OC 22.4 - SARS-CoV-2 protein ORF3a promotes release of procoagulant tissue factor-positive extracellular vesicles

Sunday, June 25, 2023

15:30 – 15:45 ET

Room: 515

Session Available As: On-Demand

Presenting Author(s)

Shabbir A. Ansari, PhD

Research Fellow in Medicine
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Boston, MA
Boston, Massachusetts, United States

Co-Author(s)

JL

Jacob Ludington

Research Fellow in Medicine
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Boston, MA
Boston, Massachusetts, United States
KE

Keiichi Enjyoji

Research Fellow in Medicine
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Boston, MA
Boston, Massachusetts, United States
PA

Papa Anderson

Research Assisstant
Division of Cardiology, Beth Israel Deaconess Medical Center, Boston, MA
Boston, Massachusetts, United States
GO

Getulio Pereira de Oliveira Junior

Associate Research Scientist
Barnett Institute for Chemical and Biological Analysis, Northeastern University, Boston, MA
Boston, Massachusetts, United States
IG

Ionita Ghiran

Associate Professor of Anaesthesia
Department of Anaesthesia, Beth Israel Deaconess Medical Center, Boston, MA
Boston, Massachusetts, United States
RS

Robert E. Schwartz

Associate Professor of Medicine at the Sanford I. Weill Medical College of Cornell University
Division of Gastroenterology and Hepatology, Weill Cornell Medicine, New York, NY
New York City, New York, United States
AS

Alec Schmaier

Instructor of Medicine
Division of Cardiology, Beth Israel Deaconess Medical Center, Boston, MA
Boston, Massachusetts, United States
Sol Schulman, MD, PhD (he/him/his)

Assistant Professor of Medicine
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Boston, MA
Boston, Massachusetts, United States

Background: SARS-CoV-2 infection is complicated by dysregulation of blood coagulation and high rates of thrombosis. Procoagulant extracellular vesicles (EVs) expressing tissue factor (TF) are elevated in COVID-19 and correlate with disease severity and D-dimer levels, which predict overall mortality. SARS-CoV-2 accessory protein ORF3a disrupts host endomembrane traffic to support viral replication and exocytosis, but virus-intrinsic mechanisms promoting elaboration of procoagulant EVs have not been described.

Aims: Our aim was to test whether ORF3a, which we previously demonstrated enhances cell surface TMEM16F-dependent phosphatidylserine (PS) externalization and procoagulant activity, regulates shedding of prothrombotic EVs.

Methods: ORF3a was expressed from an inducible promoter in A549 alveolar basal epithelial cells and via lentiviral transduction of primary HUVECs. EVs were isolated from conditioned media by ultracentrifugation and characterized using nanoparticle tracking analysis, immuno-electron microscopy, and immunoblot. Factor Xa and thrombin generation and PS content were determined biochemically.

Results: Induction of ORF3a expression in A549 cells promoted EV production by over seven-fold as compared to control (p < 0.0007) with a concomitant 25% reduction in mean EV diameter from 131 nm to 99.7 nm (p < 0.0002). ORF3a significantly enhanced EV supported factor Xa generation (over nine-fold, p< 0.0001) and prothrombinase activity (over five-fold, p< 0.0004) in a TF- and PS-dependent manner. Similar findings were observed in HUVECs. Inhibition of TMEM16-dependent phospholipid scrambling with the repurposed anti-gout therapeutic benzbromarone attenuated EV release by 60% (p < 0.004), TF-dependent factor Xa generation by 50% (p < 0.008), and prothrombinase activity by 47% (p < 0.04). Electron microscopy confirmed accumulation of irregular multivesicular bodies in ORF3a-expressing cells. Immunoblot and immuno-electron microscopy detected TF and the multivesicular body/exosome marker CD63 on purified EVs.

Conclusion(s): SARS-CoV-2 ORF3a promotes release of procoagulant EVs at least partly via exosome release, suggesting a targetable virus-intrinsic mechanism by which infection, including that of epithelial cells, could augment systemic coagulation activation.

Learning Objectives:

Upon completion, participants will be able to describe the role of the SARS-CoV-2 accessory protein ORF3a in procoagulant activation of tissue factor (TF) and thrombin generation, both on the cell surface and on extracellular vesicles.
Upon completion, participants will be able to appreciate the virus-intrinsic mechanisms by which ORF3a drives procoagulant extracellular vesicle biogenesis.
Upon completion, participants will be able to understand the contribution of key host cellular pathways and mediators that contribute to ORF3a-induced procoagulant extracelluar vesicles formation.