Research organism

elife

eLife

eLife

2050-084X

eLife Sciences Publications, Ltd

64330

10.7554/eLife.64330

Research Article

Epidemiology and Global Health

Medicine

SARS-CoV-2 suppresses anticoagulant and fibrinolytic gene expression in the lung

Mast

Alan E

https://orcid.org/0000-0003-3740-0318

1†Wolberg

Alisa S

http://orcid.org/0000-0002-2845-2303

2†Gailani

David

3†Garvin

Michael R

https://orcid.org/0000-0002-2204-7569

4Alvarez

Christiane

4Miller

J Izaak

4Aronow

Bruce

567Jacobson

Daniel

https://orcid.org/0000-0002-9822-8251

jacobsonda@ornl.gov4581Versiti Blood Research Institute, Department of Cell Biology Neurobiology and Anatomy Medical College of Wisconsin

Milwaukee

United States2Department of Pathology and Laboratory Medicine and UNC Blood Research Center

Chapel Hill

United States3Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center

Nashville

United States4Oak Ridge National Laboratory, Biosciences Division

Oak Ridge

United States5University of Tennessee Knoxville, The Bredesen Center for Interdisciplinary Research and Graduate Education

Knoxville

United States6Biomedical Informatics, Cincinnati Children’s Hospital Research Foundation

Cincinnati

United States7University of Cincinnati

Cincinnati

United States8University of Tennessee Knoxville, Department of Psychology

Knoxville

United States

Emoto

Noriaki

Reviewing EditorKobe Pharmaceutical UniversityJapanvan der Meer

Jos WM

Senior EditorRadboud University Medical CentreNetherlands

†

These authors contributed equally to this work

08032021

2021

10

e64330

2610202006032021

© 2021, Mast et al

2021

Mast et al

http://creativecommons.org/licenses/by/4.0/

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Extensive fibrin deposition in the lungs and altered levels of circulating blood coagulation proteins in COVID-19 patients imply local derangement of pathways that limit fibrin formation and/or promote its clearance. We examined transcriptional profiles of bronchoalveolar lavage fluid (BALF) samples to identify molecular mechanisms underlying these coagulopathies. mRNA levels for regulators of the kallikrein–kinin (C1-inhibitor), coagulation (thrombomodulin, endothelial protein C receptor), and fibrinolytic (urokinase and urokinase receptor) pathways were significantly reduced in COVID-19 patients. While transcripts for several coagulation proteins were increased, those encoding tissue factor, the protein that initiates coagulation and whose expression is frequently increased in inflammatory disorders, were not increased in BALF from COVID-19 patients. Our analysis implicates enhanced propagation of coagulation and decreased fibrinolysis as drivers of the coagulopathy in the lungs of COVID-19 patients.

COVID-19coagulationfibrinolysisbronchoalvelolarSARS-CoV-2

Research organismHuman

http://dx.doi.org/10.13039/100006228

Oak Ridge National Laboratory

LOIS:10074

Garvin

Michael R

Miller

J Izaak

Jacobson

Daniel

http://dx.doi.org/10.13039/100000002

National Institutes of Health

U24 HL148865

Aronow

Bruce

http://dx.doi.org/10.13039/100000002

National Institutes of Health

HL068835

Mast

Alan E

http://dx.doi.org/10.13039/100000002

National Institutes of Health

HL143403

Wolberg

Alisa S

http://dx.doi.org/10.13039/100000002

National Institutes of Health

HL126974

Wolberg

Alisa S

http://dx.doi.org/10.13039/100000002

National Institutes of Health

HL140025

Gailani

David

http://dx.doi.org/10.13039/100000049

National Institute on Aging

3RF1AG053303-01S2

Jacobson

Daniel

Garvin

Michael R

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Author impact statement

Transcription changes in cells taken from bronchoalveolar fluid of COVID-19 patients indicate severe disruption of coagulation and fibrinolytic pathways in the lung and suggest similar processes in other organs.

Introduction

The bradykinin storm model for COVID-19 pathogenesis was recently developed from our analyses of gene expression, clinical, autopsy, pathology, and ChIP-Seq data (Garvin et al., 2020). Several clinical studies by other groups have demonstrated positive results from therapeutic interventions predicted by our model (Entrenas Castillo et al., 2020; van de Veerdonk et al., 2020; WHO Rapid Evidence Appraisal for COVID-19 Therapies (REACT) Working Group et al., 2020). Here, we extend this model to include the concurrent dysregulation of the coagulation and fibrinolytic pathways.

SARS-CoV-2 displays considerable tissue tropism (Adachi et al., 2020; Barton et al., 2020; Su et al., 2020; Xu et al., 2020). The lungs are often affected with disease that can range from mild pneumonia, to severe dyspnea and hypoxia, to critical respiratory failure, shock, and multiorgan failure. The time course for the development of severe disease is typically 8–12 days, but some patients rapidly deteriorate about 7 days following development of symptoms. Lung tissue from people who died from COVID-19 has pauci-inflammatory septal capillary injury and luminal and mural fibrin deposition in alveolar septal capillaries (Lax et al., 2020; Magro et al., 2020), thrombi in small- and medium-sized arteries (Lax et al., 2020; Magro et al., 2020), and fibrinous thrombi in small pulmonary arterioles with evidence of tumefaction of the endothelium (Dolhnikoff et al., 2020; Fox et al., 2020; Geerdes-Fenge et al., 2020; Lax et al., 2020). A report that administering tissue plasminogen activator (tPA) to dissolve fibrin transiently alleviates respiratory distress in COVID-19 patients supports the premise that fibrin deposition contributes to the respiratory failure (Wang et al., 2020). Thus, it appears that COVID-19 hypoxemia stems at least partially from fibrin deposits surrounding alveoli that restrict oxygen transfer and microvascular thrombi that cause ventilation–perfusion defects.

