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Tissue factor in COVID-19-associated coagulopathy

  • Saravanan Subramaniam
    Correspondence
    Corresponding author at: Pulmonary Center, Department of Medicine, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118, USA.
    Affiliations
    Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA
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  • Hema Kothari
    Affiliations
    Carter Immunology Center, University of Virginia, Charlottesville, VA 22908, USA

    Cardiovascular Division, Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA
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  • Markus Bosmann
    Affiliations
    Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA

    Center for Thrombosis and Hemostasis, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
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Published:September 30, 2022DOI:https://doi.org/10.1016/j.thromres.2022.09.025

      Highlights

      • Autopsy lung tissues from COVID-19 patients showed higher expression of TF that correlated with areas of fibrin-enriched thrombi.
      • Antiphospholipid antibodies, TF+-EV as well as activation of PRRs and complement factors could trigger TF-dependent procoagulant activity.
      • Elevated levels of proinflammatory cytokines including IL-6, IL-1β, TNF-α can induce TF expression on immune and non-immune cells that may contribute to CAC.

      Abstract

      Evidence of micro- and macro-thrombi in the arteries and veins of critically ill COVID-19 patients and in autopsies highlight the occurrence of COVID-19-associated coagulopathy (CAC). Clinical findings of critically ill COVID-19 patients point to various mechanisms for CAC; however, the definitive underlying cause is unclear. Multiple factors may contribute to the prothrombotic state in patients with COVID-19. Aberrant expression of tissue factor (TF), an initiator of the extrinsic coagulation pathway, leads to thrombotic complications during injury, inflammation, and infections. Clinical evidence suggests that TF-dependent coagulation activation likely plays a role in CAC. Multiple factors could trigger abnormal TF expression and coagulation activation in patients with severe COVID-19 infection. Proinflammatory cytokines that are highly elevated in COVID-19 (IL-1β, IL-6 and TNF-α) are known induce TF expression on leukocytes (e.g. monocytes, macrophages) and non-immune cells (e.g. endothelium, epithelium) in other conditions. Antiphospholipid antibodies, TF-positive extracellular vesicles, pattern recognition receptor (PRR) pathways and complement activation are all candidate factors that could trigger TF-dependent procoagulant activity. In addition, coagulation factors, such as thrombin, may further potentiate the induction of TF via protease-activated receptors on cells. In this systematic review, with other viral infections, we discuss potential mechanisms and cell-type-specific expressions of TF during SARS-CoV-2 infection and its role in the development of CAC.

      Keywords

      1. Introduction

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      2. Tissue factor pathway

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      Influenza (IAV/H1N1) patients with ARDS have an activated coagulation system and increased risk of thrombosis. IAV hospitalized patients showed elevated levels of D-dimer, which was associated with a higher risk of disease progression [
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      • Baric R.S.
      Mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury.
      ] or infected with the mouse-adapted PR8/H1N1 variant displayed an increase in lung TF mRNA and TF activity [
      • Antoniak S.
      • Tatsumi K.
      • Hisada Y.
      • Milner J.J.
      • Neidich S.D.
      • Shaver C.M.
      • Pawlinski R.
      • Beck M.A.
      • Bastarache J.A.
      • Mackman N.
      Tissue factor deficiency increases alveolar hemorrhage and death in influenza A virus-infected mice.
      ]. Similarly, mice infected with SARS-CoV-1 (MA15) showed profoundly greater TF RNA expression at 2-days post infection (dpi) which remained elevated at 4 and 7-dpi [
      • Gralinski L.E.
      • Bankhead III, A.
      • Jeng S.
      • Menachery V.D.
      • Proll S.
      • Belisle S.E.
      • Matzke M.
      • Webb-Robertson B.J.
      • Luna M.L.
      • Shukla A.K.
      • Ferris M.T.
      • Bolles M.
      • Chang J.
      • Aicher L.
      • Waters K.M.
      • Smith R.D.
      • Metz T.O.
      • Law G.L.
      • Katze M.G.
      • McWeeney S.
      • Baric R.S.
      Mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury.
      ] (Table 1).
      Table 1TF expression during other viral infections.
      VirusSpeciesSourceExperimental systemFindingsReferences
      Dengue virusHumanHUVECsIn vitro↑ TF

      ↑ TM

      ↑ tPA
      • Jiang Z.
      • Tang X.
      • Xiao R.
      • Jiang L.
      • Chen X.
      Dengue virus regulates the expression of hemostasis-related molecules in human vein endothelial cells.
      ,
      • Huang Y.H.
      • Lei H.Y.
      • Liu H.S.
      • Lin Y.S.
      • Chen S.H.
      • Liu C.C.
      • Yeh T.M.
      Tissue plasminogen activator induced by dengue virus infection of human endothelial cells.
      HumanMonocytesIn vitro↑ TF

      ↑ PAI-1
      • Krishnamurti C.
      • Alving B.
      Effect of dengue virus on procoagulant and fibrinolytic activities of monocytes.
      ,
      • Krishnamurti C.
      • Wahl L.M.
      • Alving B.M.
      Stimulation of plasminogen activator inhibitor activity in human monocytes infected with dengue virus.
      ,
      • Chunhakan S.
      • Butthep P.
      • Yoksan S.
      • Tangnararatchakit K.
      • Chuansumrit A.
      Vascular leakage in dengue hemorrhagic fever is associated with dengue infected monocytes, monocyte activation/exhaustion, and cytokines production.
      HumanPlasmaClinical↑ TF

      ↑ PAI-1

      ↑ tPA
      • de Azeredo E.Leal
      • Solorzano V.E.
      • de Oliveira D.B.
      • Marinho C.F.
      • de Souza L.J.
      • da Cunha R.V.
      • Damasco P.V.
      • Kubelka C.F.
      • de-Oliveira-Pinto L.M.
      Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection.
      ,
      • Huang Y.H.
      • Liu C.C.
      • Wang S.T.
      • Lei H.Y.
      • Liu H.L.
      • Lin Y.S.
      • Wu H.L.
      • Yeh T.M.
      Activation of coagulation and fibrinolysis during dengue virus infection.
      HumanPlasmaClinical (children)↑ TF

      ↑ vWF

      ↑ PAI-1
      • Sosothikul D.
      • Seksarn P.
      • Pongsewalak S.
      • Thisyakorn U.
      • Lusher J.
      Activation of endothelial cells, coagulation and fibrinolysis in children with Dengue virus infection.
      HumanMonocytes (from patients)Clinical↑ TF
      • de Azeredo E.L.
      • Kubelka C.F.
      • Alburquerque L.M.
      • Barbosa L.S.
      • Damasco P.V.
      • Avila C.A.
      • Motta-Castro A.R.
      • da Cunha R.V.
      • Monteiro R.Q.
      Tissue factor expression on monocytes from patients with severe dengue fever.
      Ebola virusHumanMonocytes/macrophagesIn vitro↑ TF

      ↓ Plasma protein C
      • Geisbert T.W.
      • Young H.A.
      • Jahrling P.B.
      • Davis K.J.
      • Kagan E.
      • Hensley L.E.
      Mechanisms underlying coagulation abnormalities in ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event.
      Macaques1. Lymphoid macrophages

      2. Peripheral blood cell
      In vivo1. ↑ TF

      2. ↑ TF membrane microparticles
      • Geisbert T.W.
      • Hensley L.E.
      • Jahrling P.B.
      • Larsen T.
      • Geisbert J.B.
      • Paragas J.
      • Young H.A.
      • Fredeking T.M.
      • Rote W.E.
      • Vlasuk G.P.
      Treatment of ebola virus infection with a recombinant inhibitor of factor VIIa/tissue factor: a study in rhesus monkeys.
      ,
      • Geisbert T.W.
      • Young H.A.
      • Jahrling P.B.
      • Davis K.J.
      • Kagan E.
      • Hensley L.E.
      Mechanisms underlying coagulation abnormalities in ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event.
      Puumala ortho-hanta virusHumanPlasmaIn vivo1. Circulating EV-TF

      2. ↑ PAI-1

      3. ↑ tPA
      • Schmedes C.M.
      • Grover S.P.
      • Hisada Y.M.
      • Goeijenbier M.
      • Hultdin J.
      • Nilsson S.
      • Thunberg T.
      • Ahlm C.
      • Mackman N.
      • Fors Connolly A.M.
      Circulating extracellular vesicle tissue factor activity during orthohantavirus infection is associated with intravascular coagulation.
      HumanHUVECsIn vitro↑ TF, ↑ PAI-1
      • Goeijenbier M.
      • Meijers J.C.
      • Anfasa F.
      • Roose J.M.
      • van de Weg C.A.
      • Bakhtiari K.
      • Henttonen H.
      • Vaheri A.
      • Osterhaus A.D.
      • van Gorp E.C.
      • Martina B.E.
      Effect of Puumala hantavirus infection on human umbilical vein endothelial cell hemostatic function: platelet interactions, increased tissue factor expression and fibrinolysis regulator release.
      Zika virusHumanHUVECsIn vitro↑ TF

      ↑ IL-6

      ↑ IL-8
      • Anfasa F.
      • Goeijenbier M.
      • Widagdo W.
      • Siegers J.Y.
      • Mumtaz N.
      • Okba N.
      • van Riel D.
      • Rockx B.
      • Koopmans M.P.G.
      • Meijers J.C.M.
      • Martina B.E.E.
      Zika virus infection induces elevation of tissue factor production and apoptosis on human umbilical vein endothelial cells.
      CytomegalovirusHumanHUVECsIn vitro↑ TF
      • Neppelenbroek S.I.M.
      • Rootjes P.A.
      • Boxhoorn L.
      • Wagenaar J.F.P.
      • Simsek S.
      • Stam F.
      Cytomegalovirus-associated thrombosis.
      HumanHUVECsIn vitro↑ PCA
      • Visseren F.L.
      • Bouwman J.J.
      • Bouter K.P.
      • Diepersloot R.J.
      • de Groot P.H.
      • Erkelens D.W.
      Procoagulant activity of endothelial cells after infection with respiratory viruses.
      HumanMonocytesIn vitro↑ TF
      • Bouwman J.J.
      • Visseren F.L.
      • Bosch M.C.
      • Bouter K.P.
      • Diepersloot R.J.
      Procoagulant and inflammatory response of virus-infected monocytes.
      Measles virusHumanHUVECsIn vitro↑ TF

      ↑ IL-1beta
      • Mazure G.
      • Grundy J.E.
      • Nygard G.
      • Hudson M.
      • Khan K.
      • Srai K.
      • Dhillon A.P.
      • Pounder R.E.
      • Wakefield A.J.
      Measles virus induction of human endothelial cell tissue factor procoagulant activity in vitro.
      Herpes simplex virusHumanHUVECsIn vitro↑ TF

      ↓ TM

      ↓ PAI-1
      • Key N.S.
      • Vercellotti G.M.
      • Winkelmann J.C.
      • Moldow C.F.
      • Goodman J.L.
      • Esmon N.L.
      • Esmon C.T.
      • Jacob H.S.
      Infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression.
      ,
      • Bok R.A.
      • Jacob H.S.
      • Balla J.
      • Juckett M.
      • Stella T.
      • Shatos M.A.
      • Vercellotti G.M.
      Herpes simplex virus decreases endothelial cell plasminogen activator inhibitor.
      MaresPeripheral monocytesIn vitro↑ TF, ↑ FXa generation
      • Yeo W.M.
      • Osterrieder N.
      • Stokol T.
      Equine herpesvirus type 1 infection induces procoagulant activity in equine monocytes.
      VirusVirus envelopeIn vitro↑ TF

      ↑ PCA
      • Pryzdial E.L.
      • Sutherland M.R.
      • Ruf W.
      The procoagulant envelope virus surface: contribution to enhanced infection.
      ,
      • Sutherland M.R.
      • Ruf W.
      • Pryzdial E.L.
      Tissue factor and glycoprotein C on herpes simplex virus type 1 are protease-activated receptor 2 cofactors that enhance infection.
      MouseVirus envelopeIn vivo↑ PCA
      • Sutherland M.R.
      • Simon A.Y.
      • Shanina I.
      • Horwitz M.S.
      • Ruf W.
      • Pryzdial E.L.G.
      Virus envelope tissue factor promotes infection in mice.
      AdenovirusHumanHUVECsIn vitro↑ PCA
      • Visseren F.L.
      • Bouwman J.J.
      • Bouter K.P.
      • Diepersloot R.J.
      • de Groot P.H.
      • Erkelens D.W.
      Procoagulant activity of endothelial cells after infection with respiratory viruses.
      Influenza virusHumanHUVECsIn vitro↑ PCA
      • Visseren F.L.
      • Bouwman J.J.
      • Bouter K.P.
      • Diepersloot R.J.
      • de Groot P.H.
      • Erkelens D.W.
      Procoagulant activity of endothelial cells after infection with respiratory viruses.
      HumanMonocytesIn vitro↑ TF
      • Bouwman J.J.
      • Visseren F.L.
      • Bosch M.C.
      • Bouter K.P.
      • Diepersloot R.J.
      Procoagulant and inflammatory response of virus-infected monocytes.
      Mouse1. Lung mRNA

      2. BALF
      In vivo↑ TF

      ↑ BALF MV-TF
      • Gralinski L.E.
      • Bankhead III, A.
      • Jeng S.
      • Menachery V.D.
      • Proll S.
      • Belisle S.E.
      • Matzke M.
      • Webb-Robertson B.J.
      • Luna M.L.
      • Shukla A.K.
      • Ferris M.T.
      • Bolles M.
      • Chang J.
      • Aicher L.
      • Waters K.M.
      • Smith R.D.
      • Metz T.O.
      • Law G.L.
      • Katze M.G.
      • McWeeney S.
      • Baric R.S.
      Mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury.
      ,
      • Antoniak S.
      • Tatsumi K.
      • Hisada Y.
      • Milner J.J.
      • Neidich S.D.
      • Shaver C.M.
      • Pawlinski R.
      • Beck M.A.
      • Bastarache J.A.
      • Mackman N.
      Tissue factor deficiency increases alveolar hemorrhage and death in influenza A virus-infected mice.
      ,
      • Tatsumi K.
      • Antoniak S.
      • Subramaniam S.
      • Gondouin B.
      • Neidich S.D.
      • Beck M.A.
      • Mickelson J.
      • Monroe III, D.M.
      • Bastarache J.A.
      • Mackman N.
      Anticoagulation increases alveolar hemorrhage in mice infected with influenza A.
      Human immuno-deficiency virusHumanPeripheral monocytesIn vitro↑ TF and PCA
      • Schechter M.E.
      • Andrade B.B.
      • He T.
      • Richter G.H.
      • Tosh K.W.
      • Policicchio B.B.
      • Singh A.
      • Raehtz K.D.
      • Sheikh V.
      • Ma D.
      • Brocca-Cofano E.
      • Apetrei C.
      • Tracy R.
      • Ribeiro R.M.
      • Sher A.
      • Francischetti I.M.B.
      • Pandrea I.
      • Sereti I.
      Inflammatory monocytes expressing tissue factor drive SIV and HIV coagulopathy.
      HumanPeripheral monocytesClinical↑ TF

