Angiopoietin-2 Inhibition of Thrombomodulin-Mediated Anticoagulation—A Novel Mechanism That May Contribute to Hypercoagulation in Critically Ill COVID-19 Patients
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Abstract
Hypercoagulation and endothelial dysfunction play central roles in severe forms of COVID-19 infections, but the molecular mechanisms involved are unclear. Increased plasma levels of the inflammatory cytokine and TIE2 receptor antagonist Angiopoietin-2 were reported in severely ill COVID-19 patients. In vitro experiments suggest that Angiopoietin-2 bind and inhibits thrombomodulin. Thrombomodulin is expressed on the luminal surface of endothelial cells where it is an important member of the intrinsic anticoagulant pathway through activation of protein C. Using clinical data, mouse models, and in vitro assays, we tested if Angiopoietin-2 plays a causal role in COVID-19-associated hypercoagulation through direct inhibition of thrombin/thrombomodulin-mediated physiological anticoagulation. Angiopoietin-2 was measured in 61 patients at admission, and after 10 days in the 40 patients remaining in the ICU. We found that Angiopoietin-2 levels were increased in COVID-19 patients in correlation with disease severity, hypercoagulation, and mortality. In support of a direct effect of Angiopoietin-2 on coagulation, we found that injected Angiopoietin-2 in mice associated to thrombomodulin and resulted in a shortened tail bleeding time, decreased circulating levels of activated protein C, and increased plasma thrombin/antithrombin complexes. Conversely, bleeding time was increased in endothelial-specific Angiopoietin-2 knockout mice, while knockout of Tie2 had no effect on tail bleeding. Using in vitro assays, we found that Angiopoietin-2 inhibited thrombomodulin-mediated anticoagulation and protein C activation in human donor plasma. Our data suggest a novel in vivo mechanism for Angiopoietin-2 in COVID-19-associated hypercoagulation, implicating that Angiopoietin-2 inhibitors may be effective in the treatment of hypercoagulation in severe COVID-19 infection.
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SciScore for 10.1101/2021.01.13.21249429: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Informed consent was obtained from the patient, or next of kin if the patient was unable to give consent.
IACUC: All animal experiments were approved by the Uppsala Committee of Ethics of Animal Experiments (approved permit 5.8.18-04862-2020), in accordance with the Swedish Animal Protection Act (SFS 1988:534), and were conducted according to guidelines established by the Swedish Board of Agriculture.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable All experiments were performed in both female and male mice. Table 2: Resources
Antibodies Sentences Resources Lung lysates were immunoprecipitated with a … SciScore for 10.1101/2021.01.13.21249429: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Informed consent was obtained from the patient, or next of kin if the patient was unable to give consent.
IACUC: All animal experiments were approved by the Uppsala Committee of Ethics of Animal Experiments (approved permit 5.8.18-04862-2020), in accordance with the Swedish Animal Protection Act (SFS 1988:534), and were conducted according to guidelines established by the Swedish Board of Agriculture.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable All experiments were performed in both female and male mice. Table 2: Resources
Antibodies Sentences Resources Lung lysates were immunoprecipitated with a rabbit anti-mouse thrombomodulin antibody (ab230010, Abcam) attached to protein G conjugated Dynabeads (10004D, Thermo Fisher Scientific). anti-mouse thrombomodulinsuggested: NoneBlots were blocked with 5% BSA for 1 h and incubated overnight with mouse anti-6X His tag antibody (27E8, 2366, Cell Signaling). anti-6Xsuggested: NoneAfter washing and incubating with anti-mouse HRP conjugated secondary antibody (NA931, Sigma), proteins were visualized using ECL plus detection reagents (GERPN2232, Sigma). anti-mouse HRPsuggested: NoneGERPN2232suggested: NoneBlots were stripped with Re-Blot Plus Strong solution (2504, Millipore), blocked, and probed with anti-thrombomodulin antibody followed by anti-rabbit HRP conjugated secondary antibody (711-035-152, Jackson Immuno Research). anti-thrombomodulinsuggested: Noneanti-rabbit HRPsuggested: (Jackson ImmunoResearch Labs Cat# 711-035-152, RRID:AB_10015282)Total thrombomodulin in lung lysates was done as above and correlated to loading control, anti-Gapdh HRP conjugated antibody (ab9482, Abcam). anti-Gapdh HRPsuggested: (Abcam Cat# ab9482, RRID:AB_307272)Experimental Models: Organisms/Strains Sentences Resources Mice for other experiment came from in house breeding on a C57BL6/J background. C57BL6/Jsuggested: NoneSoftware and Algorithms Sentences Resources Band density was quantified with ImageJ (NIH). ImageJsuggested: (ImageJ, RRID:SCR_003070)All statistical analysis was done in GraphPad Prism 8. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:One limitation of our assay is that the measured APC levels were close to the detection limit, hence, possible differences between groups would not be detected. In contrast, a recent study did not find changes in APC in critically ill COVID-19 patients (36). However, reduced levels of APC are found in a majority of patients in sepsis and are associated with increased risk of death (42-45). APC formation may be impaired because of down-regulation or shedding of thrombomodulin induced by inflammatory cytokines (46), and, as we hypothesize herein, by ANGPT2 binding to thrombomodulin. Recent data show that circulating thrombomodulin was elevated in critically ill COVID-19 patients, suggesting shedding of thrombomodulin from the endothelium (15, 36). Furthermore, Goshua et al, reported that soluble thrombomodulin correlates with mortality in critically ill COVID-19 patients (36). In the current study, we noted that injection of ANGPT2 in mice resulted in the loss of thrombomodulin in lung tissue (Fig. 3). Further studies are needed to investigate if this is a direct or indirect effect of ANGPT2. To investigate if ANGPT2 can directly affect coagulation in vivo, we performed several experiments in mice. One simple but highly relevant experiment to evaluate coagulation is tail bleeding time (47). In these experiments, recombinant ANGPT2 and ANGPT1 fragments corresponding to the thrombomodulin and TIE2 binding region were injected before the measurement of tail bleeding time. These ex...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
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