OC 05.2 - Proteome and phosphoproteome analyses of SARS-CoV-2-infected humanized K18-ACE2 mice reveal hyperactive phenotype of circulating platelets

Saturday, July 9, 2022

1:15 PM – 1:30 PM

Location: ICC Capital Suite Room 8&9

Presentation Format: Virtual

Presenting Author(s)

Saravanan Subramaniam, M.Pharm, PhD

Research Assistant Professor
Department of Medicine, Boston University, Boston, MA, USA
Boston, Massachusetts, United States

Co-Author(s)

RM

Ryan Matthew Hekman

Research Assistant
Center for Network Systems Biology, Boston University, Boston, MA, USA
Boston, Massachusetts, United States
AJ

Archana Jayaraman

Postdoc
Pulmonary Center, Department of Medicine, Boston University, Boston, MA, USA
Boston, Massachusetts, United States
AK

Aoife Kateri O'Connell

Research Assistant
National Emerging Infectious Diseases Laboratories (NEIDL) and Department of Microbiology, Boston University, Boston, MA, USA
Boston, Massachusetts, United States
MP

Mary Paige Montanaro

Research Assistant
National Emerging Infectious Diseases Laboratories (NEIDL) and Department of Pathology and Laboratory Medicine, Boston University, Boston, USA
Boston, Massachusetts, United States
DK

Devin Kenney

Graduate student
National Emerging Infectious Diseases Laboratories (NEIDL) and Department of Microbiology, Boston University, Boston, USA
Boston, Massachusetts, United States
ME

Maria Ericsson

Director
Electron Microscopy Facility at Harvard Medical School, Harvard University
Boston, Massachusetts, United States
SW

Sarah Walachowski

Postdoc
Pulmonary Center, Department of Medicine, Boston University, Boston, MA, USA
Boston, Massachusetts, United States
BB

Benjamin Blum

Graduate student
Center for Network Systems Biology, Boston University, Boston, MA, USA
Boston, Massachusetts, United States
KR

Katya Ravid

Professor
Whitaker Cardiovascular Institute and Department of Medicine, Boston University School of Medicine, Boston, MA, USA
Boston, Massachusetts, United States
NA

Nicholas A Crossland

Assistant Professor
Boston University, National Emerging Infectious Diseases Laboratories (NEIDL), Boston MA, USA
Boston, Massachusetts, United States
FD

Florian Douam

Assistant Professor
Boston University, National Emerging Infectious Diseases Laboratories (NEIDL), Boston MA, USA
Boston, Massachusetts, United States
AE

Andrew Emili

Professor
Center for Network Systems Biology, Boston University, Boston, MA, USA
Boston, Massachusetts, United States
MB

Markus Bosmann

Associate Professor
BU Pulmonary Center, Department of Medicine and National Emerging Infectious Diseases Laboratories (NEIDL), Boston University, Boston MA, USA; Center for Thrombosis and Hemostasis, University Medical Center of the Johannes Gutenberg- University, Mainz, Germany
Boston, Massachusetts, United States

Background: Platelets are effectors of hemostasis and play a major role in coordinating immune and inflammatory activities. Suitable animal models are needed to study COVID-19-associated coagulopathy and platelet effector functions in COVID-19, which are currently poorly understood.

Aims: We aimed to characterize alterations of platelets isolated from K18-hACE2 transgenic mice infected with SARS-CoV-2.

Methods: Heterozygous K18-hACE2 (human ACE2) and C57BL/6J mice were used to study SARS-CoV-2 infectivity. Lung infection, infiltration, and platelet aggregation were characterized with histology and immunohistochemistry. Platelet response to SARS-CoV-2 infection was quantified by mass spectrometry analysis of proteomics and phosphoproteomics. Western blotting, ELISA, and multiplex plasma profiling were performed to validate the proteomics and phosphoproteomics data.

Results: SARS-CoV-2 inoculated (10E6PFU, i.n.) K18-hACE2 mice started to lose weight at 4 days post-infection (dpi) and showed 90% lethality at 7-dpi in association with viral neuroinvasion. Histopathologic findings of infected K18-hACE2 mice included progressive lymphohistiocytic interstitial pneumonia with absence of diffuse alveolar damage. Lungs of infected K18-hACE2 mice (2-/4-dpi) showed mild increase in CD61+ aggregates compared to sham mice, but no overt tissue thrombosis. Gene ontology and pathway analyses of platelet proteomics and phosphoproteomics revealed that SARS-CoV-2 infection significantly upregulates the complement-coagulation cascades (F2/12/13, Tfpi, C1ra, Cd55, C4bp) and platelet activation-adhesion-degranulation proteins (Vwf, Itgb3/5, Selp, Pecam1) and chemokine (Pf4, Cxcl5/12) signaling at 2-dpi. However, interferon (Ddx58, Trim25, Mapk3) signaling was dominant at 4-dpi. Activation of proteomics and phosphoproteomics protein markers were highly correlated with platelet activation and interferon signaling at 2-/4-dpi, respectively. Plasma chemokine (e.g., Ccl8 and Pf4) and cytokines (e.g., IL6) were significantly elevated at 2-/4-dpi. SARS-CoV-2 spike protein was abundant at 2-/4-dpi in the lungs but not in platelets and kidneys, which correlated with no infectious virus in the serum.

Conclusion(s): Platelet re-programming towards activation-degranulation-aggregation is likely attributable to a pneumonia-induced elevated circulatory factors (e.g., cytokines)-driven response rather than direct platelet infection.