      ↑ D-dimer
      • Funderburg N.T.
      • Mayne E.
      • Sieg S.F.
      • Asaad R.
      • Jiang W.
      • Kalinowska M.
      • Luciano A.A.
      • Stevens W.
      • Rodriguez B.
      • Brenchley J.M.
      • Douek D.C.
      • Lederman M.M.
      Increased tissue factor expression on circulating monocytes in chronic HIV infection: relationship to in vivo coagulation and immune activation.
      HumanPlasmaClinical↑ MP-TF
      • Baker J.V.
      • Huppler Hullsiek K.
      • Bradford R.L.
      • Prosser R.
      • Tracy R.P.
      • Key N.S.
      Circulating levels of tissue factor microparticle procoagulant activity are reduced with antiretroviral therapy and are associated with persistent inflammation and coagulation activation among HIV-positive patients.
      HumanPlatelets and platelet microparticleClinical↑ platelet MP-TF
      • Mayne E.
      • Funderburg N.T.
      • Sieg S.F.
      • Asaad R.
      • Kalinowska M.
      • Rodriguez B.
      • Schmaier A.H.
      • Stevens W.
      • Lederman M.M.
      Increased platelet and microparticle activation in HIV infection: upregulation of P-selectin and tissue factor expression.
      Respiratory syncytial virusHumanHUVECsIn vitro↑ PCA
      • Visseren F.L.
      • Bouwman J.J.
      • Bouter K.P.
      • Diepersloot R.J.
      • de Groot P.H.
      • Erkelens D.W.
      Procoagulant activity of endothelial cells after infection with respiratory viruses.
      TF, tissue factor; PCA, procoagulant activity; MP-TF, tissue factor positive microparticles; TM, thrombomodulin; vWF, von Willebrand factor; PAI-1, plasminogen activator inhibitor-1; tPA, tissue plasminogen activator.
      Blockade of TF or TF-FVIIa binary complex attenuates coagulopathy and sepsis-associated mortality in animal models [
      • Pawlinski R.
      • Mackman N.
      Cellular sources of tissue factor in endotoxemia and sepsis.
      ,
      • Creasey A.A.
      • Chang A.C.
      • Feigen L.
      • Wun T.C.
      • Taylor Jr., F.B.
      • Hinshaw L.B.
      Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock.
      ,
      • Taylor Jr., F.B.
      • Chang A.
      • Ruf W.
      • Morrissey J.H.
      • Hinshaw L.
      • Catlett R.
      • Blick K.
      • Edgington T.S.
      Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody.
      ,
      • Taylor F.B.
      • Chang A.C.
      • Peer G.
      • Li A.
      • Ezban M.
      • Hedner U.
      Active site inhibited factor VIIa (DEGR VIIa) attenuates the coagulant and interleukin-6 and -8, but not tumor necrosis factor, responses of the baboon to LD100 Escherichia coli.
      ]. Similarly, in rhesus monkeys infected with the Ebola virus, treatment with a recombinant inhibitor of FVIIa/TF achieved a prolonged survival time and attenuation of the coagulation and proinflammatory responses [
      • Geisbert T.W.
      • Hensley L.E.
      • Jahrling P.B.
      • Larsen T.
      • Geisbert J.B.
      • Paragas J.
      • Young H.A.
      • Fredeking T.M.
      • Rote W.E.
      • Vlasuk G.P.
      Treatment of ebola virus infection with a recombinant inhibitor of factor VIIa/tissue factor: a study in rhesus monkeys.
      ]. Dengue virus, Respiratory Syncytial Virus, adenovirus, Measles, cytomegalovirus, Zika virus, Puumala Hantann virus, and Herpes simplex virus (HSV) infections induce TF expression in cultured endothelial cells [
      • Key N.S.
      • Vercellotti G.M.
      • Winkelmann J.C.
      • Moldow C.F.
      • Goodman J.L.
      • Esmon N.L.
      • Esmon C.T.
      • Jacob H.S.
      Infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression.
      ,
      • Huerta-Zepeda A.
      • Cabello-Gutierrez C.
      • Cime-Castillo J.
      • Monroy-Martinez V.
      • Manjarrez-Zavala M.E.
      • Gutierrez-Rodriguez M.
      • Izaguirre R.
      • Ruiz-Ordaz B.H.
      Crosstalk between coagulation and inflammation during dengue virus infection.
      ,
      • Schmedes C.M.
      • Grover S.P.
      • Hisada Y.M.
      • Goeijenbier M.
      • Hultdin J.
      • Nilsson S.
      • Thunberg T.
      • Ahlm C.
      • Mackman N.
      • Fors Connolly A.M.
      Circulating extracellular vesicle tissue factor activity during orthohantavirus infection is associated with intravascular coagulation.
      ,
      • Goeijenbier M.
      • Meijers J.C.
      • Anfasa F.
      • Roose J.M.
      • van de Weg C.A.
      • Bakhtiari K.
      • Henttonen H.
      • Vaheri A.
      • Osterhaus A.D.
      • van Gorp E.C.
      • Martina B.E.
      Effect of Puumala hantavirus infection on human umbilical vein endothelial cell hemostatic function: platelet interactions, increased tissue factor expression and fibrinolysis regulator release.
      ,
      • Anfasa F.
      • Goeijenbier M.
      • Widagdo W.
      • Siegers J.Y.
      • Mumtaz N.
      • Okba N.
      • van Riel D.
      • Rockx B.
      • Koopmans M.P.G.
      • Meijers J.C.M.
      • Martina B.E.E.
      Zika virus infection induces elevation of tissue factor production and apoptosis on human umbilical vein endothelial cells.
      ,
      • Bouwman J.J.
      • Visseren F.L.
      • Bosch M.C.
      • Bouter K.P.
      • Diepersloot R.J.
      Procoagulant and inflammatory response of virus-infected monocytes.
      ,
      • Neppelenbroek S.I.M.
      • Rootjes P.A.
      • Boxhoorn L.
      • Wagenaar J.F.P.
      • Simsek S.
      • Stam F.
      Cytomegalovirus-associated thrombosis.
      ,
      • Visseren F.L.
      • Bouwman J.J.
      • Bouter K.P.
      • Diepersloot R.J.
      • de Groot P.H.
      • Erkelens D.W.
      Procoagulant activity of endothelial cells after infection with respiratory viruses.
      ,
      • Mazure G.
      • Grundy J.E.
      • Nygard G.
      • Hudson M.
      • Khan K.
      • Srai K.
      • Dhillon A.P.
      • Pounder R.E.
      • Wakefield A.J.
      Measles virus induction of human endothelial cell tissue factor procoagulant activity in vitro.
      ]. Microvesicular TF activity correlated with plasma IL-8 and predicted mortality from severe influenza A (H1N1) infection in a multicenter study of 12 patients [
      • Rondina M.T.
      • Tatsumi K.
      • Bastarache J.A.
      • Mackman N.
      Microvesicle tissue factor activity and Interleukin-8 levels are associated with mortality in patients with influenza A/H1N1 infection.
      ] (Table 1).
      Autopsy lung tissues from COVID-19 patients showed higher expression of TF that correlated with areas of fibrin-enriched thrombi [
      • Subrahmanian S.
      • Borczuk A.
      • Salvatore S.P.
      • Laurence J.
      • Ahamed J.
      ,
      • Subrahmanian S.
      • Borczuk A.
      • Salvatore S.
      • Fung K.M.
      • Merrill J.T.
      • Laurence J.
      • Ahamed J.
      Tissue factor upregulation is associated with SARS-CoV-2 in the lungs of COVID-19 patients.
      ]. Transcriptomic profiling of inflammatory cells from BAL fluid revealed that COVID-19 patients had increased expression of procoagulant genes (e.g. TF, vWF, thrombin, FXIII, FVII, F12) and decreased levels of anticoagulant genes (e.g. PROCR, PROS1, THBD) and fibrinolytic genes (e.g. PLAU) [
      • Mast A.E.
      • Wolberg A.S.
      • Gailani D.
      • Garvin M.R.
      • Alvarez C.
      • Miller J.I.
      • Aronow B.
      • Jacobson D.
      SARS-CoV-2 suppresses anticoagulant and fibrinolytic gene expression in the lung.
      ]. One major limitation of this study was that the transcripts from the BAL fluid were not analyzed with respect to specific-cell types. In severe COVID-19, TF expression on activated monocytes was associated with elevated fibrinogen and D-dimer levels [
      • Hottz E.D.
      • Azevedo-Quintanilha I.G.
      • Palhinha L.
      • Teixeira L.
      • Barreto E.A.
      • Pao C.R.R.
      • Righy C.
      • Franco S.
      • Souza T.M.L.
      • Kurtz P.
      • Bozza F.A.
      • Bozza P.T.
      Platelet activation and platelet-monocyte aggregates formation trigger tissue factor expression in severe COVID-19 patients.
      ]. Moreover, higher quantities of monocyte TF were present in patients who required mechanical ventilation or had a lethal course of disease [
      • Hottz E.D.
      • Azevedo-Quintanilha I.G.
      • Palhinha L.
      • Teixeira L.
      • Barreto E.A.
      • Pao C.R.R.
      • Righy C.
      • Franco S.
      • Souza T.M.L.
      • Kurtz P.
      • Bozza F.A.
      • Bozza P.T.
      Platelet activation and platelet-monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19.
      ]. It was recently reported that SARS-CoV-2 infection induces the activation of TF-mediated coagulation via activation of acid sphingomyelinase [
      • Wang J.
      • Pendurthi U.R.
      • Yi G.
      • Rao L.V.M.
      SARS-CoV-2 infection induces the activation of tissue factor-mediated coagulation via activation of acid sphingomyelinase.
      ].
      A recent prospective study showed a positive correlation between increased circulating levels of TF, IL-6, IL-8, VCAM-1, complement anaphylatoxin C5a, growth arrest specific (GAS) gene 6, pentraxin-3, TNF-α and mortality in COVID-19 patients in the ICU [
      • de Bruin S.
      • Bos L.D.
      • van Roon M.A.
      • Tuip-de Boer A.M.
      • Schuurman A.R.
      • Koel-Simmelinck M.J.A.
      • Bogaard H.J.
      • Tuinman P.R.
      • van Agtmael M.A.
      • Hamann J.
      • Teunissen C.E.
      • Wiersinga W.J.
      • Koos Zwinderman A.H.
      • Brouwer M.C.
      • van de Beek D.
      • Vlaar A.P.J.
      • Amsterdam U.M.C.C.-B.I.
      Clinical features and prognostic factors in Covid-19: a prospective cohort study.
      ]. SARS-CoV-2 infection of human lung epithelial cells, and pulmonary endothelial cells overexpressing hACE2, induced expression of TF and led to procoagulant and proinflammatory responses [
      • FitzGerald E.S.
      • Chen Y.
      • Fitzgerald K.A.
      • Jamieson A.M.
      Lung epithelial cell transcriptional regulation as a factor in COVID-19 associated coagulopathies.
      ,
      • Nascimento Conde J.
      • Schutt W.R.
      • Gorbunova E.E.
      • Mackow E.R.
      Recombinant ACE2 expression is required for SARS-CoV-2 to infect primary human endothelial cells and induce inflammatory and procoagulative responses.
      ,
      • Amraei R.
      • Xia C.
      • Olejnik J.
      • White M.R.
      • Napoleon M.A.
      • Lotfollahzadeh S.
      • Hauser B.M.
      • Schmidt A.G.
      • Chitalia V.
      • Muhlberger E.
      • Costello C.E.
      • Rahimi N.
      Extracellular vimentin is an attachment factor that facilitates SARS-CoV-2 entry into human endothelial cells.
      ] (Table 2).
      Table 2Evidence for a role of TF in SARS-CoV infections.
      VirusSpeciesSourceExperimental systemFindingsReferences
      SARS-CoV-1 (SARS MA15- mouse-adapted)MouseLung mRNAIn vivo↑ TF
      • Gralinski L.E.
      • Bankhead III, A.
      • Jeng S.
      • Menachery V.D.
      • Proll S.
      • Belisle S.E.
      • Matzke M.
      • Webb-Robertson B.J.
      • Luna M.L.
      • Shukla A.K.
      • Ferris M.T.
      • Bolles M.
      • Chang J.
      • Aicher L.
      • Waters K.M.
      • Smith R.D.
      • Metz T.O.
      • Law G.L.
      • Katze M.G.
      • McWeeney S.
      • Baric R.S.
      Mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury.
      SARS-CoV-2HumanBALF cells (bulk-RNA seq)Clinical↑ TF
      • Xiong Y.
      • Liu Y.
      • Cao L.
      • Wang D.
      • Guo M.
      • Jiang A.
      • Guo D.
      • Hu W.
      • Yang J.
      • Tang Z.
      • Wu H.
      • Lin Y.
      • Zhang M.
      • Zhang Q.
      • Shi M.
      • Liu Y.
      • Zhou Y.
      • Lan K.
      • Chen Y.
      Transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in COVID-19 patients.
      HumanBALF cells (single-cell RNA seq)Clinical↑ TF in lung epithelial cell population
      • Liao M.
      • Liu Y.
      • Yuan J.
      • Wen Y.
      • Xu G.
      • Zhao J.
      • Cheng L.
      • Li J.
      • Wang X.
      • Wang F.
      • Liu L.
      • Amit I.
      • Zhang S.
      • Zhang Z.
      Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19.
      ,
      • FitzGerald E.S.
      • Chen Y.
      • Fitzgerald K.A.
      • Jamieson A.M.
      Lung epithelial cell transcriptional regulation as a factor in COVID-19-associated coagulopathies.
      HumanPBMCs (bulk-RNA seq)Clinical↑ TF
      • Xiong Y.
      • Liu Y.
      • Cao L.
      • Wang D.
      • Guo M.
      • Jiang A.
      • Guo D.
      • Hu W.
      • Yang J.
      • Tang Z.
      • Wu H.
      • Lin Y.
      • Zhang M.
      • Zhang Q.
      • Shi M.
      • Liu Y.
      • Zhou Y.
      • Lan K.
      • Chen Y.
      Transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in COVID-19 patients.
      ,
      • FitzGerald E.S.
      • Chen Y.
      • Fitzgerald K.A.
      • Jamieson A.M.
      Lung epithelial cell transcriptional regulation as a factor in COVID-19-associated coagulopathies.
      HumanWhole blood (monocytes, granulocytes, and platelets)Clinical↑ TF
      • Hottz E.D.
      • Azevedo-Quintanilha I.G.
      • Palhinha L.
      • Teixeira L.
      • Barreto E.A.
      • Pao C.R.R.
      • Righy C.
      • Franco S.
      • Souza T.M.L.
      • Kurtz P.
      • Bozza F.A.
      • Bozza P.T.
      Platelet activation and platelet-monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19.
      ,
      • Canzano P.
      • Brambilla M.
      • Porro B.
      • Cosentino N.
      • Tortorici E.
      • Vicini S.
      • Poggio P.
      • Cascella A.
      • Pengo M.F.
      • Veglia F.
      • Fiorelli S.
      • Bonomi A.
      • Cavalca V.
      • Trabattoni D.
      • Andreini D.
      • Omodeo Sale E.
      • Parati G.
      • Tremoli E.
      • Camera M.
      Platelet and endothelial activation as potential mechanisms behind the thrombotic complications of COVID-19 patients.
      HumanSerumClinical↑ EV-TF and activity
      • Balbi C.
      • Burrello J.
      • Bolis S.
      • Lazzarini E.
      • Biemmi V.
      • Pianezzi E.
      • Burrello A.
      • Caporali E.
      • Grazioli L.G.
      • Martinetti G.
      • Fusi-Schmidhauser T.
      • Vassalli G.
      • Melli G.
      • Barile L.
      Circulating extracellular vesicles are endowed with enhanced procoagulant activity in SARS-CoV-2 infection.
      HumanPlasmaClinical↑ EV-TF

