Prothrombotic Transformation of the Endothelium Is Markedly Augmented By Cytokine Priming and Inhibited By Tie2 Activation

The endothelium is an intrinsically anticoagulant surface that upon viral or bacterial infection promotes thrombosis via the upregulation of pro-coagulatory mechanisms and loss of anticoagulant properties. The COVID-19 pandemic has underscored the association between respiratory infection and thrombosis by highlighting the importance of host cytokine responses in mediating systemic vasculopathy. Immune activation within the alveolar-capillary unit precedes systemic pathogen spread and induces marked increases in plasma cytokine levels prior to pathogen circulation. We therefore tested the hypothesis that cytokine exposure primes ECs toward a pro-coagulatory state that is subsequently activated by pathogen-associated molecular patterns (PAMPs). In initial studies, human ECs were exposed to cytokines elevated in the setting of pneumonia including TNFα, IFNα, IFNβ, IFNγ, IL-1β, IL-6, IL-10, and MCP for 24 hours prior to induction with poly(I:C), a viral PAMP mimetic and TLR3 agonist, and subsequently evaluated for thrombin generation in a plasma-based assay. Pre-incubation of ECs with several candidate cytokines enhanced responses to poly(I:C) by 10-20-fold. Focusing on the antiviral type-II interferon cytokine IFNγ, we found that its pre-incubation with ECs enhanced thrombin generation induced by poly(I:C) in a dose-dependent manner. IFNγ followed by poly(I:C) also enhanced factor VIIa-dependent factor Xa (FXa) generation, implicating upregulation of TF activity. SARS-CoV-2 nucleocapsid and spike proteins showed little prothrombotic effects in ECs by themselves. However, following incubation of ECs with IFNγ, both proteins stimulated FXa generation. These observations were not limited to viral PAMPs, as pre-incubation with IFNγ enhanced thrombin and FXa generation in response to lipopolysaccharide (LPS), a bacterial PAMP. Exposure of ECs to IFNγ also resulted in a reduction in total thrombomodulin, even prior to PAMP exposure. To better replicate the alveolar-capillary functional unit, we used an alveolus-on-chip model. The chip includes primary pulmonary microvascular ECs cultured under flow conditions in a vascular channel and small airway epithelial cells in an alveolus channel. The two channels are separated by a collagen matrix and the chip is subject to cyclic stretch to mimic breathing. Exposure of pneumocytes in the pulmonary channel to IFNγ and either poly(I:C) or LPS resulted in decreased production of thrombomodulin in the vascular channel, validating the monoculture results. One strategy for preventing the prothrombotic transformation of ECs is to stimulate cytoprotective endothelial pathways. Tie2 is a cytoprotective receptor found primarily on ECs. Incubation of ECs with the Tie2 ligand angiopoietin-1 (Angpt-1) significantly reversed the marked thrombin generation observed with IFNγ-priming followed by poly(I:C) exposure. Angpt-1 did not directly inhibit thrombin or FXa generation, as measured in standard thrombin time and aPTT assays. However, exposure to Angpt-1 did reverse depletion of protein C generation induced by poly(I:C) or LPS. Angpt-1 also reversed thrombomodulin reduction, providing an explanation for the ability of Angpt-1 to suppress cytokine/PAMP-induced prothrombotic changes on the ECs. To test the effect of Tie2 activation on thrombus formation in vivo, we used an LPS model since LPS was the most thrombotic of the PAMPs that we tested in mice. We used the small molecule [4-[(2S)-2-[[(2S)-2-(methoxycarbonylamino)-3-phenylpropanoyl]amino]-2-(2-thiophen-2-yl-1,3-thiazol-4-yl)ethyl]phenyl]sulfamic acid to promote Tie2 activation. Infusion of this Tie2 activating compound reduced LPS-induced accumulation of fibrin in both lung and liver. Taken together, these studies show that cytokines prime ECs for prothrombotic changes in response to PAMP exposure. Decreased thrombomodulin expression upon cytokine priming with resultant reduction in activated protein C production is essential to these prothrombotic changes. Tie2 activation inhibits the prothrombotic transformation and represents a potential strategy for reversing thrombosis associated with respiratory infection.

Flaumenhaft:Platelet Diagnostics: Consultancy, Current equity holder in private company, Other: Founder, Patents & Royalties; Function Therapeutics: Consultancy, Current holder of stock options in a privately-held company; Xap Therapeutics: Consultancy.