Abnormal levels of circulating coagulation proteins are present in patients with COVID-19. Changes include increased levels of coagulation proteins associated with the acute phase response (e.g., fibrinogen and factor VIII) and endothelial activation (e.g., von Willebrand factor), as well as elevated biomarkers of coagulation activation (e.g., D-dimer) (Chen et al., 2020; Gattinoni et al., 2020; Helms et al., 2020; Middeldorp et al., 2020; Panigada et al., 2020; Tang et al., 2020; Zhou et al., 2020a; Helms et al., 2020; Middeldorp et al., 2020; Panigada et al., 2020). Accordingly, thrombotic events have been detected in up to 30% of COVID-19 patients (Helms et al., 2020; Middeldorp et al., 2020; Panigada et al., 2020) including large vessel occlusions such as deep vein thrombosis, pulmonary embolism, and ischemic stroke, as well as microvascular thrombosis and extravascular fibrin deposition in a variety of tissues including lung and skin (Helms et al., 2020; Middeldorp et al., 2020; Panigada et al., 2020). Inappropriate fibrin deposition and thrombosis are thought to stem from the interaction of systemic (blood) changes with tissue-specific dysfunction. However, mechanisms that contribute to prevalence of thrombi and fibrin in the pulmonary vasculature and extravascular space, thought to be a major cause of morbidity and mortality in COVID-19, have remained elusive.

The goal of the present study was to identify changes in the lungs of patients with severe COVID-19 that could contribute to local derangement of hemostatic mechanisms. Bronchoalveolar lavage fluid (BALF) contains lung parenchymal, epithelial, and alveolar cells, as well as immune cells that infiltrate epithelial and luminal spaces. RNA sequencing of BALF provides a snapshot of the transcriptome at the interface where capillary gas exchange occurs and has been used to characterize lung function in many diseases (Gu et al., 2019; Jakieła et al., 2018; Kahn et al., 2015; Sengupta et al., 2019; Sun et al., 2019; Weigt et al., 2019; Zhang et al., 2019). Here, we compared the transcriptional signatures of BALF from patients with COVID-19 and uninfected controls. Our analyses focused on transcripts for proteins that function in coagulation, fibrinolysis, and kinin formation in the lung to identify dysregulated mechanisms that may contribute to COVID-19 pathophysiology.

Results and discussionChanges in transcripts encoding proteins in the kallikrein–kinin/bradykinin systemOverview of pathways examined

The plasma kallikrein–kinin system is comprised of the protease precursors factor XII (FXII, encoded by F12) and prekallikrein (encoded by KLKB1) and the cofactor high-molecular-weight kininogen (HK, encoded by KNG1) (Schmaier, 2016; Schmaier et al., 2019). In healthy individuals, FXII and prekallikrein undergo reciprocal activation to the proteases FXIIa and kallikrein (Revenko et al., 2011; Schmaier, 2016). Kallikrein cleaves HK to liberate bradykinin, which contributes to setting vascular tone and permeability by interacting with bradykinin receptors (encoded by BDKRB1 and BDKRB2) (Marceau et al., 2020). This process is regulated by C1-Inhibitor (encoded by SERPING1) (Marceau et al., 2020). Congenital C1-Inhibitor deficiency causes hereditary angioedema, which is characterized by bouts of bradykinin-induced soft tissue swelling (Busse and Christiansen, 2020; Cicardi and Zuraw, 2018). C1-Inhibitor in blood is primarily of hepatocyte origin, although it also is produced by other cell types including vascular endothelium (Prada et al., 1998).

Study findings

Transcripts encoding C1-Inhibitor were decreased 80-fold in BALF from COVID-19 patients (Table 1), raising the possibilities that contact activation-initiated thrombin generation is locally dysregulated and control of bradykinin production is compromised. Furthermore, the angiotensin-converting enzyme (ACE) that degrades bradykinin (Cicardi and Zuraw, 2018; Davin et al., 2019) was downregulated eightfold in COVID-19 BALF.

Table 1.Differentially expressed coagulation genes.