      ↑ D-dimer
      • Campello E.
      • Radu C.M.
      • Simion C.
      • Spiezia L.
      • Bulato C.
      • Gavasso S.
      • Tormene D.
      • Perin N.
      • Turatti G.
      • Simioni P.
      Longitudinal trend of plasma concentrations of extracellular vesicles in patients hospitalized for COVID-19.
      ,
      • Guervilly C.
      • Bonifay A.
      • Burtey S.
      • Sabatier F.
      • Cauchois R.
      • Abdili E.
      • Arnaud L.
      • Lano G.
      • Pietri L.
      • Robert T.
      • Velier M.
      • Papazian L.
      • Albanese J.
      • Kaplanski G.
      • Dignat-George F.
      • Lacroix R.
      Dissemination of extreme levels of extracellular vesicles: tissue factor activity in patients with severe COVID-19.
      ,
      • Rosell A.
      • Havervall S.
      • von Meijenfeldt F.
      • Hisada Y.
      • Aguilera K.
      • Grover S.P.
      • Lisman T.
      • Mackman N.
      • Thalin C.
      Patients with COVID-19 have elevated levels of circulating extracellular vesicle tissue factor activity that is associated with severity and mortality-brief report.
      ,
      • Campbell R.A.
      • Hisada Y.
      • Denorme F.
      • Grover S.P.
      • Bouck E.G.
      • Middleton E.A.
      • Wolberg A.S.
      • Rondina M.T.
      • Mackman N.
      Comparison of the coagulopathies associated with COVID-19 and sepsis.
      ,
      • Francischetti I.M.B.
      • Toomer K.
      • Zhang Y.
      • Jani J.
      • Siddiqui Z.
      • Brotman D.J.
      • Hooper J.E.
      • Kickler T.S.
      Upregulation of pulmonary tissue factor, loss of thrombomodulin and immunothrombosis in SARS-CoV-2 infection.
      HumanPMECs (hACE2 overexpressed)In vitro↑ TF
      • Nascimento Conde J.
      • Schutt W.R.
      • Gorbunova E.E.
      • Mackow E.R.
      Recombinant ACE2 expression is required for SARS-CoV-2 to infect primary human endothelial cells and induce inflammatory and procoagulative responses.
      HumanMonocytes-derived macrophagesIn vitro↑ TF and PCA via sphingomyelinase
      • Wang J.
      • Pendurthi U.R.
      • Yi G.
      • Rao L.V.M.
      SARS-CoV-2 infection induces the activation of tissue factor-mediated coagulation via activation of acid sphingomyelinase.
      HumanLung epithelial cells (bulk-RNA seq)In vitro↑ TF
      • Jha P.K.
      • Vijay A.
      • Halu A.
      • Uchida S.
      • Aikawa M.
      Gene expression profiling reveals the shared and distinct transcriptional signatures in human lung epithelial cells infected with SARS-CoV-2, MERS-CoV, or SARS-CoV: potential implications in cardiovascular complications of COVID-19.
      Mouse (K18-hACE2)Lung mRNAIn vivo↑ TF
      • Qin Z.
      • Liu F.
      • Blair R.
      • Wang C.
      • Yang H.
      • Mudd J.
      • Currey J.M.
      • Iwanaga N.
      • He J.
      • Mi R.
      • Han K.
      • Midkiff C.C.
      • Alam M.A.
      • Aktas B.H.
      • Heide R.S.V.
      • Veazey R.
      • Piedimonte G.
      • Maness N.J.
      • Ergun S.
      • Mauvais-Jarvis F.
      • Rappaport J.
      • Kolls J.K.
      • Qin X.
      Endothelial cell infection and dysfunction, immune activation in severe COVID-19.
      TF, tissue factor; PCA, procoagulant activity; MP-TF, tissue factor positive microparticles.
      These findings support the concept that induced overexpression of TF on circulating monocytes, endothelial cells, and lung epithelium may act as a trigger for CAC.
      Alternatively, virus surface–host cell communication via TF in the viral envelope and lipid bilayers (as demonstrated for HSV type-1) could also directly trigger the procoagulant response in COVID-19 [
      • Pryzdial E.L.
      • Sutherland M.R.
      • Ruf W.
      The procoagulant envelope virus surface: contribution to enhanced infection.
      ,
      • Sutherland M.R.
      • Simon A.Y.
      • Shanina I.
      • Horwitz M.S.
      • Ruf W.
      • Pryzdial E.L.G.
      Virus envelope tissue factor promotes infection in mice.
      ,
      • Schoeman D.
      • Fielding B.C.
      Coronavirus envelope protein: current knowledge.
      ] (Table 1). Further studies are needed to understand SARS-CoV-2 viral envelope and glycoproteins interactions with host cells and the subsequent thrombo-inflammatory responses.

