SARS-CoV-2 infection of K18-HACE2 transgenic mice induces an early hyperactive phenotype in circulating platelets

Background: Coronavirus disease 2019 (COVID-19) is associated with a hypercoagulable state and increased incidence of cardiovascular complications. 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 proteome. Results: SARS-CoV-2 inoculated ( 10 6 PFU, 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. SARSCoV-2 RNA copies peaked at 4-dpi in K18-hACE2 mouse lungs, but not in C57BL/6J mice, confirming the need for human ACE2 receptor for viral entry. Histopathologic findings of infected K18-hACE2 mice included progressive lymphohistiocytic interstitial pneumonia with absence of diffuse alveolar damage. Lungs and kidneys 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 analysis of platelet proteome 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, Gp9, Selp, Pecam1 ) and chemokine ( Pf4, Cxcl5/12, Ccl8 ) signaling at 2-dpi. However, interferon ( Ddx58, Trim25, Mapk3 ) signaling was dominant at 4-dpi. Abundance of SARS-CoV-2 spike protein at 2-/4-dpi in the lungs, but not in the kidneys and platelets of K18-hACE2 mice, suggests that platelet re-programming towards activation-degranulation-aggregation is likely attributable to pneumonia-induced cytokine-driven response rather than direct platelet infection. Conclusions: Circulating platelets in SARS-CoV-2 infected K18-hACE2 mice demonstrate a specific early hyperactive phenotype consistent with procoagulant platelets.