Gene	Protein product	Mean COVID-19	Mean control	Fold change	Log2FC	FDR
A2M	α2-Macroglubulin	3.9	177.6	−43.5	−5.4	5.2E-15
BDKRB1	Bradykinin receptor B1	3.3	0.0	258.9	8.0	4.3E-91
BDKRB2	Bradykinin receptor B2	8.9	0.2	49.1	5.6	1.9E-40
F13A1	Factor XIII-A subunit	2.5	9.2	−3.6	−1.8	2.6E-05
F13B	Factor XIII-B subunit	0.6	0.0	117.8	6.9	2.7E-31
F12	Factor XII	0.5	3.2	−4.4	−2.1	5.7E-10
F11	Factor XI	6.6	0.1	81.2	6.3	7.9E-79
F10	Factor X	3.2	0.0	169.9	7.4	7.9E-140
F9	Factor IX	0.9	0.0	189.6	7.6	2.2E-39
F8	Factor VIII	3.9	15.8	−4.6	−2.2	1.6E-40
F7	Factor VII	10.1	0.0	363.5	8.5	2.9E-73
F5	Factor V	9.7	6.8	1.4	0.5	0.13
F3	Tissue Factor	4.2	3.8	-1.0	-0.01	1
F2	Prothrombin	0.9	0.0	188.8	7.6	7.8E-67
FGA	Fibrinogen Aα chain	3.4	0.0	322.5	8.3	3.4E-206
FGB	Fibrinogen Bβ chain	3.7	0.0	303.9	8.2	2.4E-151
FGG	Fibrinogen γ chain	0.7	0.0	85.4	6.4	4.8E-17
KLKB1	Kallikrein B1	1.3	1.8	−1.4	−0.5	2.1E-01
KNG1	Kininogen	3.3	0.0	190.7	7.6	4.2E-163
PLAT	Tissue plasminogen activator	1.8	0.4	5.3	2.4	7.1E-24
PLAU	Urokinase	3.8	158.3	−37.1	−5.2	8.2E-172
PLAUR	Urokinase receptor	6.2	313.3	−42.1	−5.4	6.4E-286
PLG	Plasminogen	2.2	0.0	75.3	6.2	7.3E-38
PROC	Protein C	2.0	0.0	226.5	7.8	4.1E-158
PROCR	Endothelial protein C receptor	1.7	57.9	−33.8	−5.1	1.9E-50
PROS1	Protein S	3.2	195.3	−54.2	−5.8	1.1E-174
SERPINA5	Protein C Inhibitor	2.7	0.0	786.8	9.6	5.4E-145
SERPINB2	Plasminogen activator inhibitor-2	1.0	0.5	2.3	1.2	2.1E-01
SERPINC1	Antithrombin	1.9	0.1	13.6	3.8	3.1E-31
SERPIND1	Heparin cofactor II	3.0	0.0	94.0	6.6	2.2E-72
SERPINE1	Plasminogen activator inhibitor-1	2.7	5.3	−1.8	−0.9	8.8E-02
SERPINF2	α2-antiplasmin	5.4	8.2	−1.4	−0.5	1.1E-02
SERPING1	C-1 inhibitor	23.3	1923.2	−80.1	−6.3	5.4E-33
TFPI	Tissue factor pathway inhibitor	3.4	0.4	7.7	2.9	8.7E-19
THBD	Thrombomodulin	9.6	224.0	−22.2	−4.5	3.2E-48
VWF	Von Willebrand factor	14.5	7.7	2.0	1.0	2.0E-05

Changes in transcripts encoding extrinsic pathway proteins that initiate thrombin generationOverview of pathways examined

The integral cytoplasmic membrane protein tissue factor (TF, encoded by F3) is the primary initiator of thrombin generation in vivo. In many inflammatory conditions, cellular TF expression is upregulated (Edwards et al., 1979), providing a mechanistic explanation for hypercoagulable states associated with a variety of bacterial and viral infections (Grover and Mackman, 2018; Levi and van der Poll, 2017). Tissue factor pathway inhibitor (TFPI, encoded by TFPI) is a Kunitz-type protease inhibitor produced by vascular endothelium (Bajaj et al., 1990) and is the major inhibitor of the factor VIIa (FVIIa)/TF complex (Wood et al., 2014).

Study findings

Surprisingly, transcripts encoding TF were similar in COVID-19 and control BALF samples (Figure 1; Table 1), whereas transcripts for TFPI were increased eightfold in COVID-19 BALF. On balance, these data indicate that pulmonary fibrin deposition does not stem from enhanced local TF production and that counterintuitively, COVID-19 may dampen TF-dependent mechanisms in the lungs.

Figure 1.Transcriptional changes in lung induced by COVID-19 infection did not alter <italic>F3</italic>, but decreased aPC anticoagulant capacity, suggesting decreased inhibition of the propagation phase of coagulation.

Figure shows differential gene expression (log₂ fold change) of coagulation pathway transcripts in BALF of COVID-19 patients; the image illustrates mechanistic relationships of the protein products of the identified transcripts during coagulation. Shading indicates relative expression in COVID-19 patients compared to controls: increased (red) or decreased (blue). There was minimal change in F3 (encoding tissue factor ) and increased TFPI (encoding the major inhibitor of tissue factor activity). There was decreased THBD, PROCR, and PROS1 (encoding proteins that enhance anticoagulant activity) and increased SERPINA5 (encoding protein C inhibitor). There was also decreased SERPING1 (encoding C1-Inhibitor). Other transcripts showing changes (e.g., F11, F10, F7, F2) encode proteins typically produced in the liver; local expression of these proteins is unclear.

Changes in transcripts encoding intrinsic pathway proteins that initiate thrombin generationOverview of pathways examined

Increased thrombin generation can also be initiated by the plasma protease FXIIa (Maas and Renné, 2018; Stavrou, 2018). While FXIIa does not contribute to hemostasis, it is implicated in thrombo-inflammatory conditions. FXIIa converts factor XI (FXI, encoded by F11) to the protease FXIa (Ivanov et al., 2017; Long et al., 2016; Schmaier, 2016; Wang et al., 2019), a potent activator of the coagulation protease factor IX (FIX, encoded by F9) (Mohammed et al., 2018).