      4. Pattern recognition receptors and TF in COVID-19

      Activation of inflammatory and coagulation pathways is an integral part of host defense responses against infections. These mechanisms have evolved to limit pathogen dissemination, and to orchestrate pathogen killing and tissue repair. On the other hand, dysregulation and excessive activation of these pathways can contribute to thrombosis and tissue damage [
      • Antoniak S.
      • Mackman N.
      Multiple roles of the coagulation protease cascade during virus infection.
      ,
      • Antoniak S.
      The coagulation system in host defense.
      ,
      • Beristain-Covarrubias N.
      • Perez-Toledo M.
      • Thomas M.R.
      • Henderson I.R.
      • Watson S.P.
      • Cunningham A.F.
      Understanding infection-induced thrombosis: lessons learned from animal models.
      ,
      • Sumbria D.
      • Berber E.
      • Rouse B.T.
      Factors affecting the tissue damaging consequences of viral infections.
      ].
      Pattern recognition receptors (PRRs) sense conserved molecular structures known as pathogen-/damage-associated molecular patterns which are released from pathogens (PAMPs) or damaged host cells (DAMPs). Toll-like receptors (TLRs) belong to the family of PRRs. Substantial evidence indicates that TLR activation during viral infections induce TF gene expression with a subsequent imbalance of the pro- and anti-coagulant states [
      • Subramaniam S.
      • Scharrer I.
      Procoagulant activity during viral infections.
      ,
      • Khanmohammadi S.
      • Rezaei N.
      Role of toll-like receptors in the pathogenesis of COVID-19.
      ,
      • Alessandri L.M.
      • Read A.W.
      • Burton P.R.
      • Stanley F.J.
      An analysis of sudden infant death syndrome in aboriginal infants.
      ]. The TLR3 agonist polyinosinic:polycytidylic acid [poly(I:C)], which mimics viral double stranded RNA, triggers TF expression in human endothelial cells and in mice [
      • Shibamiya A.
      • Hersemeyer K.
      • Schmidt Woll T.
      • Sedding D.
      • Daniel J.M.
      • Bauer S.
      • Koyama T.
      • Preissner K.T.
      • Kanse S.M.
      A key role for toll-like receptor-3 in disrupting the hemostasis balance on endothelial cells.
      ,
      • Subramaniam S.
      • Ogoti Y.
      • Hernandez I.
      • Zogg M.
      • Botros F.
      • Burns R.
      • Mackman N.
      • Antoniak S.
      • Fletcher C.
      • W. H.
      Thrombin-PAR1/2 signaling axis modulates TLR3-mediated procoagulant, proinflammatory, and proadhesive responses in vascular endothelial cells.
      ,
      • Subramaniam S.
      • Ogoti Y.
      • Hernandez I.
      • Zogg M.
      • Botros F.
      • Burns R.
      • DeRousse J.T.
      • Dockendorff C.
      • Mackman N.
      • Antoniak S.
      • Fletcher C.
      • Weiler H.
      A thrombin-PAR1/2 feedback loop amplifies thromboinflammatory endothelial responses to the viral RNA analogue poly(I:C).
      ]. Viruses, such as Dengue, Hantaan, Marburg, Lassa, and Ebola, induce expression of TF on infected endothelial cells and monocytes [
      • Huerta-Zepeda A.
      • Cabello-Gutierrez C.
      • Cime-Castillo J.
      • Monroy-Martinez V.
      • Manjarrez-Zavala M.E.
      • Gutierrez-Rodriguez M.
      • Izaguirre R.
      • Ruiz-Ordaz B.H.
      Crosstalk between coagulation and inflammation during dengue virus infection.
      ,
      • Goeijenbier M.
      • Meijers J.C.
      • Anfasa F.
      • Roose J.M.
      • van de Weg C.A.
      • Bakhtiari K.
      • Henttonen H.
      • Vaheri A.
      • Osterhaus A.D.
      • van Gorp E.C.
      • Martina B.E.
      Effect of Puumala hantavirus infection on human umbilical vein endothelial cell hemostatic function: platelet interactions, increased tissue factor expression and fibrinolysis regulator release.
      ,
      • Geisbert T.W.
      • Young H.A.
      • Jahrling P.B.
      • Davis K.J.
      • Larsen T.
      • Kagan E.
      • Hensley L.E.
      Pathogenesis of ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells.
      ,
      • Geisbert T.W.
      • Young H.A.
      • Jahrling P.B.
      • Davis K.J.
      • Kagan E.
      • Hensley L.E.
      Mechanisms underlying coagulation abnormalities in ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event.
      ,
      • Funderburg N.T.
      • Mayne E.
      • Sieg S.F.
      • Asaad R.
      • Jiang W.
      • Kalinowska M.
      • Luciano A.A.
      • Stevens W.
      • Rodriguez B.
      • Brenchley J.M.
      • Douek D.C.
      • Lederman M.M.
      Increased tissue factor expression on circulating monocytes in chronic HIV infection: relationship to in vivo coagulation and immune activation.
      ]. In line with these findings, studies have suggested that SARS-CoV-2 virions can be present in/on endothelial cells and might induce vascular damage [
      • Varga Z.
      • Flammer A.J.
      • Steiger P.
      • Haberecker M.
      • Andermatt R.
      • Zinkernagel A.S.
      • Mehra M.R.
      • Schuepbach R.A.
      • Ruschitzka F.
      • Moch H.
      Endothelial cell infection and endotheliitis in COVID-19.
      ,
      • Qanadli S.D.
      • Beigelman-Aubry C.
      • Rotzinger D.C.
      Vascular changes detected with thoracic CT in coronavirus disease (COVID-19) might be significant determinants for accurate diagnosis and optimal patient management.
      ]. However, the extent of direct and productive infection of endothelial cells by SARS-CoV-2 remains controversial [
      • Theopold U.
      • Pinter M.
      • Daffre S.
      • Tryselius Y.
      • Friedrich P.
      • Nassel D.R.
      • Hultmark D.
      CalpA, a drosophila calpain homolog specifically expressed in a small set of nerve, midgut, and blood cells.
      ]. An increased TF expression in endothelial cells and monocytes could contribute to the prothrombotic events in patients infected with COVID-19. Moreover, SARS-CoV-2 genomic RNA activates TLR7/TLR8 (ssRNA) and TLR3 (by dsRNA intermediates during viral replication) that mount an acute inflammatory response through the activation of nuclear factor kappa B (NF-κB) and the interferon regulatory factors (IRFs), leading to the synthesis and release of proinflammatory cytokines including IL-1, IL-6 and TNF-α [
      • Wong C.K.
      • Lam C.W.
      • Wu A.K.
      • Ip W.K.
      • Lee N.L.
      • Chan I.H.
      • Lit L.C.
      • Hui D.S.
      • Chan M.H.
      • Chung S.S.
      • Sung J.J.
      Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome.
      ]. In line with this notion, recent studies have suggested the involvement of TLR7/TLR8 and TLR3 in the inflammatory and interferon (IFN) responses induced by SARS-CoV-2 in plasmacytoid dendritic cells and epithelial cells [
      • Moreno-Eutimio M.A.
      • Lopez-Macias C.
      • Pastelin-Palacios R.
      Bioinformatic analysis and identification of single-stranded RNA sequences recognized by TLR7/8 in the SARS-CoV-2,SARS-CoV, and MERS-CoV genomes.
      ,
      • Choudhury A.
      • Mukherjee S.
      In silico studies on the comparative characterization of the interactions of SARS-CoV-2 spike glycoprotein with ACE-2 receptor homologs and human TLRs.
      ,
      • Sohn K.M.
      • Lee S.G.
      • Kim H.J.
      • Cheon S.
      • Jeong H.
      • Lee J.
      • Kim I.S.
      • Silwal P.
      • Kim Y.J.
      • Paik S.
      • Chung C.
      • Park C.
      • Kim Y.S.
      • Jo E.K.
      COVID-19 patients upregulate toll-like receptor 4-mediated inflammatory signaling that mimics bacterial sepsis.
      ,
      • Cuevas A.M.
      • Clark J.M.
      • Potter J.J.
      Increased TLR/MyD88 signaling in patients with obesity: is there a link to COVID-19 disease severity?.
      ,
      • Salvi V.
      • Nguyen H.O.
      • Sozio F.
      • Schioppa T.
      • Laffranchi M.
      • Scapini P.
      • Passari M.
      • Barbazza I.
      • Tiberio L.
      • Tamassia N.
      • Garlanda C.
      • Prete A.Del
      • Cassatella M.A.
      • Mantovani A.
      • Sozzani S.
      • Bosisio D.
      SARS-CoV-2-associated ssRNAs Activate Inflammation and Immunity via TLR7/8.
      ,
      • Li Y.
      • Renner D.M.
      • Comar C.E.
      • Whelan J.N.
      • Reyes H.M.
      • Cardenas-Diaz F.L.
      • Truitt R.
      • Tan L.H.
      • Dong B.
      • Alysandratos K.D.
      • Huang J.
      • Palmer J.N.
      • Adappa N.D.
      • Kohanski M.A.
      • Kotton D.N.
      • Silverman R.H.
      • Yang W.
      • Morrisey E.E.
      • Cohen N.A.
      • Weiss S.R.
      SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes.
      ]. Similarly, SARS-CoV-2 replication induces a delayed IFN response that begins after sensing of viral RNA by the cytosolic MDA5 and RIG-I sensors in lung epithelial cells [
      • Yin X.
      • Riva L.
      • Pu Y.
      • Martin-Sancho L.
      • Kanamune J.
      • Yamamoto Y.
      • Sakai K.
      • Gotoh S.
      • Miorin L.
      • De Jesus P.D.
      • Yang C.C.
      • Herbert K.M.
      • Yoh S.
      • Hultquist J.F.
      • Garcia-Sastre A.
      • Chanda S.K.
      MDA5 governs the innate immune response to SARS-CoV-2 in lung epithelial cells.
      ,
      • Sharma A.
      • Kontodimas K.
      • Bosmann M.
      The MAVS immune recognition pathway in viral infection and sepsis.
      ]. Interestingly, SARS-CoV-2 spike protein, as well as mouse-adapted coronavirus MHV-A59, interact with TLR4 for subsequent initiation of IL-1β expression in THP-1 cells [
      • Zhao Y.
      • Kuang M.
      • Li J.
      • Zhu L.
      • Jia Z.
      • Guo X.
      • Hu Y.
      • Kong J.
      • Yin H.
      • Wang X.
      • You F.
      Publisher correction: SARS-CoV-2 spike protein interacts with and activates TLR4.
      ]. This response promotes inflammation, local influx of neutrophils and macrophages to the alveoli and activation of the adaptive immune response. IL-1β and other proinflammatory cytokines are likely to induce TF expression on endothelial and epithelial cells and alter vascular permeability [
      • Szotowski B.
      • Antoniak S.
      • Poller W.
      • Schultheiss H.P.
      • Rauch U.
      Procoagulant soluble tissue factor is released from endothelial cells in response to inflammatory cytokines.
      ,
      • Hou T.
      • Tieu B.C.
      • Ray S.
      • Recinos Iii A.
      • Cui R.
      • Tilton R.G.
      • Brasier A.R.
      Roles of IL-6-gp130 signaling in vascular inflammation.
      ,
      • Ishibashi T.
      • Kimura H.
      • Shikama Y.
      • Uchida T.
      • Kariyone S.
      • Hirano T.
      • Kishimoto T.
      • Takatsuki F.
      • Akiyama Y.
      Interleukin-6 is a potent thrombopoietic factor in vivo in mice.
      ,
      • Puhlmann M.
      • Weinreich D.M.
      • Farma J.M.
      • Carroll N.M.
      • Turner E.M.
      • Alexander Jr., H.R.
      Interleukin-1beta induced vascular permeability is dependent on induction of endothelial tissue factor (TF) activity.
      ,
      • Kirchhofer D.
      • Tschopp T.B.
      • Hadvary P.
      • Baumgartner H.R.
      Endothelial cells stimulated with tumor necrosis factor-alpha express varying amounts of tissue factor resulting in inhomogenous fibrin deposition in a native blood flow system.Effects of thrombin inhibitors.
      ].
      Inflammasomes are key components of the innate immune system. Inflammasome activation is initiated by several cytosolic PPRs (e.g. NOD-like receptors) that respond to either microbe-derived PAMPs or DAMPs generated by injured host cells [
      • Schroder K.
      • Tschopp J.
      The inflammasomes.
      ]. Canonical inflammasome activation of caspase-1 (CASP1) has been shown to induce the generation of highly procoagulant TF-bearing extracellular vesicles from macrophages [
      • Rothmeier A.S.
      • Marchese P.
      • Petrich B.G.
      • Furlan-Freguia C.
      • Ginsberg M.H.
      • Ruggeri Z.M.
      • Ruf W.
      Caspase-1-mediated pathway promotes generation of thromboinflammatory microparticles.
      ]. Moreover, endogenous DAMPs, such as high mobility group box 1 (HMGB1), also activate the non-canonical caspase 11 (CASP11). CASP11 can increase the procoagulant activity of TF on innate immune cells through gasdermin D (GSDMD) and transmembrane protein 16F (TMEM16F)-mediated phosphatidylserine translocation [
      • Yang X.
      • Cheng X.
      • Tang Y.
      • Qiu X.
      • Wang Z.
      • Fu G.
      • Wu J.
      • Kang H.
      • Wang J.
      • Wang H.
      • Chen F.
      • Xiao X.
      • Billiar T.R.
      • Lu B.
      The role of type 1 interferons in coagulation induced by gram-negative bacteria.
      ]. Recent clinical findings demonstrated that NLRP3 inflammasome-derived products, such as Casp1p20, IL-1β, and IL-18, correlated with markers of disease severity in the sera of COVID-19 patients, including IL-6 and lactate dehydrogenase [
      • Rodrigues T.S.
      • de Sa K.S.G.
      • Ishimoto A.Y.
      • Becerra A.
      • Oliveira S.
      • Almeida L.
      • Goncalves A.V.
      • Perucello D.B.
      • Andrade W.A.
      • Castro R.
      • Veras F.P.
      • Toller-Kawahisa J.E.
      • Nascimento D.C.
      • de Lima M.H.F.
      • Silva C.M.S.
      • Caetite D.B.
      • Martins R.B.
      • Castro I.A.
      • Pontelli M.C.
      • de Barros F.C.
      • do Amaral N.B.
      • Giannini M.C.
      • Bonjorno L.P.
      • Lopes M.I.F.
      • Santana R.C.
      • Vilar F.C.
      • Auxiliadora-Martins M.
      • Luppino-Assad R.
      • de Almeida S.C.L.
      • de Oliveira F.R.
      • Batah S.S.
      • Siyuan L.
      • Benatti M.N.
      • Cunha T.M.
      • Alves-Filho J.C.
      • Cunha F.Q.
      • Cunha L.D.
      • Frantz F.G.
      • Kohlsdorf T.
      • Fabro A.T.
      • Arruda E.
      • de Oliveira R.D.R.
      • Louzada-Junior P.
      • Zamboni D.S.
      Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients.
      ,
      • Freeman T.L.
      • Swartz T.H.
      Targeting the NLRP3 inflammasome in severe COVID-19.
      ]. Similarly, Ferreira et al. reported that infection severity in COVID-19 may be associated with inflammasome activation in monocytes and downstream production of large amounts of IL-1β, IL-6 and TNF-α [
      • Ferreira A.C.
      • Soares V.C.
      • de Azevedo-Quintanilha I.G.
      • Dias S.
      • Fintelman-Rodrigues N.
      • Sacramento C.Q.
      • Mattos M.
      • de Freitas C.S.
      • Temerozo J.R.
      • Teixeira L.
      • Damaceno Hottz E.
      • Barreto E.A.
      • Pao C.R.R.
      • Palhinha L.
      • Miranda M.
      • Bou-Habib D.C.
      • Bozza F.A.
      • Bozza P.T.
      • Souza T.M.L.
      SARS-CoV-2 engages inflammasome and pyroptosis in human primary monocytes.
      ]. Monocyte TF expression and platelet activation are correlated with markers, such as fibrinogen and D-dimers, in patients with invasive mechanical ventilation or a lethal outcome [
      • Hottz E.D.
      • Azevedo-Quintanilha I.G.
      • Palhinha L.
      • Teixeira L.
      • Barreto E.A.
      • Pao C.R.R.
      • Righy C.
      • Franco S.
      • Souza T.M.L.
      • Kurtz P.
      • Bozza F.A.
      • Bozza P.T.
      Platelet activation and platelet-monocyte aggregates formation trigger tissue factor expression in severe COVID-19 patients.
      ]. While it is unclear whether inflammasome-mediated pyroptosis pathways contribute to coagulation activation in severe COVID-19 infection [
      • Wu C.
      • Lu W.
      • Zhang Y.
      • Zhang G.
      • Shi X.
      • Hisada Y.
      • Grover S.P.
      • Zhang X.
      • Li L.
      • Xiang B.
      • Shi J.
      • Li X.A.
      • Daugherty A.
      • Smyth S.S.
      • Kirchhofer D.
      • Shiroishi T.
      • Shao F.
      • Mackman N.
      • Wei Y.
      • Li Z.
      Inflammasome activation triggers blood clotting and host death through pyroptosis.
      ], a pyroptosis and Gasdermin D inhibitor (Disulfiram) was associated with a lower risk of COVID-19 in a retrospective cohort study [
      • Fillmore N.
      • Bell S.
      • Shen C.
      • Nguyen V.
      • La J.
      • Dubreuil M.
      • Strymish J.
      • Brophy M.
      • Mehta G.
      • Wu H.
      • Lieberman J.
      • Do N.
      • Sander C.
      Disulfiram use is associated with lower risk of COVID-19: a retrospective cohort study.
      ], and is currently tested in clinical trials. Higher expression of caspase-1 in the endothelium of COVID-19 patients as compared with H1N1 and control groups revealed the occurrence of pyroptosis in capillary-alveolar endothelial cells [
      • Nagashima S.
      • Mendes M.C.
      • Camargo Martins A.P.
      • Borges N.H.
      • Godoy T.M.
      • Miggiolaro A.
      • da Silva Deziderio F.
      • Machado-Souza C.
      • de Noronha L.
      Endothelial dysfunction and thrombosis in patients with COVID-19-brief report.
      ].
      High levels of TF, D-dimers and thrombin in BAL fluids of patients with ARDS coincided with a strong activation of coagulation [
      • Bautista-Vargas M.
      • Bonilla-Abadia F.
      • Canas C.A.
      Potential role for tissue factor in the pathogenesis of hypercoagulability associated with in COVID-19.
      ]. Fibrin deposition in alveolar spaces is a key feature of acute lung injury. The mechanisms that contribute to disturbed bronchoalveolar fibrin turnover are localized TF-mediated thrombin generation and downregulation of fibrinolysis pathway due to increased activity of fibrinolytic inhibitors, in particular PAI-1 [
      • Schultz M.J.
      • Haitsma J.J.
      • Zhang H.
      • Slutsky A.S.
      Pulmonary coagulopathy as a new target in therapeutic studies of acute lung injury or pneumonia–a review.
      ].
      Overall, activation of PRRs contributes to the propagation of inflammation and initial coagulation activation in viral infection. Further investigations of PRR activation in COVID-19 and TF signaling are needed to advance our understanding of the pathogenesis of COVID-19 and CAC.

