Immunothrombotic Dysregulation in COVID-19 Pneumonia Is Associated With Respiratory Failure and Coagulopathy

Abstract

Background: Severe acute respiratory syndrome corona virus 2 infection causes severe pneumonia (coronavirus disease 2019 [COVID-19]), but the mechanisms of subsequent respiratory failure and complicating renal and myocardial involvement are poorly understood. In addition, a systemic prothrombotic phenotype has been reported in patients with COVID-19.

Methods: A total of 62 subjects were included in our study (n=38 patients with reverse transcriptase polymerase chain reaction-confirmed COVID-19 and n=24 non-COVID-19 controls). We performed histopathologic assessment of autopsy cases, surface marker-based phenotyping of neutrophils and platelets, and functional assays for platelet, neutrophil functions, and coagulation tests, as well.

Results: We provide evidence that organ involvement and prothrombotic features in COVID-19 are linked by immunothrombosis. We show that, in COVID-19, inflammatory microvascular thrombi are present in the lung, kidney, and heart, containing neutrophil extracellular traps associated with platelets and fibrin. Patients with COVID-19 also present with neutrophil-platelet aggregates and a distinct neutrophil and platelet activation pattern in blood, which changes with disease severity. Whereas cases of intermediate severity show an exhausted platelet and hyporeactive neutrophil phenotype, patients severely affected with COVID-19 are characterized by excessive platelet and neutrophil activation in comparison with healthy controls and non-COVID-19 pneumonia. Dysregulated immunothrombosis in severe acute respiratory syndrome corona virus 2 pneumonia is linked to both acute respiratory distress syndrome and systemic hypercoagulability.

Conclusions: Taken together, our data point to immunothrombotic dysregulation as a key marker of disease severity in COVID-19. Further work is necessary to determine the role of immunothrombosis in COVID-19.

Keywords: COVID-19; blood platelets; disseminated intravascular coagulation; neutrophils; respiratory insufficiency; severe acute respiratory syndrome coronavirus 2; thrombosis.

Figure 1.

COVID-19–associated coagulopathy in the lung, kidney, and heart presents as microvascular immunothrombosis. A, COVID-19 disease progression of autopsy case. Horowitz oxygenation index (Pao₂/Fio₂), and interleukin-6 (IL-6), high sensitive Troponin T and glomerular filtration rate (GFR) are plotted. B, Axial and coronal computed tomographic scans of the chest of the patient at presentation with COVID-19 defining bipulmonary infiltrates and ground glass opacities. C, Hematoxylin-eosin staining of a pulmonary microthrombus. Immune cells, most notably cells with segmented nuclei, can be observed in the thrombus (black arrows). D, Elastica van Gieson, fibrinogen, and platelet (CD42b) immunohistochemistry of the lung microthrombus. E, Exemplary immunofluorescence of a cut through a lung microthrombus showing of neutrophils (MPO) and fibrinogen. Dashed lines indicate vessel borders. Scale bars, 100 µm. F, Percentage of vessels with immunothrombosis (platelets, fibrinogen, neutrophils) in the lung vasculature. n=5 controls, n=5 COVID-19 autopsy cases. Two-tailed unpaired t test. G, Immunofluorescence staining of intravascular microthrombi with spatial association of neutrophils (MPO), platelets (Plt, CD42b) and fibrin. Scale bars, 10 µm; dashed lines indicate vessel borders. **P<0.01. COVID-19 indicates coronavirus disease 2019; and MPO, myeloperoxidase.

Figure 2.