Study findings

Transcripts encoding FXII were slightly decreased (−4-fold) in COVID-19 and control BALF samples (Figure 1; Table 1), but the expression of the C1 inhibitor (encoded by SERPING1), which normally inhibits the FXII protein activity, is downregulated 80-fold. While it is not known if pulmonary tissues normally produce FXI or FIX, transcripts for FXI (81-fold) and FIX (190-fold) were both increased in BALF from COVID-19 patients. Numerous studies have reported correlations between plasma FIX and FXI levels and risk for venous thromboembolism and ischemic stroke (Ammollo et al., 2014; Folsom et al., 2015; Mohammed et al., 2018; Preis et al., 2017; Salomon et al., 2011, Salomon et al., 2008; Siegerink et al., 2010; Suri et al., 2010). Perhaps local increases in FIX or FXI expression enhance thrombin generation and promote thrombus formation.

Changes in transcripts encoding anticoagulant proteinsOverview of pathways examined

Healthy lung endothelium has membrane-associated anticoagulant proteins that inhibit different points in the thrombin generation pathway. These include TFPI, which inhibits the initiation of coagulation as described above, and thrombomodulin (encoded by THBD) and endothelial protein C receptor (EPCR, encoded by PROCR), which coordinate to inhibit the propagation of coagulation. Thrombomodulin captures thrombin and converts it from a procoagulant enzyme to an activator of protein C (encoded by PROC) in a process enhanced by binding of PC to EPCR. Activated PC with its cofactor protein S (encoded by PROS1) downregulates thrombin generation by inactivating factors VIIIa and Va and produces a cytoprotective/anti-inflammatory effect when bound to EPCR through cleavage of protease activated receptor-1 on endothelial cells (Ito et al., 2019; Riewald et al., 2002).

Study findings

Transcripts encoding thrombomodulin (−22-fold) and EPCR (−33-fold) were each reduced in BALF from COVID-19 patients compared with controls (Figure 1; Table 1), suggesting SARS-CoV-2 is associated with reduced expression of these proteins on vascular endothelium. We also observed a marked increase in protein C transcripts in BALF from COVID-19 patients (227-fold), but a significant reduction in PS transcripts (−54-fold). Since protein C and protein S in blood are primarily synthesized by hepatocytes, the importance of local synthesis of these proteins is not known. However, expression patterns in BALF are consistent with the observation that the plasma concentration of protein C is moderately elevated, and protein S moderately reduced, in COVID-19 patients (Panigada et al., 2020). In COVID-19 BALF, there was also a substantial increase (786-fold) in mRNA for protein C inhibitor (encoded by SERPINA5), a serpin regulator of activated protein C. Taken as a whole, the transcript pattern in COVID-19 BALF suggests a diminished capacity of the endothelial anticoagulant system to downregulate local thrombin generation.

Changes in transcripts that encode fibrinogen and proteins in the fibrinolytic pathwayOverview of pathways examined

The fibrinolytic system is responsible for enzymatic degradation of fibrin. Plasminogen activators convert plasminogen to plasmin, which cleaves fibrin and generates fibrin degradation products, including the circulating biomarker D-dimer. Cells within the lung express plasminogen activators (e.g., [tPA, encoded by PLAT], urokinase plasminogen activator [uPA, encoded by PLAU], and the uPA receptor [uPAR, encoded by PLAUR]) that prevent fibrin accumulation in small airways and the alveolar compartment and that maintain blood vessel patency (Shetty et al., 2008). uPA and uPAR are expressed by lung epithelial cells, alveolar macrophages, and fibroblasts, and reduced expression of these proteins contributes to acute lung injury (Shetty et al., 2008). The plasminogen activators are inhibited by plasminogen activator inhibitor-1 (PAI-1, encoded by SERPINE1). Plasma fibrinogen, a dimer of trimers (Aα₂Bβ₂γ₂) encoded by three genes, FGA, FGB, and FGG, is synthesized primarily by hepatocytes (Pieters and Wolberg, 2019); however, synthesis has been reported in stimulated cultured lung alveolar epithelial cells and in alveolar epithelium in an animal model of bacterial pneumonia (Guadiz et al., 1997; Simpson-Haidaris et al., 1998).

Study findings

Transcripts encoding the fibrinogen chains were increased in COVID-19 BALF (Table 1, Figure 2; ). Our findings suggest that local fibrinogen expression in the lungs of COVID-19 patients, like fibrinogen expression by hepatocytes, is upregulated during the acute phase response and may provide additional substrate for local thrombin-mediated fibrin production. Our analysis uncovered evidence of reduced expression of transcripts encoding uPA (−37-fold) and uPAR (−42-fold) (Table 1, Figure 2) in BALF from COVID-19 patients. Expression of transcripts encoding tPA was very low in BALF from healthy individuals. Transcripts encoding tPA were elevated (fivefold) in COVID-19 BALF, but absolute expression was low compared to uPA and uPAR transcripts in uninfected controls (Table 1, Figure 2). Similarly, expression of transcript for plasminogen (encoded by PLG) was elevated in COVID-19 BALF but remained low compared to other transcripts. The combination of increased fibrinogen expression and reduced production of uPA and uPAR indicates a loss of local fibrinolytic capacity, consistent with a lung environment permissive of the accumulation of fibrin deposits within pulmonary vessels and the alveolar space (Lax et al., 2020; Magro et al., 2020).