      5. Cytokine storm and TF in COVID-19

      The immunopathology of the SARS-CoV-2-induced cytokine release is complex, and the underlying molecular mechanisms are not completely characterized. IL-1β, IL-6, IL-18, IFN-γ, and TNF-α are key proinflammatory cytokines that are thought to play central immunopathologic roles in the development of the cytokine storm [
      • Fajgenbaum D.C.
      • June C.H.
      Cytokine storm.
      ]. A recent report proposed that IL-6 signaling causes harmful changes to liver sinusoidal endothelial cells and may promote blood clotting and contribute to liver injury [
      • McConnell M.J.
      • Kawaguchi N.
      • Kondo R.
      • Sonzogni A.
      • Licini L.
      • Valle C.
      • Bonaffini P.A.
      • Sironi S.
      • Alessio M.G.
      • Previtali G.
      • Seghezzi M.
      • Zhang X.
      • Lee A.
      • Pine A.B.
      • Chun H.J.
      • Zhang X.
      • Fernandez-Hernando C.
      • Qing H.
      • Wang A.
      • Price C.
      • Sun Z.
      • Utsumi T.
      • Hwa J.
      • Strazzabosco M.
      • Iwakiri Y.
      Liver injury in COVID-19 and IL-6 trans-signaling-induced endotheliopathy.
      ]. In line with this, targeted inhibition of IL-1β and IL-6 has been shown to reduce inflammatory and thrombosis biomarkers and moderately improve survival rates in COVID-19 patients [
      • Perrone F.
      • Piccirillo M.C.
      • Ascierto P.A.
      • Salvarani C.
      • Parrella R.
      • Marata A.M.
      • Popoli P.
      • Ferraris L.
      • Marrocco-Trischitta M.M.
      • Ripamonti D.
      • Binda F.
      • Bonfanti P.
      • Squillace N.
      • Castelli F.
      • Muiesan M.L.
      • Lichtner M.
      • Calzetti C.
      • Salerno N.D.
      • Atripaldi L.
      • Cascella M.
      • Costantini M.
      • Dolci G.
      • Facciolongo N.C.
      • Fraganza F.
      • Massari M.
      • Montesarchio V.
      • Mussini C.
      • Negri E.A.
      • Botti G.
      • Cardone C.
      • Gargiulo P.
      • Gravina A.
      • Schettino C.
      • Arenare L.
      • Chiodini P.
      • Gallo C.
      I. Tocivid-19 investigators
      Tocilizumab for patients with COVID-19 pneumonia. The single-arm TOCIVID-19 prospective trial.
      ,
      • Price C.C.
      • Altice F.L.
      • Shyr Y.
      • Koff A.
      • Pischel L.
      • Goshua G.
      • Azar M.M.
      • McManus D.
      • Chen S.C.
      • Gleeson S.E.
      • Britto C.J.
      • Azmy V.
      • Kaman K.
      • Gaston D.C.
      • Davis M.
      • Burrello T.
      • Harris Z.
      • Villanueva M.S.
      • Aoun-Barakat L.
      • Kang I.
      • Seropian S.
      • Chupp G.
      • Bucala R.
      • Kaminski N.
      • Lee A.I.
      • LoRusso P.M.
      • Topal J.E.
      • Dela Cruz C.
      • Malinis M.
      Tocilizumab treatment for cytokine release syndrome in hospitalized patients with coronavirus disease 2019: survival and clinical outcomes.
      ,
      • Toniati P.
      • Piva S.
      • Cattalini M.
      • Garrafa E.
      • Regola F.
      • Castelli F.
      • Franceschini F.
      • Airo P.
      • Bazzani C.
      • Beindorf E.A.
      • Berlendis M.
      • Bezzi M.
      • Bossini N.
      • Castellano M.
      • Cattaneo S.
      • Cavazzana I.
      • Contessi G.B.
      • Crippa M.
      • Delbarba A.
      • De Peri E.
      • Faletti A.
      • Filippini M.
      • Filippini M.
      • Frassi M.
      • Gaggiotti M.
      • Gorla R.
      • Lanspa M.
      • Lorenzotti S.
      • Marino R.
      • Maroldi R.
      • Metra M.
      • Matteelli A.
      • Modina D.
      • Moioli G.
      • Montani G.
      • Muiesan M.L.
      • Odolini S.
      • Peli E.
      • Pesenti S.
      • Pezzoli M.C.
      • Pirola I.
      • Pozzi A.
      • Proto A.
      • Rasulo F.A.
      • Renisi G.
      • Ricci C.
      • Rizzoni D.
      • Romanelli G.
      • Rossi M.
      • Salvetti M.
      • Scolari F.
      • Signorini L.
      • Taglietti M.
      • Tomasoni G.
      • Tomasoni L.R.
      • Turla F.
      • Valsecchi A.
      • Zani D.
      • Zuccala F.
      • Zunica F.
      • Foca E.
      • Andreoli L.
      • Latronico N.
      Tocilizumab for the treatment of severe COVID-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of 100 patients in Brescia, Italy.
      ,
      • Eimer J.
      • Vesterbacka J.
      • Svensson A.K.
      • Stojanovic B.
      • Wagrell C.
      • Sonnerborg A.
      • Nowak P.
      Tocilizumab shortens time on mechanical ventilation and length of hospital stay in patients with severe COVID-19: a retrospective cohort study.
      ,
      • Potere N.
      • Di Nisio M.
      • Cibelli D.
      • Scurti R.
      • Frattari A.
      • Porreca E.
      • Abbate A.
      • Parruti G.
      Interleukin-6 receptor blockade with subcutaneous tocilizumab in severe COVID-19 pneumonia and hyperinflammation: a case-control study.
      ,
      • Gupta S.
      • Wang W.
      • Hayek S.S.
      • Chan L.
      • Mathews K.S.
      • Melamed M.L.
      • Brenner S.K.
      • Leonberg-Yoo A.
      • Schenck E.J.
      • Radbel J.
      • Reiser J.
      • Bansal A.
      • Srivastava A.
      • Zhou Y.
      • Finkel D.
      • Green A.
      • Mallappallil M.
      • Faugno A.J.
      • Zhang J.
      • Velez J.C.Q.
      • Shaefi S.
      • Parikh C.R.
      • Charytan D.M.
      • Athavale A.M.
      • Friedman A.N.
      • Redfern R.E.
      • Short S.A.P.
      • Correa S.
      • Pokharel K.K.
      • Admon A.J.
      • Donnelly J.P.
      • Gershengorn H.B.
      • Douin D.J.
      • Semler M.W.
      • Hernan M.A.
      • Leaf D.E.
      • Investigators S.-C.
      Association between early treatment with tocilizumab and mortality among critically ill patients with COVID-19.
      ,
      • Cavalli G.
      • Larcher A.
      • Tomelleri A.
      • Campochiaro C.
      • Della-Torre E.
      • De Luca G.
      • Farina N.
      • Boffini N.
      • Ruggeri A.
      • Poli A.
      • Scarpellini P.
      • Rovere-Querini P.
      • Tresoldi M.
      • Salonia A.
      • Montorsi F.
      • Landoni G.
      • Castagna A.
      • Ciceri F.
      • Zangrillo A.
      • Dagna L.
      Interleukin-1 and interleukin-6 inhibition compared with standard management in patients with COVID-19 and hyperinflammation: a cohort study.
      ]. Inflammation-induced coagulation is the net result of the overexpression/activation of coagulant factors (e.g. TF), decreased production of anti-coagulant factors (e.g. TF pathway inhibitor, thrombomodulin, protein C) and suppression of fibrinolytic proteins [
      • Lipinski S.
      • Bremer L.
      • Lammers T.
      • Thieme F.
      • Schreiber S.
      • Rosenstiel P.
      Coagulation and inflammation. Molecular insights and diagnostic implications.
      ]. Activation of TLRs on alveolar epithelial cells, macrophages, and circulating blood monocytes plays an important role in innate immunity and inflammatory response during viral infections. A recent study revealed that SARS-CoV-2 is internalized by human monocytes and macrophages [
      • Boumaza A.
      • Gay L.
      • Mezouar S.
      • Bestion E.
      • Diallo A.B.
      • Michel M.
      • Desnues B.
      • Raoult D.
      • La Scola B.
      • Halfon P.
      • Vitte J.
      • Olive D.
      • Mege J.L.
      Monocytes and macrophages, targets of SARS-CoV-2: the clue for Covid-19 immunoparalysis.
      ,
      • Kenney D.J.
      • O’Connell A.K.
      • Turcinovic J.
      • Montanaro P.
      • Hekman R.M.
      • Tamura T.
      • Berneshawi A.R.
      • Cafiero T.R.
      • Abdullatif S.A.
      • Blum B.
      • Goldstein S.I.
      • Heller B.L.
      • Gertje H.P.
      • Bullitt E.
      • Trachtenberg A.J.
      • Chavez E.
      • Sheikh A.
      • Kurnick S.
      • Grosz K.
      • Bosmann M.
      • Ericsson M.
      • Huber B.R.
      • Saeed M.
      • Balazs A.B.
      • Francis K.P.
      • Klose A.
      • Paragas N.
      • Campbell J.D.
      • Connor J.H.
      • Emili A.
      • Crossland N.A.
      • Ploss A.
      • Douam F.
      Macrophages Govern Antiviral Responses in Human Lung Tissues Protected From SARS-CoV-2 Infection.
      ]. TLR activation by viral products (e.g. spike protein) [
      • Zhao Y.
      • Kuang M.
      • Li J.
      • Zhu L.
      • Jia Z.
      • Guo X.
      • Hu Y.
      • Kong J.
      • Yin H.
      • Wang X.
      • You F.
      Publisher correction: SARS-CoV-2 spike protein interacts with and activates TLR4.
      ] and single stranded RNA (ssRNA) [
      • Salvi V.
      • Nguyen H.O.
      • Sozio F.
      • Schioppa T.
      • Laffranchi M.
      • Scapini P.
      • Passari M.
      • Barbazza I.
      • Tiberio L.
      • Tamassia N.
      • Garlanda C.
      • Prete A.Del
      • Cassatella M.A.
      • Mantovani A.
      • Sozzani S.
      • Bosisio D.
      SARS-CoV-2-associated ssRNAs Activate Inflammation and Immunity via TLR7/8.
      ] activates downstream signaling leading to the production of proinflammatory cytokines and chemokines. These cytokines and chemokines orchestrate in the recruitment of immune cells, such as neutrophils, monocytes and T cells, to the lungs resulting in widespread lung inflammation [
      • Hojyo S.
      • Uchida M.
      • Tanaka K.
      • Hasebe R.
      • Tanaka Y.
      • Murakami M.
      • Hirano T.
      How COVID-19 induces cytokine storm with high mortality.
      ,
      • Ozato K.
      • Tsujimura H.
      • Tamura T.
      Toll-like receptor signaling and regulation of cytokine gene expression in the immune system.
      ,
      • Gustine J.N.
      • Jones D.
      Immunopathology of hyperinflammation in COVID-19.
      ]. Accumulating clinical data also suggest that cytokine release is associated with COVID-19 severity and that cytokines are mediators of death from COVID-19 [
      • Hu B.
      • Huang S.
      • Yin L.
      The cytokine storm and COVID-19.
      ]. Pro-inflammatory cytokines, including IL-1α, IL-1β, IL-6, IL-8, MCP-1, IFN-γ, TNF-α, [
      • Huang C.
      • Wang Y.
      • Li X.
      • Ren L.
      • Zhao J.
      • Hu Y.
      • Zhang L.
      • Fan G.
      • Xu J.
      • Gu X.
      • Cheng Z.
      • Yu T.
      • Xia J.
      • Wei Y.
      • Wu W.
      • Xie X.
      • Yin W.
      • Li H.
      • Liu M.
      • Xiao Y.
      • Gao H.
      • Guo L.
      • Xie J.
      • Wang G.
      • Jiang R.
      • Gao Z.
      • Jin Q.
      • Wang J.
      • Cao B.
      Clinical features of patients infected with 2019 novel coronavirus in Wuhan,China.
      ,
      • Chen G.
      • Wu D.
      • Guo W.
      • Cao Y.
      • Huang D.
      • Wang H.
      • Wang T.
      • Zhang X.
      • Chen H.
      • Yu H.
      • Zhang X.
      • Zhang M.
      • Wu S.
      • Song J.
      • Chen T.
      • Han M.
      • Li S.
      • Luo X.
      • Zhao J.
      • Ning Q.
      Clinical and immunological features of severe and moderate coronavirus disease 2019.
      ] may induce CAC via expression of TF on endothelial cells, monocytes, macrophages and T cells [
      • Szotowski B.
      • Antoniak S.
      • Poller W.
      • Schultheiss H.P.
      • Rauch U.
      Procoagulant soluble tissue factor is released from endothelial cells in response to inflammatory cytokines.
      ,
      • Nawroth P.P.
      • Stern D.M.
      Modulation of endothelial cell hemostatic properties by tumor necrosis factor.
      ,
      • Nawroth P.P.
      • Handley D.A.
      • Esmon C.T.
      • Stern D.M.
      Interleukin 1 induces endothelial cell procoagulant while suppressing cell-surface anticoagulant activity.
      ,
      • Herbert J.M.
      • Savi P.
      • Laplace M.C.
      • Lale A.
      IL-4 inhibits LPS-, IL-1 beta- and TNF alpha-induced expression of tissue factor in endothelial cells and monocytes.
      ,
      • Schwager I.
      • Jungi T.W.
      Effect of human recombinant cytokines on the induction of macrophage procoagulant activity.
      ,
      • Schecter A.D.
      • Rollins B.J.
      • Zhang Y.J.
      • Charo I.F.
      • Fallon J.T.
      • Rossikhina M.
      • Giesen P.L.
      • Nemerson Y.
      • Taubman M.B.
      Tissue factor is induced by monocyte chemoattractant protein-1 in human aortic smooth muscle and THP-1 cells.
      ,
      • Neumann F.J.
      • Ott I.
      • Marx N.
      • Luther T.
      • Kenngott S.
      • Gawaz M.
      • Kotzsch M.
      • Schomig A.
      Effect of human recombinant interleukin-6 and interleukin-8 on monocyte procoagulant activity.
      ,
      • De Palma R.
      • Cirillo P.
      • Ciccarelli G.
      • Barra G.
      • Conte S.
      • Pellegrino G.
      • Pasquale G.
      • Nassa G.
      • Pacifico F.
      • Leonardi A.
      • Insabato L.
      • Cali G.
      • Golino P.
      • Cimmino G.
      Expression of functional tissue factor in activated T-lymphocytes in vitro and in vivo: a possible contribution of immunity to thrombosis?.
      ] and may promote blood clotting. In contrast, a recent study demonstrated that the baseline levels of IFN-γ were negatively associated with increased lung fibrosis in COVID-19 patients at discharge. However, this study was performed with a relatively small number of patients. The authors speculate that IFN-γ is anti-fibrotic because it mediates a more rapid clearance of SARS-CoV-2. Thus, low circulating IFN-γ and its risk associated with fibrosis in COVID-19 patients need further validation [
      • Hu Z.J.
      • Xu J.
      • Yin J.M.
      • Li L.
      • Hou W.
      • Zhang L.L.
      • Zhou Z.
      • Yu Y.Z.
      • Li H.J.
      • Feng Y.M.
      • Jin R.H.
      Lower circulating interferon-gamma is a risk factor for lung fibrosis in COVID-19 patients.
      ].
      Activated coagulation factors, including FXa, thrombin and fibrin, promote the synthesis of pro-inflammatory cytokines (IL-6, IL-8, MCP-1) and cell adhesion molecules (E-selectin, ICAM-1, and VCAM-1) in endothelial and epithelial cells [
      • Sower L.E.
      • Froelich C.J.
      • Carney D.H.
      • Fenton II, J.W.
      • Klimpel G.R.
      Thrombin induces IL-6 production in fibroblasts and epithelial cells. Evidence for the involvement of the seven-transmembrane domain (STD) receptor for alpha-thrombin.
      ,
      • Qi J.
      • Goralnick S.
      • Kreutzer D.L.
      Fibrin regulation of interleukin-8 gene expression in human vascular endothelial cells.
      ,
      • Ueno A.
      • Murakami K.
      • Yamanouchi K.
      • Watanabe M.
      • Kondo T.
      Thrombin stimulates production of interleukin-8 in human umbilical vein endothelial cells.
      ,
      • Anrather D.
      • Millan M.T.
      • Palmetshofer A.
      • Robson S.C.
      • Geczy C.
      • Ritchie A.J.
      • Bach F.H.
      • Ewenstein B.M.
      Thrombin activates nuclear factor-kappaB and potentiates endothelial cell activation by TNF.
      ]. Moreover, the TF-thrombin-PAR1/2 signaling axis further potentiates TLR3-mediated expression of TF, IL-8, E-selectin, ICAM-1, and VCAM-1 in endothelial cells [
      • Subramaniam S.
      • Ogoti Y.
      • Hernandez I.
      • Zogg M.
      • Botros F.
      • Burns R.
      • Mackman N.
      • Antoniak S.
      • Fletcher C.
      • W. H.
      Thrombin-PAR1/2 signaling axis modulates TLR3-mediated procoagulant, proinflammatory, and proadhesive responses in vascular endothelial cells.
      ,
      • Subramaniam S.
      • Ogoti Y.
      • Hernandez I.
      • Zogg M.
      • Botros F.
      • Burns R.
      • DeRousse J.T.
      • Dockendorff C.
      • Mackman N.
      • Antoniak S.
      • Fletcher C.
      • Weiler H.
      A thrombin-PAR1/2 feedback loop amplifies thromboinflammatory endothelial responses to the viral RNA analogue poly(I:C).
      ,
      • Subramaniam S.
      • Ruf W.
      • Bosmann M.
      Advocacy of targeting protease-activated receptors in severe coronavirus disease 2019.
      ]. Thus, elevated levels of thrombin during SARS-CoV-2 infection may play a crucial role in propagation of cytokine release and TF-dependent activation of coagulation. In addition, SARS-CoV-2 infection of endothelial cells also causes cell death, which leads to vascular leakage and induces a cytopathic effect on airway epithelial cells and multiorgan failure [
      • Fajgenbaum D.C.
      • June C.H.
      Cytokine storm.
      ,
      • Kenney D.J.
      • O’Connell A.K.
      • Turcinovic J.
      • Montanaro P.
      • Hekman R.M.
      • Tamura T.
      • Berneshawi A.R.
      • Cafiero T.R.
      • Abdullatif S.A.
      • Blum B.
      • Goldstein S.I.
      • Heller B.L.
      • Gertje H.P.
      • Bullitt E.
      • Trachtenberg A.J.
      • Chavez E.
      • Sheikh A.
      • Kurnick S.
      • Grosz K.
      • Bosmann M.
      • Ericsson M.
      • Huber B.R.
      • Saeed M.
      • Balazs A.B.
      • Francis K.P.
      • Klose A.
      • Paragas N.
      • Campbell J.D.
      • Connor J.H.
      • Emili A.
      • Crossland N.A.
      • Ploss A.
      • Douam F.
      Macrophages Govern Antiviral Responses in Human Lung Tissues Protected From SARS-CoV-2 Infection.
      ,
      • Fara A.
      • Mitrev Z.
      • Rosalia R.A.
      • Assas B.M.
      Cytokine storm and COVID-19: a chronicle of pro-inflammatory cytokines.
      ,
      • Ragab D.
      • Salah Eldin H.
      • Taeimah M.
      • Khattab R.
      • Salem R.
      The COVID-19 cytokine storm; what we know so far.
      ,
      • Park W.B.
      • Kwon N.J.
      • Choi S.J.
      • Kang C.K.
      • Choe P.G.
      • Kim J.Y.
      • Yun J.
      • Lee G.W.
      • Seong M.W.
      • Kim N.J.
      • Seo J.S.
      • Oh M.D.
      Virus isolation from the first patient with SARS-CoV-2 in Korea.
      ].
      Overall, the current data support the concept that an early and excessive release of cytokines as well as dysregulated coagulation activity (FXa, thrombin, and fibrin) may contribute to the thrombo-inflammatory responses in COVID-19. Further studies on cytokine-mediated expression of TF in COVID-19 could advance our understanding of the molecular mechanisms of CAC.