High-dimensional analysis of neutrophil markers reveals a distinct neutrophil signature, characterized by overshooting, global neutrophil activation in severe disease. A, Neutrophil count of patients with COVID-19 from white blood cell differential: n=19 CoV_int, n=10 CoV_sev, 2-tailed unpaired Student t test. Reference range for neutrophil counts is shown in gray (1.85–6.8 G/L). B, Linear regression of neutrophil count with Horowitz index (Pao₂/Fio₂) of patients at blood draw. n=19 CoV_int, n=10 CoV_sev. C through F, t-Distributed Stochastic Neighbor Embedding (t-SNE) of neutrophil heterogeneity panel of Ctrl, Ctrl_pneu, CoV_int, and CoV_sev (n=25 000 downsampled cells per group). n=7 Ctrl, n=4 Ctrl_pneu, n=11 CoV_int, n=5 CoV_sev patients. C, t-SNE plot. D, t-SNE density plot displaying polarization of each group. E, Color-coded Phenograph-based subclusters of the t-SNE plot are shown. F, Heat map of the mean fluorescence intensity (MFI) for neutrophil subclusters relative to the maximum MFI. See Methods for the exact clustering of the heat map. For PMN 3 and PMN 6, the percentages of total neutrophils of each patient within this subcluster are annotated in violin plots. Percentages of neutrophils in each subcluster are shown in gray above the heat map. Kruskal-Wallis test and post hoc Dunn multiple comparison test. Line denotes significant Kruskal-Wallis test but nonsignificant post hoc test. G, Linear regression of CD177 expression of neutrophils with Horowitz index. n=10 CoV_int, n=5 CoV_sev. H, Percent of activated neutrophils in COVID-19 and control blood assessed by citH3 and MPO membrane blebbing in 6 high-power fields per patient. Representative micrographs are shown on the left. n=5 controls, n=3 CoV_sev. Two-tailed unpaired Student t test. I, Linear regression of neutrophil platelet aggregates as percentage of total blood leukocytes with Horowitz index. n=10 CoV_int, n=5 CoV_sev. *P<0.05, **P<0.01. citH3 indicates citrullinated histone H3; COVID-19, coronavirus disease 2019; CoV_int, patient group with intermediate COVID-19; CoV_sev, patient group with severe COVID-19; Ctrl_pneu, non–COVID-19 pneumonia; MFI, mean fluorescence intensity; MPO, myeloperoxidase; and PMN, polymorphonuclear cells.

Figure 3.

High-dimensional analysis of platelet markers and platelet function tests reveal a distinct platelet phenotype. A, Platelet count on the day of admission for patients with intermediate (CoV_int n=19) and severe COVID-19 (CoV_sev n=7). Reference range for platelet counts is shown in gray (160–360 ×1000/µL). B, Time course of platelet count (red spheres) and interleukin-6 (IL-6; white triangles) of patients with CoV_sev normalized to the day of ICU admission. Number of patients for each time point shown above. Gray: reference range for platelet counts. C, Changes in platelet count and IL-6 measurements over time (days) in patients with CoV_sev. Gray: reference range for platelet counts (160–360 ×1000/µL). n=8 for PLTC, n=9 for IL-6. Two-tailed paired t test. D, t-Distributed Stochastic Neighbor Embedding (t-SNE) of platelet heterogeneity panel of Ctrl, Ctrl_pneu, CoV_int, and CoV_sev (n=40 000 downsampled cells per group). E, t-SNE plot displayed separately for each group, represented as a density plot. F, Color-coded Phenograph platelet subclusters. G, Heat map of the mean fluorescence intensity (MFI) for each platelet subcluster relative to the maximum MFI of the surface marker. See Methods for the exact clustering of the heat map. For subcluster population Plt 1, 4, 9 and 10, the percentages of total platelets of each patient within this subcluster are annotated in violin plots. Percentages of platelets in each subcluster are shown in gray above the heat map. Kruskal-Wallis test and a post hoc Dunn multiple comparison test. Line denotes significant Kruskal-Wallis test but nonsignificant post hoc test. D through G: n=7 Ctrl, n=4 Ctrl_pneu, n=11 CoV_int, n=5 CoV_sev patients. H, Platelet function analyzer-200 (PFA) results for collagen/epinephrine (EPI) and collagen/ADP (ADP). Gray: reference ranges for the PFA test (PFA EPI: 84–160 s PFA ADP: 68–121 s). n=9 CoV_int, n=8 CoV_sev, 2-tailed unpaired Student t test. I, Whole-blood impedance aggregometry (Multiplate, MP) results for thrombin-receptor agonist peptide (TRAP) and adenosine diphosphate (ADP) stimulation. Gray: reference ranges for the MP test (MP TRAP: 76–154 U, MP ADP: 53–122 U). n=9 CoV_int, n=8 CoV_sev. *P<0.05, **P<0.01, ***P<0.001. COVID-19 indicates coronavirus disease 2019; Ctrl_pneu, non–COVID-19 pneumonia; ICU, intensive care unit; and PLTC, platelet count.