Figure 2.Transcriptional changes in lung induced by COVID-19 infection decreased <italic>PLAU</italic> and <italic>PLAUR</italic>, suggesting diminished fibrinolytic activity.

Figure shows differential gene expression (log₂ fold change) of fibrinolytic pathway transcripts in BALF of COVID-19 patients; the image illustrates mechanistic relationships of the protein products of the identified transcripts during fibrinolysis. Shading indicates relative expression in COVID-19 patients compared to controls: increased (red) or decreased (blue). There was a moderate increase in PLAT (encoding tPA). There was also enhanced expression of FGB, FGA, and FGG (encoding fibrinogen chains) and decreased expression of PLAU and PLAUR (encoding uPA and uPAR, respectively). Other transcripts showing changes (e.g., F2, PLG) encode proteins typically produced in the liver; local expression of these proteins is unclear.

Conclusions

We detected pronounced changes in mRNA levels encoding proteins involved in regulation of coagulation, fibrinolysis, and inflammation in the lungs of COVID-19 patients. These changes include reductions in transcripts for proteins known to be expressed in normal pulmonary tissue (thrombomodulin, EPCR, uPA, uPAR) that would compromise the functions of anticoagulant and fibrinolytic pathways. In concert with enhanced production of bradykinin and vascular permeability, these changes are likely to create an environment in which plasma proteins are exposed to the extravascular space, resulting in increased fibrin production and reduced fibrin degradation, enabling the fibrin deposits in pulmonary vessels and alveolar spaces observed in autopsies of COVID-19 patients (Dolhnikoff et al., 2020; Fox et al., 2020; Geerdes-Fenge et al., 2020; Lax et al., 2020), and contributing to the virulence of the virus.

An unexpected finding was the lack of change in transcripts for F3, which encodes TF, the integral membrane protein responsible for triggering thrombin generation during hemostasis. TF expression is enhanced in a wide range of thrombo-inflammatory disorders, and its over-expression is thought to be important for driving consumptive coagulation. The apparent lack of increase in F3, coupled with an apparent increase in TFPI transcripts, raises the possibility that pathways unrelated to the FVIIa/TF complex may play a significant role in COVID-19-associated thrombosis. In this regard, thrombomodulin and EPCR transcripts were substantially decreased, suggesting reduced capacity of the lung endothelium to downregulate the propagation of thrombin generation. In addition, changes in regulation or activation of the plasma kallikrein–kinin system and contact activation may be important. This is consistent with our hypothesis that COVID-19 triggers a bradykinin storm (Garvin et al., 2020), the net effect of increased bradykinin production, reduced BK degradation, and increased local expression of bradykinin receptors, is expected to enhance vasodilation and vascular permeability, exposing blood components to extravascular proteins that promote fibrin formation and contribute to tissue damage.

Fibrin deposits and thrombi have been observed in organs other than the lungs in COVID-19 patients, including kidney (Su et al., 2020) and spleen (Mestres et al., 2020). It is tempting to speculate that transcriptional changes observed in BALF may occur in these other tissues. Currently, it is not clear whether fibrin deposition in specific organs indicates localized infection of those organs, perhaps stemming from differential distribution of the SARS-CoV-2 receptor ACE2, or reflects organ susceptibility to systemic changes induced by the infection. Analysis of transcriptional and proteomic changes in different organs in concert with information on virus entry into parenchymal cells may help explain the pattern of organ-specific thrombosis during SARS-CoV-2 infection.

Our study has limitations. Although we detected changes in multiple gene transcripts, we did not identify specific cell types for each transcript, which may include lung parenchymal cells, leukocytes, megakaryocytes (Fox et al., 2020), and other cells present in native and inflammatory states that undergo transcriptional changes in response to infection. Single-cell analyses will be required to better understand these changes during SARS-CoV-2 infection. As with all transcriptional data, we could not determine whether changes in mRNA manifest as changes in protein expression. While histologic analyses clearly demonstrate fibrin deposition in the lung, further studies using agnostic and targeted proteomics, as well as biochemical analyses will be needed to confirm causation. We focused on changes in coagulation and fibrinolytic pathways in the present study; however, the pathologic processes observed in the lungs of COVID-19 patients are likely to be influenced by changes in other pathways, such as those involving complement. Crosstalk between coagulation, fibrinolytic, and complement pathways is well documented (Ewald and Eisenberg, 1995; Foley et al., 2016; Fox et al., 2020; Kleniewski and Donaldson, 1987). Despite these limitations, it is instructive to consider how the changes in coagulation and fibrinolytic gene expression documented in our study of BALF may represent common events occurring in multiple vascular beds in COVID-19, as identification of a common dysfunction may reveal a targetable mechanism to prevent thrombosis in these patients.