      6. Antiphospholipid (aPL) antibodies and TF in COVID-19

      Anti-phospholipid syndrome (APS) is an autoimmune prothrombotic disease characterized by persistent presence of aPL antibodies, leading to recurrent arterial and venous thromboembolic events [
      • Giannakopoulos B.
      • Krilis S.A.
      The pathogenesis of the antiphospholipid syndrome.
      ]. Patients with APS produce high-avidity autoantibodies to phospholipids and phospholipid-binding proteins (aPL antibodies), including prothrombin, plasminogen, cardiolipin, and β2 glycoprotein I (β2GPI) [
      • Giannakopoulos B.
      • Krilis S.A.
      The pathogenesis of the antiphospholipid syndrome.
      ]. The prevalence of APS varies with the presence of aPL antibodies in ∼1–5 % of healthy young individuals to ∼50 % in elderly populations with chronic diseases [
      • Petri M.
      Epidemiology of the antiphospholipid antibody syndrome.
      ,
      • Juby A.G.
      • Davis P.
      Prevalence and disease associations of certain autoantibodies in elderly patients.
      ]. These aPL antibodies bind to cell surfaces and activate endothelial cells, platelets, monocytes, and neutrophils [
      • Zuo Y.
      • Shi H.
      • Li C.
      • Knight J.S.
      Antiphospholipid syndrome: a clinical perspective.
      ,
      • Yalavarthi S.
      • Gould T.J.
      • Rao A.N.
      • Mazza L.F.
      • Morris A.E.
      • Nunez-Alvarez C.
      • Hernandez-Ramirez D.
      • Bockenstedt P.L.
      • Liaw P.C.
      • Cabral A.R.
      • Knight J.S.
      Release of neutrophil extracellular traps by neutrophils stimulated with antiphospholipid antibodies: a newly identified mechanism of thrombosis in the antiphospholipid syndrome.
      ]. aPL antibodies induce expression of cell adhesion molecules in endothelial cells, and of TF in endothelial cells and monocytes. Additionally, aPL antibodies may also induce shedding of TF-positive microparticles, which contribute to thrombotic events [
      • Lopez-Pedrera C.
      • Buendia P.
      • Barbarroja N.
      • Siendones E.
      • Velasco F.
      • Cuadrado M.J.
      Antiphospholipid-mediated thrombosis: interplay between anticardiolipin antibodies and vascular cells.
      ,
      • Dobado-Berrios P.M.
      • Lopez-Pedrera C.
      • Velasco F.
      • Aguirre M.A.
      • Torres A.
      • Cuadrado M.J.
      Increased levels of tissue factor mRNA in mononuclear blood cells of patients with primary antiphospholipid syndrome.
      ,
      • Reverter J.C.
      • Tassies D.
      • Font J.
      • Monteagudo J.
      • Escolar G.
      • Ingelmo M.
      • Ordinas A.
      Hypercoagulable state in patients with antiphospholipid syndrome is related to high induced tissue factor expression on monocytes and to low free protein s.
      ,
      • Boles J.
      • Mackman N.
      Role of tissue factor in thrombosis in antiphospholipid antibody syndrome.
      ]. Transient aPL antibodies occur in patients with viral infections, which include human immunodeficiency virus (HIV), varicella zoster virus, hepatitis C virus, cytomegalovirus (CMV), Epstein–Barr virus (EBV), adenovirus, and parvovirus B19 [
      • Sene D.
      • Piette J.C.
      • Cacoub P.
      Antiphospholipid antibodies, antiphospholipid syndrome and viral infections.
      ,
      • Uthman I.W.
      • Gharavi A.E.
      Viral infections and antiphospholipid antibodies.
      ]. Notably, a recent case study found high levels of aPL antibodies (β2GPI IgA, β2GPI IgG; anticardiolipin IgA) in three patients with severe COVID-19 with coagulopathy and preexisting comorbidities [
      • Zhang Y.
      • Xiao M.
      • Zhang S.
      • Xia P.
      • Cao W.
      • Jiang W.
      • Chen H.
      • Ding X.
      • Zhao H.
      • Zhang H.
      • Wang C.
      • Zhao J.
      • Sun X.
      • Tian R.
      • Wu W.
      • Wu D.
      • Ma J.
      • Chen Y.
      • Zhang D.
      • Xie J.
      • Yan X.
      • Zhou X.
      • Liu Z.
      • Wang J.
      • Du B.
      • Qin Y.
      • Gao P.
      • Qin X.
      • Xu Y.
      • Zhang W.
      • Li T.
      • Zhang F.
      • Zhao Y.
      • Li Y.
      • Zhang S.
      Coagulopathy and antiphospholipid antibodies in patients with Covid-19.
      ]. However, the relevance of elevated aPL antibodies in COVID-19 is hard to evaluate because such antibodies can also arise transiently in patients with other critical illnesses and life-threatening infections [
      • Asherson R.A.
      • Cervera R.
      Antiphospholipid antibodies and infections.
      ,
      • Vila P.
      • Hernandez M.C.
      • Lopez-Fernandez M.F.
      • Batlle J.
      Prevalence, follow-up and clinical significance of the anticardiolipin antibodies in normal subjects.
      ,
      • Connell N.T.
      • Battinelli E.M.
      • Connors J.M.
      Coagulopathy of COVID-19 and antiphospholipid antibodies.
      ].
      β2GPI is a plasma protein that is crucial in maintaining hemostasis and the most common target of pathogenic aPL antibodies [
      • McDonnell T.
      • Wincup C.
      • Buchholz I.
      • Pericleous C.
      • Giles I.
      • Ripoll V.
      • Cohen H.
      • Delcea M.
      • Rahman A.
      The role of beta-2-glycoprotein I in health and disease associating structure with function: more than just APS.
      ,
      • Salmon J.E.
      • de Groot P.G.
      Pathogenic role of antiphospholipid antibodies.
      ,
      • Andreoli L.
      • Fredi M.
      • Nalli C.
      • Franceschini F.
      • Meroni P.L.
      • Tincani A.
      Antiphospholipid antibodies mediate autoimmunity against dying cells.
      ]. aPL antibodies can also activate platelets that express high levels of glycoprotein(Gp) IIb/IIIa [
      • Espinola R.G.
      • Pierangeli S.S.
      • Gharavi A.E.
      • Harris E.N.
      Hydroxychloroquine reverses platelet activation induced by human IgG antiphospholipid antibodies.
      ] and thromboxane A2 [
      • Robbins D.L.
      • Leung S.
      • Miller-Blair D.J.
      • Ziboh V.
      Effect of anticardiolipin/beta2-glycoprotein I complexes on production of thromboxane A2 by platelets from patients with the antiphospholipid syndrome.
      ]. Furthermore, aPL antibodies can induce complement activation via the classical pathway to generate complement fragments which propagate inflammatory cells to initiate thrombosis and tissue injury via the membrane attack complex (C5b-9) and anaphylatoxins (C3a, C5a) receptors-mediated responses [
      • Tegla C.A.
      • Cudrici C.
      • Patel S.
      • Trippe III, R.
      • Rus V.
      • Niculescu F.
      • Rus H.
      Membrane attack by complement: the assembly and biology of terminal complement complexes.
      ,
      • Wetsel R.A.
      Structure, function and cellular expression of complement anaphylatoxin receptors.
      ,
      • Tung M.L.
      • Tan B.
      • Cherian R.
      • Chandra B.
      Anti-phospholipid syndrome and COVID-19 thrombosis: connecting the dots.
      ]. Muller-Calleja et al. showed that monoclonal cofactor-independent aPL antibodies rapidly activate TF on myelomonocytic cells in mice [
      • Muller-Calleja N.
      • Ritter S.
      • Hollerbach A.
      • Falter T.
      • Lackner K.J.
      • Ruf W.
      Complement C5 but not C3 is expendable for tissue factor activation by cofactor-independent antiphospholipid antibodies.
      ]. Moreover, aPL antibodies from serum of hospitalized COVID-19 patients are potently thrombogenic [
      • Zuo Y.
      • Estes S.K.
      • Ali R.A.
      • Gandhi A.A.
      • Yalavarthi S.
      • Shi H.
      • Sule G.
      • Gockman K.
      • Madison J.A.
      • Zuo M.
      • Yadav V.
      • Wang J.
      • Woodard W.
      • Lezak S.P.
      • Lugogo N.L.
      • Smith S.A.
      • Morrissey J.H.
      • Kanthi Y.
      • Knight J.S.
      Prothrombotic autoantibodies in serum from patients hospitalized with COVID-19.
      ]. In our ongoing proteomics studies, β2GPI, GpIIb/IIIa, and thromboxane A2 receptor were increased in platelets of SARS-CoV-2-infected K18-hACE2 mice [
      • Subramaniam S.
      • Hekman R.M.
      • Jayaraman A.
      • O’Connell A.K.
      • Montanaro P.
      • Blum B.
      • Kenney D.
      • Ericsson M.
      • Ravid K.
      • Crossland N.A.
      • Emili A.
      • Douam F.
      • Bosmann M.
      Platelet Proteome Analysis Reveals an Early Hyperactive Phenotype in SARS-CoV-2-infected Humanized ACE2 Mice.
      ]. Higher antigen levels of these proteins could further instigate the production of aPL antibodies in COVID-19, since platelets can bind to professional antigen presenting cells in blood followed by phagocytosis for antigen processing and presentation [
      • Ali R.A.
      • Wuescher L.M.
      • Worth R.G.
      Platelets: essential components of the immune system.
      ].
      In recent years, studies on Neutrophil Extracellular Traps (NETs) have shown evidence that autoantibodies against β2GPI induce NETs and enhance thrombosis. NETosis is a unique form of cell death and the formation of NETs is characterized by the release of decondensed chromatin and granular contents to the extracellular space [
      • Vorobjeva N.V.
      • Chernyak B.V.
      NETosis: molecular mechanisms, role in physiology and pathology.
      ]. COVID-19 patients often have high amounts of NETs in their blood [
      • Zuo Y.
      • Zuo M.
      • Yalavarthi S.
      • Gockman K.
      • Madison J.A.
      • Shi H.
      • Woodard W.
      • Lezak S.P.
      • Lugogo N.L.
      • Knight J.S.
      • Kanthi Y.
      Neutrophil extracellular traps and thrombosis in COVID-19.
      ,
      • Shi H.
      • Zuo Y.
      • Yalavarthi S.
      • Gockman K.
      • Zuo M.
      • Madison J.A.
      • Blair C.
      • Woodward W.
      • Lezak S.P.
      • Lugogo N.L.
      • Woods R.J.
      • Lood C.
      • Knight J.S.
      • Kanthi Y.
      Neutrophil calprotectin identifies severe pulmonary disease in COVID-19.
      ,
      • Barnes B.J.
      • Adrover J.M.
      • Baxter-Stoltzfus A.
      • Borczuk A.
      • Cools-Lartigue J.
      • Crawford J.M.
      • Dassler-Plenker J.
      • Guerci P.
      • Huynh C.
      • Knight J.S.
      • Loda M.
      • Looney M.R.
      • McAllister F.
      • Rayes R.
      • Renaud S.
      • Rousseau S.
      • Salvatore S.
      • Schwartz R.E.
      • Spicer J.D.
      • Yost C.C.
      • Weber A.
      • Zuo Y.
      • Egeblad M.
      Targeting potential drivers of COVID-19: neutrophil extracellular traps.
      ], which many contribute to the procoagulant response. Folco et al. reported that NETs induce VCAM-1, ICAM-1, and TF expression towards an increased procoagulant state of endothelial cells, which is further augmented by IL-1α and Cathepsin G [
      • Folco E.J.
      • Mawson T.L.
      • Vromman A.
      • Bernardes-Souza B.
      • Franck G.
      • Persson O.
      • Nakamura M.
      • Newton G.
      • Luscinskas F.W.
      • Libby P.
      Neutrophil extracellular traps induce endothelial cell activation and tissue factor production through interleukin-1alpha and cathepsin G.
      ].
      On one hand, aPL antibodies can directly activate monocytes, which in turn interact with the endothelium via MCP-1, resulting in TF-dependent pro-thrombotic events [
      • Kinev A.V.
      • Roubey R.A.
      Tissue factor in the antiphospholipid syndrome.
      ,
      • Shantsila E.
      • Lip G.Y.
      The role of monocytes in thrombotic disorders. Insights from tissue factor, monocyte-platelet aggregates and novel mechanisms.
      ,
      • Cho C.S.
      • Cho M.L.
      • Chen P.P.
      • Min S.Y.
      • Hwang S.Y.
      • Park K.S.
      • Kim W.U.
      • Min D.J.
      • Min J.K.
      • Park S.H.
      • Kim H.Y.
      Antiphospholipid antibodies induce monocyte chemoattractant protein-1 in endothelial cells.
      ,
      • Cuadrado M.J.
      • Lopez-Pedrera C.
      • Khamashta M.A.
      • Camps M.T.
      • Tinahones F.
      • Torres A.
      • Hughes G.R.
      • Velasco F.
      Thrombosis in primary antiphospholipid syndrome: a pivotal role for monocyte tissue factor expression.
      ]. On the other hand, aPL antibodies can directly activate β2GPI expressing monocytes, which subsequently upregulates NF-κB-mediated TF expression via activation of mitogen-activated protein kinases (MAPK) [
      • Yasuda S.
      • Bohgaki M.
      • Atsumi T.
      • Koike T.
      Pathogenesis of antiphospholipid antibodies: impairment of fibrinolysis and monocyte activation via the p38 mitogen-activated protein kinase pathway.
      ].
      Additional studies confirmed an increased expression of TF and elevated procoagulant activity in circulating monocytes of patients with aPL antibodies [
      • Dobado-Berrios P.M.
      • Lopez-Pedrera C.
      • Velasco F.
      • Aguirre M.A.
      • Torres A.
      • Cuadrado M.J.
      Increased levels of tissue factor mRNA in mononuclear blood cells of patients with primary antiphospholipid syndrome.
      ,
      • Cuadrado M.J.
      • Lopez-Pedrera C.
      • Khamashta M.A.
      • Camps M.T.
      • Tinahones F.
      • Torres A.
      • Hughes G.R.
      • Velasco F.
      Thrombosis in primary antiphospholipid syndrome: a pivotal role for monocyte tissue factor expression.
      ]. aPL antibodies can also mediate inflammatory response of endothelial cells through the activation of innate immune receptors TLR2 and TLR4. These TLRs serve as binding sites for dimeric β2GPI, which results in endothelial dysfunction by increased expression of TF and adhesion molecules [
      • Alard J.E.
      • Gaillard F.
      • Daridon C.
      • Shoenfeld Y.
      • Jamin C.
      • Youinou P.
      TLR2 is one of the endothelial receptors for beta 2-glycoprotein I.
      ,
      • Raschi E.
      • Chighizola C.B.
      • Grossi C.
      • Ronda N.
      • Gatti R.
      • Meroni P.L.
      • Borghi M.O.
      beta2-glycoprotein I, lipopolysaccharide and endothelial TLR4: three players in the two hit theory for anti-phospholipid-mediated thrombosis.
      ,
      • Raschi E.
      • Testoni C.
      • Bosisio D.
      • Borghi M.O.
      • Koike T.
      • Mantovani A.
      • Meroni P.L.
      Role of the MyD88 transduction signaling pathway in endothelial activation by antiphospholipid antibodies.
      ,
      • Velasquez M.
      • Rojas M.
      • Abrahams V.M.
      • Escudero C.
      • Cadavid A.P.
      Mechanisms of endothelial dysfunction in antiphospholipid syndrome: association with clinical manifestations.
      ]. Treatment of endothelial cells with IgG-aPL antibodies from clinically active APS induced expression of TF, IL-6, and IL-8 via activation of p38 MAPK and NF-kB pathways [
      • Vega-Ostertag M.
      • Casper K.
      • Swerlick R.
      • Ferrara D.
      • Harris E.N.
      • Pierangeli S.S.
      Involvement of p38 MAPK in the up-regulation of tissue factor on endothelial cells by antiphospholipid antibodies.
      ]. Similar to this observation, human sera from n = 118 hospitalized COVID-19 patients contained anticardiolipin IgG/IgM and anti-phosphatidlyserine/prothrombin (anti-PS/PT) IgG/IgM. These aPL antibodies in the COVID-19 patients' sera upregulated the expression of surface adhesion markers (E-selectin, VCAM-1, and ICAM-1) in cultured endothelial cells [
      • Shi H.
      • Zuo Y.
      • Gandhi A.A.
      • Sule G.
      • Yalavarthi S.
      • Gockman K.
      • Madison J.A.
      • Wang J.
      • Zuo M.
      • Shi Y.
      • Knight J.S.
      • Kanthi Y.
      Endothelial Cell-activating Antibodies in COVID-19.
      ]. In contrast, Borghi et al. reported a low prevalence of aPL antibodies in COVID-19 patients and no association with major thrombotic events [
      • Borghi M.O.
      • Beltagy A.
      • Garrafa E.
      • Curreli D.
      • Cecchini G.
      • Bodio C.
      • Grossi C.
      • Blengino S.
      • Tincani A.
      • Franceschini F.
      • Andreoli L.
      • Lazzaroni M.G.
      • Piantoni S.
      • Masneri S.
      • Crisafulli F.
      • Brugnoni D.
      • Muiesan M.L.
      • Salvetti M.
      • Parati G.
      • Torresani E.
      • Mahler M.
      • Heilbron F.
      • Pregnolato F.
      • Pengo M.
      • Tedesco F.
      • Pozzi N.
      • Meroni P.L.
      Anti-phospholipid antibodies in COVID-19 are different from those detectable in the anti-phospholipid syndrome.
      ].
      In conclusion, the aPL antibodies may contribute to the development of arterial and venous thrombosis through various mechanisms in severe COVID-19 patients. However, more data is needed on the occurrence of aPL antibodies during SARS-CoV-2 infection. The mechanisms underlying aPL antibody-mediated coagulopathy are understudied. Further clinical and experimental studies will help to better assess the role of APS in the pathogenesis of COVID-19 and CAC.