Figure 4.

A systemic procoagulant state and neutrophil extracellular trap (NET) formation in COVID-19. A, International normalized ratio (INR) and activated partial thromboplastin time (aPTT lupus anticoagulant-sensitive) of patients with COVID-19 at the time of blood draw. Reference ranges are shown in gray (INR, 0.8–1.2; aPTT, 22–34 s), n=19 CoV_int, n=11 CoV_sev. B, Fibrinogen (Clauss) plasma levels of patients with COVID-19 at the time of blood draw. Reference ranges are shown in gray (210–400 mg/dL), n=20 CoV_int, n=11 CoV_sev. C, Linear regression of fibrinogen plasma level and Horowitz index (Pao₂/Fio₂) of patients with COVID-19. n=19 CoV_int (orange), n=11 CoV_sev (red). D, d-Dimer plasma levels of patients with COVID-19. Reference ranges are shown in gray (<0.5 µg/mL), n=20 CoV_int, n=11 CoV_sev. E,EXTEM and INTEM maximum clot firmness (MCF). Reference ranges are shown in gray (EXTEM, 50–72 mm; INTEM, 50–72 mm) F, EXTEM and INTEM clot formation time (CFT). Reference ranges are shown in gray (EXTEM, 34–159 mm; INTEM, 30–110 mm). G, Linear regression of INTEM MCF and Horowitz index. H, EXTEM maximum lysis (ML). Reference ranges are shown in gray (<15%). I, Linear regression of EXTEM ML and Horowitz index. J, FIBTEM MCF, Reference ranges are shown in gray (9–25 mm). K, Linear regression of FIBTEM MCF and fibrinogen plasma levels. E through K, CoV_int n=9 and CoV_sev n=8. E, F, H,and J, Two-tailed unpaired Student t test. L, Schematic of the platelet-rich plasma (PRP) stimulation assay. M, Representative micrograph of platelets isolated from a patient with CoV_sev binding to a neutrophil undergoing NETosis in vitro and quantification of control or COVID-19 platelets associated with neutrophils (see L). Scale bar, 10 µm. N, Representative micrographs and quantification of NETs formed by neutrophils stimulated with control or COVID-19 PRP (see L). Scale bar, 20 µm. M and N, n=3 COVID-19 patients, n=5 controls. Two-tailed unpaired Student t test. O, Immunofluorescence staining of a representative COVID-19 lung autopsy specimen. Arrow indicate activated neutrophil; and Stars, neutrophil extracellular traps. Top right, Three-dimensional reconstruction if NETing neutrophil. Arrow indicates citH3 and DNA signal in MPO+ NET structure. Bottom right, pseudocolored fibrinogen costaining. Scale bar, 10 µm, dashed lines indicate vessel borders. P, NETs per vessels of COVID-19 and non–COVID-19 lung autopsy specimens. n=5 patients with COVID-19, n=5 controls. Two-tailed unpaired t test with Welch correction. Q, Immunofluorescence staining of COVID-19 autopsy specimens of the lung, kidney, and heart. Top, Staining for neutrophils (myeloperoxidase, MPO), DNA, and citrullinated histone H3 (citH3). Rectangles show areas of interest with NET-like structures in all 3 organs that are enlarged in R. Bottom, Fibrinogen staining is pseudocolored. Scale bar, 10 µm, dashed lines indicate vessel borders. R, Enlarged areas from Q with NET-like structures (arrows). Scale bar, 10 µm. *P<0.05, **P<0.01, ***P<0.001. CoV_int indicates patient group with intermediate COVID-19; CoV_sev, patient group with severe COVID-19; and COVID-19, coronavirus disease 2019.