Materials and methodsStudy participants

The nine BALF samples from 5 patients used in this study were collected from patients in Wuhan, China. All patients displayed pneumonia and other severe symptoms upon admission to the hospital in Wuhan, China, in late December 2019 and were therefore admitted to the ICU (Table 2). They were originally used for RNA sequencing to identify the etiological agent for COVID-19 and to determine the genomic sequence of SARS-CoV-2. Human mRNA sequences from these samples were used to develop our bradykinin storm model (Garvin et al., 2020), but have not previously been analyzed to understand localized coagulopathy in the lungs of patients with COVID-19 (Zhou et al., 2020b). We compared mRNA transcripts for proteins involved in coagulation, fibrinolysis, and kinin formation in BALF from these samples with those from 40 controls from a study on the contribution of obesity to disease severity in asthmatics (Michalovich et al., 2019). The 40 control individuals included smokers and non-smokers, asthmatics and non-asthmatics, as well as obese and non-obese individuals. Given this diverse set of morbidities and the resulting variance in gene expression, they represent an informative control for patients with COVID-19 as statistically significant changes detected due to COVID-19 are not just representing subtle shifts in variance (Table 1). Expression datasets are available from the NCBI Sequence Read Archive under the accession numbers PRJNA605983 (severe acute respiratory syndrome coronavirus 2 raw sequence reads) and PRJNA434133 (Microbiome and Inflammatory Interactions in Obese and Severe Asthmatic Adults).

Table 2.Clinical data for patients from which BALF was extracted and analyzed for this study.

GISAID accession	Isolate	NCBI accession	SRA accessions	Swab date	Age	Sex	Patient no	Date onset	Symptoms_admission	Status 1/13/20	Diagnosis history
EPI_ISL_402127	WIV02	MN996527	SRR11092058, SRR11092063	12/30/19	32	Male	ICU-04	12/19/19	Fever, cough, dyspnea	Fever, intermitttent cough	Negative
EPI_ISL_402124	WIV04	MN996528	SRR11092057, SRR11092062	12/30/19	49	Female	ICU-06	12/27/19	Fever (37.9C), palpitation	Fever, malaise, cough	Coronavirus (nt)
EPI_ISL_402128	WIV05	MN996529	SRR11092061	12/30/19	52	Female	ICU-08	12/22/19	Fever (38C), expectoration, malaise, dyspnea	Recovered, disharged	Streptococcus pneumoniae (nt)
EPI_ISL_402129	WIV06	MN996530	SRR11092056, SRR11092060	12/30/19	40	Male	ICU-09	12/28/20	Fever (38C), expectoration	Fever (38C), expectoration, dizziness	Negative
EPI_ISL_402130	WIV07	MN996531	SRR11092059, SRR11092064	12/30/19	56	Male	ICU-10	12/20/19	Fever, dyspnea, chest tightness	Fever, malaise, cough, dyspnea	Negative

Gene expression analysis

The CLC Genomics Workbench (20.0.3) was used to trim FASTQ files downloaded from the NCBI Sequence Read Archive to remove any adapter sequences or other artifacts from processing. The RNA reads were mapped to the GRCh38_latest_rna.fna version of the human transcriptome. Mapping parameters included a cost penalty of two for mismatches and three for small insertions or deletions (INDEL), and similarity and length fraction were both set to 0.95. The resulting transcript profiles were manually inspected to account for expression artifacts, such as reads mapping solely to repetitive elements such as the Alu transposable element. Transcripts whose counts came solely from (or were dominated by) reads at repetitive elements were removed from the analysis. Where transcripts per million (TPM) values were zero (transcripts were not detected), we used the lowest TPM across samples to two decimal places. If all samples in a group had no detected transcripts, we used the lowest TPM to two decimal places from the entire set of genes queried. The edgeR package (McCarthy et al., 2012; Robinson et al., 2010) was used to identify genes that were differentially expressed in COVID-19 patient samples compared to controls. Count data were scaled to normalize for library size and normalization factors determined as instructed in edgeR documentation. Dispersion was estimated, and the read counts for each gene were fit with a negative binomial model. Each gene was tested for differential expression. The Benjamini–Hochberg method was used to determine the false discovery rate.

Acknowledgements

We would like to acknowledge funding from the Oak Ridge National Laboratory, Laboratory Directed Research and Development Fund, LOIS:10074 (which supported the systems biology work), and the National Institutes of Health, U24 HL148865: the LungMap Consortium (which supported tissue and cell-based expression conceptualization). Funding was also provided by the National Institutes of Health grants HL068835 (AM), HL143403 (AW), HL126974 (AW), 3RF1AG053303-01S2 (DJ), and HL140025 (DG) to support analyses and interpretation of the coagulation and fibrinolytic pathways. This research used resources of the Oak Ridge Leadership Computing Facility, which is a DOE Office of Science User Facility supported under Contract DE-AC05-00OR22725. This research used resources of the Compute and Data Environment for Science (CADES) at the Oak Ridge National Laboratory.

Additional information

Competing interests

receives research funding from Novo Nordisk and has received honoraria for serving on Novo Nordisk advisory boards.

receives research funding from Takeda and Bristol Myers Squibb

receives research funding from Bayer and has received honoraria for serving on Anthos, Bristol-Myers Squibb, Ionis and Janssen advisory boards.