      7. Complement activation and TF in COVID-19

      Complement activation products (e.g. C5b-9, C5a, C3a) can enhance neutrophil/monocyte activation and their recruitment to the infected lungs. Several complement effectors, acting in concert with platelets, can fuel thrombo-inflammation and endothelial dysfunction [
      • Skendros P.
      • Mitsios A.
      • Chrysanthopoulou A.
      • Mastellos D.C.
      • Metallidis S.
      • Rafailidis P.
      • Ntinopoulou M.
      • Sertaridou E.
      • Tsironidou V.
      • Tsigalou C.
      • Tektonidou M.
      • Konstantinidis T.
      • Papagoras C.
      • Mitroulis I.
      • Germanidis G.
      • Lambris J.D.
      • Ritis K.
      Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis.
      ]. A complement-driven prothrombotic state is observed in diseases such as paroxysmal nocturnal hemoglobinuria, glomerulonephritis, and vasculitis [
      • Davalos D.
      • Akassoglou K.
      Fibrinogen as a key regulator of inflammation in disease.
      ,
      • Lee J.W.
      • Jang J.H.
      • Kim J.S.
      • Yoon S.S.
      • Lee J.H.
      • Kim Y.K.
      • Jo D.Y.
      • Chung J.
      • Sohn S.K.
      Clinical signs and symptoms associated with increased risk for thrombosis in patients with paroxysmal nocturnal hemoglobinuria from a Korean Registry.
      ,
      • Hill A.
      • Kelly R.J.
      • Hillmen P.
      Thrombosis in paroxysmal nocturnal hemoglobinuria.
      ]. Complement activation induces TF expression in various cell types, and skews mast cells and basophils towards a prothrombotic phenotype [
      • Wong E.K.S.
      • Kavanagh D.
      Diseases of complement dysregulation-an overview.
      ]. Activation of the complement and kallikrein/kinin system in critically ill COVID-19 patients is linked to thromboinflammation [
      • Lipcsey M.
      • Persson B.
      • Eriksson O.
      • Blom A.M.
      • Fromell K.
      • Hultstrom M.
      • Huber-Lang M.
      • Ekdahl K.N.
      • Frithiof R.
      • Nilsson B.
      The outcome of critically ill COVID-19 patients is linked to thromboinflammation dominated by the Kallikrein/Kinin system.
      ]. Spike protein of SARS-CoV-2 is recognized by Mannose-binding lectin, which results in both viral inhibition and complement activation [
      • Stravalaci M.
      • Pagani I.
      • Paraboschi E.M.
      • Pedotti M.
      • Doni A.
      • Scavello F.
      • Mapelli S.N.
      • Sironi M.
      • Perucchini C.
      • Varani L.
      • Matkovic M.
      • Cavalli A.
      • Cesana D.
      • Gallina P.
      • Pedemonte N.
      • Capurro V.
      • Clementi N.
      • Mancini N.
      • Invernizzi P.
      • Bayarri-Olmos R.
      • Garred P.
      • Rappuoli R.
      • Duga S.
      • Bottazzi B.
      • Uguccioni M.
      • Asselta R.
      • Vicenzi E.
      • Mantovani A.
      • Garlanda C.
      Recognition and inhibition of SARS-CoV-2 by humoral innate immunity pattern recognition molecules.
      ]. The alternative pathway (down-regulation of cellular complement inhibitors [CD46, CD55, CD59] in infected cells) and the classical pathway (after anti-SARS-CoV-2 or auto-antibody production ensues) also contribute to complement activation [
      • Afzali B.
      • Noris M.
      • Lambrecht B.N.
      • Kemper C.
      The state of complement in COVID-19.
      ,
      • Henry B.M.
      • Szergyuk I.
      • de Oliveira M.H.S.
      • Lippi G.
      • Benoit J.L.
      • Vikse J.
      • Benoit S.W.
      Complement levels at admission as a reflection of coronavirus disease 2019 (COVID-19) severity state.
      ,
      • Bosmann M.
      Complement activation during critical illness: current findings and an outlook in the era of COVID-19.
      ,
      • Bosmann M.
      Complement control for COVID-19.
      ].
      The anaphylatoxins, C3a and C5a, play critical roles in immune modulation and regulation of cellular adaptation via their cell surface receptors (C3aR, C5aR1, C5aR2) [
      • Bosmann M.
      • Ward P.A.
      Role of C3, C5 and anaphylatoxin receptors in acute lung injury and in sepsis.
      ]. Anaphylatoxins promote TF-dependent activation of coagulation pathways [
      • Chauhan A.J.
      • Wiffen L.J.
      • Brown T.P.
      COVID-19: a collision of complement, coagulation and inflammatory pathways.
      ,
      • Carvelli J.
      • Demaria O.
      • Vely F.
      • Batista L.
      • Benmansour N.Chouaki
      • Fares J.
      • Carpentier S.
      • Thibult M.L.
      • Morel A.
      • Remark R.
      • Andre P.
      • Represa A.
      • Piperoglou C.
      • Explore C.-I.P.H.g.
      • Explore C.-M.I.g.
      • Cordier P.Y.
      • Dault E.Le
      • Guervilly C.
      • Simeone P.
      • Gainnier M.
      • Morel Y.
      • Ebbo M.
      • Schleinitz N.
      • Vivier E.
      Association of COVID-19 inflammation with activation of the C5a-C5aR1 axis.
      ,
      • Ritis K.
      • Doumas M.
      • Mastellos D.
      • Micheli A.
      • Giaglis S.
      • Magotti P.
      • Rafail S.
      • Kartalis G.
      • Sideras P.
      • Lambris J.D.
      A novel C5a receptor-tissue factor cross-talk in neutrophils links innate immunity to coagulation pathways.
      ,
      • Redecha P.
      • Tilley R.
      • Tencati M.
      • Salmon J.E.
      • Kirchhofer D.
      • Mackman N.
      • Girardi G.
      Tissue factor: a link between C5a and neutrophil activation in antiphospholipid antibody induced fetal injury.
      ,
      • Foley J.H.
      • Conway E.M.
      Cross talk pathways between coagulation and inflammation.
      ]. The plasma/serum concentrations of sC5b-9 and C5a are elevated in hospitalized COVID-19 patients [
      • Cugno M.
      • Meroni P.L.
      • Gualtierotti R.
      • Griffini S.
      • Grovetti E.
      • Torri A.
      • Panigada M.
      • Aliberti S.
      • Blasi F.
      • Tedesco F.
      • Peyvandi F.
      Complement activation in patients with COVID-19: a novel therapeutic target.
      ,
      • Giudice V.
      • Pagliano P.
      • Vatrella A.
      • Masullo A.
      • Poto S.
      • Polverino B.M.
      • Gammaldi R.
      • Maglio A.
      • Sellitto C.
      • Vitale C.
      • Serio B.
      • Cuffa B.
      • Borrelli A.
      • Vecchione C.
      • Filippelli A.
      • Selleri C.
      Combination of ruxolitinib and eculizumab for treatment of severe SARS-CoV-2-related acute respiratory distress syndrome: a controlled study.
      ,
      • Ma L.
      • Sanjaya K.S.
      • Cano M.
      • Kuppuswamy V.
      • Bajwa J.
      Increased complement activation is a distinctive feature of severe SARS-CoV-2 infection.
      ]. In line with this, two cases of COVID-19 patients treated with an anti-C5a antibody showed a clinical improvement, as measured by increased lung oxygenation and decreased systemic inflammation [
      • Gao T.
      • Hu M.
      • Zhang X.
      • Li H.
      • Zhu L.
      • Liu H.
      • Dong Q.
      • Zhang Z.
      • Wang Z.
      • Hu Y.
      • Fu Y.
      • Jin Y.
      • Li K.
      • Zhao S.
      • Xiao Y.
      • Luo S.
      • Li L.
      • Zhao L.
      • Liu J.
      • Zhao H.
      • Liu Y.
      • Yang W.
      • Peng J.
      • Chen X.
      • Li P.
      • Liu Y.
      • Xie Y.
      • Song J.
      • Zhang L.
      • Ma Q.
      • Bian X.
      • Chen W.
      • Liu X.
      • Mao Q.
      • Cao C.
      Highly Pathogenic Coronavirus N Protein Aggravates Lung Injury by MASP-2-mediated Complement Over-activation.
      ]. An association of endothelial dysfunction with COVID-19 and enhancement by complement fragments, C5a and C3a, has been verified in patients with severe COVID-19. C5a-mediated activation of endothelial C5aR1 induces TF production [
      • Ikeda K.
      • Nagasawa K.
      • Horiuchi T.
      • Tsuru T.
      • Nishizaka H.
      • Niho Y.
      C5a induces tissue factor activity on endothelial cells.
      ]. Three clinical trials have been registered for eculizumab, a complement C5 inhibitor, as a treatment for patients with COVID-19 (ClinicalTrials.gov Identifiers: NCT04288713, NCT04346797, and NCT04355494) [
      • Noris M.
      • Benigni A.
      • Remuzzi G.
      The case of complement activation in COVID-19 multiorgan impact.
      ,
      • Li J.
      • Liu B.
      The roles and potential therapeutic implications of C5a in the pathogenesis of COVID-19-associated coagulopathy.
      ]. Recently, a combination of ruxolitinib (a JAK1/2 inhibitor) and eculizumab was administered to severely ill COVID-19 patients with a hypercoagulable state and ARDS. This resulted in significant improvements in respiratory symptoms, pulmonary lesions, and decreased D-dimer concentrations [
      • Giudice V.
      • Pagliano P.
      • Vatrella A.
      • Masullo A.
      • Poto S.
      • Polverino B.M.
      • Gammaldi R.
      • Maglio A.
      • Sellitto C.
      • Vitale C.
      • Serio B.
      • Cuffa B.
      • Borrelli A.
      • Vecchione C.
      • Filippelli A.
      • Selleri C.
      Combination of ruxolitinib and eculizumab for treatment of severe SARS-CoV-2-related acute respiratory distress syndrome: a controlled study.
      ,
      • Laurence J.
      • Mulvey J.J.
      • Seshadri M.
      • Racanelli A.
      • Harp J.
      • Schenck E.J.
      • Zappetti D.
      • Horn E.M.
      • Magro C.M.
      Anti-complement C5 therapy with eculizumab in three cases of critical COVID-19.
      ].
      A dysregulation of the complement system could promote thrombotic events in patients with severe COVID-19 [
      • Magro C.
      • Mulvey J.J.
      • Berlin D.
      • Nuovo G.
      • Salvatore S.
      • Harp J.
      • Baxter-Stoltzfus A.
      • Laurence J.
      Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: a report of five cases.
      ]. A deposition of membrane attack complex pores in vascular cell membranes is a key feature of several microthrombotic syndromes and has also been demonstrated in COVID-19 [
      • Amor S.
      • Fernandez Blanco L.
      • Baker D.
      Innate immunity during SARS-CoV-2: evasion strategies and activation trigger hypoxia and vascular damage.
      ]. Initial genetic evidence suggests that TF as well as regulators of the complement and coagulation pathways are associated with the development of severe COVID-19 pathologies [
      • Subramaniam S.
      • Hekman R.M.
      • Jayaraman A.
      • O’Connell A.K.
      • Montanaro P.
      • Blum B.
      • Kenney D.
      • Ericsson M.
      • Ravid K.
      • Crossland N.A.
      • Emili A.
      • Douam F.
      • Bosmann M.
      Platelet Proteome Analysis Reveals an Early Hyperactive Phenotype in SARS-CoV-2-infected Humanized ACE2 Mice.
      ,
      • Ramlall V.
      • Thangaraj P.M.
      • Meydan C.
      • Foox J.
      • Butler D.
      • Kim J.
      • May B.
      • De Freitas J.K.
      • Glicksberg B.S.
      • Mason C.E.
      • Tatonetti N.P.
      • Shapira S.D.
      Immune complement and coagulation dysfunction in adverse outcomes of SARS-CoV-2 infection.
      ]. Complement activation has also been shown to directly regulate TF activity on monocytes by activating thiol-isomerase pathways required for TF functions in thrombosis [
      • Muller-Calleja N.
      • Ritter S.
      • Hollerbach A.
      • Falter T.
      • Lackner K.J.
      • Ruf W.
      Complement C5 but not C3 is expendable for tissue factor activation by cofactor-independent antiphospholipid antibodies.
      ,
      • Langer F.
      • Spath B.
      • Fischer C.
      • Stolz M.
      • Ayuk F.A.
      • Kroger N.
      • Bokemeyer C.
      • Ruf W.
      Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase.
      ,
      • Subramaniam S.
      • Jurk K.
      • Hobohm L.
      • Jackel S.
      • Saffarzadeh M.
      • Schwierczek K.
      • Wenzel P.
      • Langer F.
      • Reinhardt C.
      • Ruf W.
      Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development.
      ].
      In summary, complement activation and anaphylatoxins may regulate TF-mediated thrombosis, which would support the approach of testing complement inhibitors for beneficial therapeutic effects on CAC.

