the PSI domain has been associated with greater risk of cardiovascular disease and displays increased platelet adhesion and aggregation in-vitro. However, L33P impact within the blood coagulation system has yet to be explored.

Aims: Examine the thiol isomerase activity of L33P and the mechanism whereby this polymorphism affects the blood coagulation system.

Methods: Using a combination of reduced RNase, insulin β-chain reduction and MPB binding assays, the thiol isomerase potential of recombinant L33P PSI domain (rL33P) was examined. Thromboelastography, clot retraction, prothrombin time (PT) and activated partial thromboplastin time (APTT) assays were employed to test L33P impact on the coagulation system.

Results: rL33P protein exhibited increased thiol-isomerase activity in comparison to wild-type PSI (rPSI) in a scrambled and reduced RNase assay. These results were confirmed through insulin β-chain reduction and MPB assays. Clot formation time was decreased for rL33P-treated blood in thromboelastography and clot retraction assays. Bacitracin, a thiol-isomerase inhibitor, attenuated these changes. Interestingly, APTT tests, an intrinsic coagulation pathway test, showed no significant difference between rL33P or rPSI-treated platelet-poor-plasma clot formation. However, PT assays, an extrinsic coagulation pathway-specific test, displayed significantly faster clotting times for rL33P-treated platelet-poor-plasma. Increased addition of recombinant tissue factor, further exacerbated rL33P effects. Preliminary isothermal titration calorimetry results further suggest direct interaction between tissue factor and rL33P.

Conclusion(s): Our results demonstrate that through enhanced thiol isomerase activity and interaction with extrinsic coagulation pathway factors, L33P upregulates blood coagulation. This pro-coagulant state may contribute to the increased risk of cardiovascular events in patients carrying this integrin polymorphism.