No competing interests declared

Author contributions

Conceptualization, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review and editing

Conceptualization, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review and editing

Conceptualization, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review and editing

Conceptualization, Data curation, Formal analysis, Visualization, Writing - original draft, Writing - review and editing

Visualization

Formal analysis, Investigation, Methodology

Conceptualization, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review and editing

Conceptualization, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

Additional files

Transparent reporting form

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Data for control and COVID-19 bronchoalveolar lavage samples are available in the Sequence Read Archive at NCBI.

The following previously published datasets were used:

ZhouPYangXLWangXGHuBZhangLZhangWSiHRZhuYLiBHuangCLChenHDChenJLuoYGuoHJiangRDLiuMQChenYShenXRWangXZhengXSZhaoKChenQJDengFLiuLLYanBZhanFXWangYYXiaoGFShiZL2020Severe acute respiratory syndrome coronavirus 2 Raw sequence readsNCBI BioProjectPRJNA605983

GlaxoSmithKline2018Microbiome and Inflammatory Interactions in Obese and Severe Asthmatic AdultsNCBI BioProjectPRJNA434133

References

Adachi

T

Chong

JM

Nakajima

N

Sano

M

Yamazaki

J

Miyamoto

I

Nishioka

H

Akita

H

Sato

Y

Kataoka

M

Katano

H

Tobiume

M

Sekizuka

T

Itokawa

K

Kuroda

M

Suzuki

T

2020

Clinicopathologic and immunohistochemical findings from autopsy of patient with COVID-19, japan

Emerging Infectious Diseases2621572161

10.3201/eid2609.201353

32412897

Ammollo

CT

Semeraro

F

Colucci

M

Simioni

P

2014

Factor IX-Padua enhances the fibrinolytic resistance of plasma clots

Thrombosis and Haemostasis111226232

10.1160/TH13-06-0489

24136406

Bajaj

MS

Kuppuswamy

MN

Saito

H

Spitzer

SG

Bajaj

SP

1990

Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis

PNAS8788698873

10.1073/pnas.87.22.8869

2247459

Barton

LM

Duval

EJ

Stroberg

E

Ghosh

S

Mukhopadhyay

S

2020

COVID-19 autopsies, Oklahoma, USA

American Journal of Clinical Pathology153725733

10.1093/ajcp/aqaa062

32275742

Busse

PJ

Christiansen

SC

2020

Hereditary angioedema

New England Journal of Medicine38211361148

10.1056/NEJMra1808012

32187470

Chen

N

Zhou

M

Dong

X

Qu

J

Gong

F

Han

Y

Qiu

Y

Wang

J

Liu

Y

Wei

Y

Xia

J

Yu

T

Zhang

X

Zhang

L

2020

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

The Lancet395507513

10.1016/S0140-6736(20)30211-7

Cicardi

M

Zuraw

BL

2018

Angioedema due to bradykinin dysregulation

The Journal of Allergy and Clinical Immunology: In Practice611321141

10.1016/j.jaip.2018.04.022

30033914

Davin

L

Marechal

P

Lancellotti

P

Martinez

C

Pierard

L

Radermecker

R

2019

Angioedema: a rare and sometimes delayed side effect of angiotensin-converting enzyme inhibitors

Acta Cardiologica74277281

10.1080/00015385.2018.1507477

30328786

Dolhnikoff

M

Duarte-Neto

AN

de Almeida Monteiro

RA

da Silva

LFF

de Oliveira

EP

Saldiva

PHN

Mauad

T

Negri

EM

2020

Pathological evidence of pulmonary thrombotic phenomena in severe COVID-19

Journal of Thrombosis and Haemostasis1815171519

10.1111/jth.14844

32294295

Edwards

RL

Rickles

FR

Bobrove

AM

1979

Mononuclear cell tissue factor: cell of origin and requirements for activation

Blood54359370

10.1182/blood.V54.2.359.359

454843

Entrenas Castillo

M

Entrenas Costa

LM

Vaquero Barrios

JM

Alcalá Díaz

JF

López Miranda

J

Bouillon

R

Quesada Gomez

JM

2020

"Effect of calcifediol treatment and best available therapy versus best available therapy on intensive care unit admission and mortality among patients hospitalized for COVID-19: A pilot randomized clinical study"

The Journal of Steroid Biochemistry and Molecular Biology203

105751

10.1016/j.jsbmb.2020.105751

32871238

Ewald

GA

Eisenberg

PR

1995

Plasmin-mediated activation of contact system in response to pharmacological thrombolysis

Circulation912836

10.1161/01.CIR.91.1.28

7805216

Foley

JH

Walton

BL

Aleman

MM

O'Byrne

AM

Lei

V

Harrasser

M

Foley

KA

Wolberg

AS

Conway

EM

2016

Complement activation in arterial and venous thrombosis is mediated by plasmin

EBioMedicine5175182

10.1016/j.ebiom.2016.02.011

27077125

Folsom

AR

Tang

W

Roetker

NS

Heckbert

SR

Cushman

M

Pankow

JS

2015

Prospective study of circulating factor XI and incident venous thromboembolism: the longitudinal investigation of thromboembolism etiology (LITE)