      8. Extracellular vesicles and TF in COVID-19

      Extracellular vesicles (EV) are a heterogeneous group of cell-derived membranous structures containing exosomes and microvesicles, which originate from the endosomal system or which are shed from the plasma membrane, respectively [
      • van Niel G.
      • D'Angelo G.
      • Raposo G.
      Shedding light on the cell biology of extracellular vesicles.
      ]. EVs are released by various cells during lung inflammation and innate immune responses [
      • Chen Z.
      • Larregina A.T.
      • Morelli A.E.
      Impact of extracellular vesicles on innate immunity.
      ,
      • Kouwaki T.
      • Okamoto M.
      • Tsukamoto H.
      • Fukushima Y.
      • Oshiumi H.
      Extracellular vesicles deliver host and virus RNA and regulate innate immune response.
      ].
      Several studies have shown that TF is often incorporated into microparticles and EVs, where it exerts critical pathophysiological activities and propagates inflammation, including during COVID-19 [
      • McFadyen J.D.
      • Stevens H.
      • Peter K.
      The emerging threat of (micro)thrombosis in COVID-19 and its therapeutic implications.
      ,
      • Marchandot B.
      • Sattler L.
      • Jesel L.
      • Matsushita K.
      • Schini-Kerth V.
      • Grunebaum L.
      • Morel O.
      COVID-19 related coagulopathy: a distinct entity?.
      ]. Zaid et al. showed increased levels of platelet-derived CD41+-EVs in the plasma of COVID-19 patients [
      • Zaid Y.
      • Puhm F.
      • Allaeys I.
      • Naya A.
      • Oudghiri M.
      • Khalki L.
      • Limami Y.
      • Zaid N.
      • Sadki K.
      • Ben El Haj R.
      • Mahir W.
      • Belayachi L.
      • Belefquih B.
      • Benouda A.
      • Cheikh A.
      • Langlois M.A.
      • Cherrah Y.
      • Flamand L.
      • Guessous F.
      • Boilard E.
      Platelets can associate with SARS-Cov-2 RNA and are hyperactivated in COVID-19.
      ]. Balbi et al. demonstrated elevated levels of 37 EV antigens involved in inflammation, platelet activation, coagulation processes, and endothelial dysfunction, including CD62P, CD142 (TF), and CD41, in serum samples from COVID-19 patients [
      • Balbi C.
      • Burrello J.
      • Bolis S.
      • Lazzarini E.
      • Biemmi V.
      • Pianezzi E.
      • Burrello A.
      • Caporali E.
      • Grazioli L.G.
      • Martinetti G.
      • Fusi-Schmidhauser T.
      • Vassalli G.
      • Melli G.
      • Barile L.
      Circulating extracellular vesicles are endowed with enhanced procoagulant activity in SARS-CoV-2 infection.
      ]. Another study demonstrated association of elevated levels of circulating EV-TF activity with disease severity and mortality in COVID-19 patients [
      • Rosell A.
      • Havervall S.
      • von Meijenfeldt F.
      • Hisada Y.
      • Aguilera K.
      • Grover S.P.
      • Lisman T.
      • Mackman N.
      • Thalin C.
      Patients with COVID-19 have elevated levels of circulating extracellular vesicle tissue factor activity that is associated with severity and mortality.
      ]. Longitudinal evaluation of MVs in COVID-19 patients plasma samples revealed that a significant decreased plasma concentrations of endothelium-derived EVs (E-Selectin+), endothelium-derived bearing TF (E-Selectin+TF+), endothelium-derived bearing ACE2 (E-Selectin+ACE2+) and leukocyte-EVs bearing TF (CD45+TF+) at 30-days post-discharge. Interestingly, platelet- and leukocyte-EVs further increased 30-days after post discharge, indicating that cellular activation persists long after the acute phase [
      • Campello E.
      • Radu C.M.
      • Simion C.
      • Spiezia L.
      • Bulato C.
      • Gavasso S.
      • Tormene D.
      • Perin N.
      • Turatti G.
      • Simioni P.
      Longitudinal trend of plasma concentrations of extracellular vesicles in patients hospitalized for COVID-19.
      ]. A comparative study of septic shock patients revealed that increased presence of distinct EVs with a higher EV-TF correlated with the thrombotic risk in severe COVID-19 patients [
      • Guervilly C.
      • Bonifay A.
      • Burtey S.
      • Sabatier F.
      • Cauchois R.
      • Abdili E.
      • Arnaud L.
      • Lano G.
      • Pietri L.
      • Robert T.
      • Velier M.
      • Papazian L.
      • Albanese J.
      • Kaplanski G.
      • Dignat-George F.
      • Lacroix R.
      Dissemination of extreme levels of extracellular vesicles: tissue factor activity in patients with severe COVID-19.
      ].
      These findings support the concept that circulating EV-associated TF may contribute to CAC and microthrombi formation. More prospective and retrospective clinical studies could enhance our understanding of the EV-TF-mediated hypercoagulatory state in COVID-19.

      9. Perspectives

      Arterial and venous thrombosis are frequently encountered in patients with severe COVID-19 and contribute to increased morbidity and mortality. In the absence of a monocausal explanation, it seems likely that the coagulopathy of COVID-19 is a cumulative result of several dysregulated pathways. Emerging evidence provides support for the concept that increased expression of TF is quite likely to contribute to thrombosis in severe COVID-19 (Fig. 1). High expression of TF on endothelial cells and monocytes may command increased fibrin formation and platelet activation during COVID-19 infection. Based on the existing evidence, we speculate that endothelial procoagulant and inflammatory responses could be due to the combination of aPL-, complement-, and cytokine-mediated expression of TF, rather than direct infection of the endothelium or blood cells. TF-coated EVs may specifically contribute to the development of CAC. Of note, the induction of TF expression is not necessarily restricted to PRR pathways with a direct role in viral immune recognition (TLR7, TLR8, RIG-I/MDA5), but may be related to DAMPs released during infection-associated tissue injury. In fact, it is a fluid scientific discussion to what extent endothelial cells, platelets and leukocytes are active replication site for SARS-CoV-2; or if an infrequent uptake of virions into these cells is better termed ‘abortive infection’. The suggested correlation of aPL antibodies with an increased risk of CAC demands further preclinical investigations and controlled clinical studies. Systematic approaches will be most suitable to test the hypothesis if TF is essential or redundant for the pathogenesis of CAC.
      Fig. 1
      Fig. 1Current concepts of TF in COVID-19-associated coagulopathy (CAC). Multiple mechanisms may contribute to TF expression, including direct infection of type I/II epithelial cells and monocytes, pattern-recognition receptors activation (TLR-3/-7/-8), complement-mediated MAC (C5b-C9) and anaphylatoxins (C5a, C3a), excessive cytokine release (IL-1, IL-6, IL-8, TNF-α) from immune and non-immune cells. These events subsequently lead to barrier dysfunction, increased vascular permeability, and activation of blood coagulation. Antiphospholipid antibodies may contribute to the activation of coagulation and endothelial cell-leukocyte interactions. TF-dependent activation of Xa/thrombin and excessive PAI-1 (which inhibits fibrinolysis) during SARS-CoV-2 infection results in the formation of fibrin-rich thrombi. IL, interleukin; NETs, neutrophil extracellular traps; vWF, von Willebrand factor; PAI-1, plasminogen activator inhibitor-1; TF, tissue factor; TNF-α, tumor necrosis factor-alpha; E-SELE, E-selectin; VCAM-1, vascular cell adhesion protein 1; ICAM-1, intercellular adhesion molecule-1; C3, complement 3; C5, complement 5; aPL antibody, antiphospholipid antibody.
      Transgenic mice with tissue-specific TF deficiency in lung epithelial cells showed increased lung hemorrhage and mortality when infected with IAV/H1N1, highlighting the essential role of TF for hemostasis during lung infection [
      • Antoniak S.
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      • Beck M.A.
      • Bastarache J.A.
      • Mackman N.
      Tissue factor deficiency increases alveolar hemorrhage and death in influenza A virus-infected mice.
      ]. On the other hand, excessive activation of TF may cause thrombotic complications [
      • Antoniak S.
      • Tatsumi K.
      • Hisada Y.
      • Milner J.J.
      • Neidich S.D.
      • Shaver C.M.
      • Pawlinski R.
      • Beck M.A.
      • Bastarache J.A.
      • Mackman N.
      Tissue factor deficiency increases alveolar hemorrhage and death in influenza A virus-infected mice.
      ,
      • Yang Y.
      • Tang H.
      Aberrant coagulation causes a hyper-inflammatory response in severe influenza pneumonia.
      ]. This doubled edged sword response of TF could be further studied using existing TF-low or cell-type specific TF-deficient mouse models.
      Further translational studies would especially benefit from tailored animal models that recapitulate the coagulation abnormalities and micro-/macro-vascular thrombosis of human COVID-19. While numerous animal species can be exploited to better understand COVID-19 pathology [
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      • Dowling W.E.
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      • Riveros-Balta A.X.
      • Albrecht R.A.
      • Andersen H.
      • Baric R.S.
      • Carroll M.W.
      • Cavaleri M.
      • Qin C.
      • Crozier I.
      • Dallmeier K.
      • de Waal L.
      • de Wit E.
      • Delang L.
      • Dohm E.
      • Duprex W.P.
      • Falzarano D.
      • Finch C.L.
      • Frieman M.B.
      • Graham B.S.
      • Gralinski L.E.
      • Guilfoyle K.
      • Haagmans B.L.
      • Hamilton G.A.
      • Hartman A.L.
      • Herfst S.
      • Kaptein S.J.F.
      • Klimstra W.B.
      • Knezevic I.
      • Krause P.R.
      • Kuhn J.H.
      • Le Grand R.
      • Lewis M.G.
      • Liu W.C.
      • Maisonnasse P.
      • McElroy A.K.
      • Munster V.
      • Oreshkova N.
      • Rasmussen A.L.
      • Rocha-Pereira J.
      • Rockx B.
      • Rodriguez E.
      • Rogers T.F.
      • Salguero F.J.
      • Schotsaert M.
      • Stittelaar K.J.
      • Thibaut H.J.
      • Tseng C.T.
      • Vergara-Alert J.
      • Beer M.
      • Brasel T.
      • Chan J.F.W.
      • Garcia-Sastre A.
      • Neyts J.
      • Perlman S.
      • Reed D.S.
      • Richt J.A.
      • Roy C.J.
      • Segales J.
      • Vasan S.S.
      • Henao-Restrepo A.M.
      • Barouch D.H.
      Animal models for COVID-19.
      ], a further refinement is needed for studying CAC. For example, humanized hACE2 mice do not seem to develop macrovascular thrombosis and pulmonary embolism from SARS-CoV-2 infection alone. Additional vascular insults (e.g. FeCl3-induced vascular injury, subtotal V. cava ligation) may be needed to precipitate major thrombotic events and screen for the efficacy of TF blocking drug candidates in small animals. Targeting TF could be a therapeutic approach in selected COVID-19 patients, who show abnormal clinical markers associated with thrombotic coagulopathy (e.g. ↑D-dimers). In conclusion, a better understanding of TF-dependent prothrombotic mechanisms could greatly facilitate the development of novel therapies to reduce the occurrence and severe consequences of CAC.

      Funding sources

      This work was supported by the Aniara Diagnostica (Coagulation Research Grant 2020-2021 , and the National Institutes of Health ( 1UL1TR001430 ) to S.S.), the National Institutes of Health ( 1UL1TR001430 , 1R01HL141513 , 1R01HL139641 to M.B.), and the Deutsche Forschungsgemeinschaft ( BO3482/3-3 , BO3482/4-1 to M.B.). We thank the Clinical & Translational Science Institute (CTSI) and Evans Center for Interdisciplinary Biomedical Research at Boston University School of Medicine for their support of the Affinity Research Collaborative on ‘Respiratory Viruses: A Focus on COVID-19’.

      CRediT authorship contribution statement

      Contribution: S.S. wrote and revised the manuscript. H.K., and M.B., provided critical feedback and contributed to writing the manuscript. All the authors are responsible for the content of this publication.

      Declaration of competing interest

      None of the authors declare any conflict of interest.

      Acknowledgments

      The authors thanks to Kara Farquharson for proof-reading the final draft of the manuscript.

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