American Journal of Hematology9010471051

10.1002/ajh.24168

26260105

Fox

SE

Akmatbekov

A

Harbert

JL

Li

G

Quincy Brown

J

Vander Heide

RS

2020

Pulmonary and cardiac pathology in Covid-19: the first autopsy series from new orleans

medRxiv10.1101/2020.04.06.20050575

Garvin

MR

Alvarez

C

Miller

JI

Prates

ET

Walker

AM

Amos

BK

Mast

AE

Justice

A

Aronow

B

Jacobson

D

2020

A mechanistic model and therapeutic interventions for COVID-19 involving a RAS-mediated bradykinin storm

eLife9

e59177

10.7554/eLife.59177

32633718

Gattinoni

L

Coppola

S

Cressoni

M

Busana

M

Rossi

S

Chiumello

D

2020

COVID-19 does not lead to a “Typical” Acute Respiratory Distress Syndrome

American Journal of Respiratory and Critical Care Medicine20112991300

10.1164/rccm.202003-0817LE

Geerdes-Fenge

HF

Reisinger

EC

Arndt

H

2020

Pulmonary artery embolism in COVID-19 despite thrombosis prophylaxis

Deutsches Aerzteblatt Online117

297

10.3238/arztebl.2020.0297

Grover

SP

Mackman

N

2018

Tissue factor: an essential mediator of hemostasis and trigger of thrombosis

Arteriosclerosis, Thrombosis, and Vascular Biology38709725

10.1161/ATVBAHA.117.309846

29437578

Gu

L

Deng

H

Ren

Z

Zhao

Y

Yu

S

Guo

Y

Dai

J

Chen

X

Li

K

Li

R

Wang

G

2019

Dynamic changes in the microbiome and mucosal immune microenvironment of the lower respiratory tract by influenza virus infection

Frontiers in Microbiology10

2491

10.3389/fmicb.2019.02491

31736922

Guadiz

G

Sporn

LA

Goss

RA

Lawrence

SO

Marder

VJ

Simpson-Haidaris

PJ

1997

Polarized secretion of fibrinogen by lung epithelial cells

American Journal of Respiratory Cell and Molecular Biology176069

10.1165/ajrcmb.17.1.2730

9224210

Helms

J

Tacquard

C

Severac

F

Leonard-Lorant

I

Ohana

M

Delabranche

X

Merdji

H

Clere-Jehl

R

Schenck

M

Fagot Gandet

F

Fafi-Kremer

S

Castelain

V

Schneider

F

Grunebaum

L

Anglés-Cano

E

Sattler

L

Mertes

P-M

Meziani

F

2020

High risk of thrombosis in patients with severe SARS-CoV-2 infection: a multicenter prospective cohort study

Intensive Care Medicine4610891098

10.1007/s00134-020-06062-x

Ito

T

Thachil

J

Asakura

H

Levy

JH

Iba

T

2019

Thrombomodulin in disseminated intravascular coagulation and other critical conditions-a multi-faceted anticoagulant protein with therapeutic potential

Critical Care23

280

10.1186/s13054-019-2552-0

31416465

Ivanov

I

Shakhawat

R

Sun

MF

Dickeson

SK

Puy

C

McCarty

OJ

Gruber

A

Matafonov

A

Gailani

D

2017

Nucleic acids as cofactors for factor XI and prekallikrein activation: different roles for high-molecular-weight kininogen

Thrombosis and Haemostasis117671681

10.1160/TH16-09-0691

28124063

Jakieła

B

Gielicz

A

Plutecka

H

Przybyszowski

M

Soja

J

Sładek

K

Bochenek

G

2018

Bronchoalveolar lavage transcriptome and eicosanoid profiles reveal heterogeneity of lower airway inflammation in aspirin-exacerbated respiratory disease (AERD)

Allergy and Immunology52

PA5010

10.1183/13993003.congress-2018.pa5010

Kahn

N

Granzow

M

Meister

M

Muley

T

Herth

FJF

Kreuter

M

2015

Transcriptome analysis in endobronchial epithelial lining fluid compared to bronchoalveolar lavage in idiopathic pulmonary fibrosis

Pneumologie69

1556653

10.1055/s-0035-1556653

Kleniewski

J

Donaldson

VH

1987

Comparison of human high molecular weight kininogen digestion by plasma kallikrein and by plasmin A revised method of purification of high molecular weight kininogen

The Journal of Laboratory and Clinical Medicine109469479

2950194

Lax

SF

Skok

K

Zechner

P

Kessler

HH

Kaufmann

N

Koelblinger

C

Vander

K

Bargfrieder

U

Trauner

M

2020

Pulmonary arterial thrombosis in COVID-19 with fatal outcome : results from a prospective, Single-Center, clinicopathologic case series

Annals of Internal Medicine173350361

10.7326/M20-2566

32422076

Levi

M

van der Poll

T

2017

Coagulation and sepsis

Thrombosis Research1493844

10.1016/j.thromres.2016.11.007

27886531

Long

AT

Kenne

E

Jung

R

Fuchs

TA

Renné

T

2016

Contact system revisited: an interface between inflammation, coagulation, and innate immunity

Journal of Thrombosis and Haemostasis14427437

10.1111/jth.13235

26707513

Maas

C

Renné

T

2018

Coagulation factor XII in thrombosis and inflammation

Blood13119031909

10.1182/blood-2017-04-569111

29483100

Magro

C

Mulvey

JJ

Berlin

D

Nuovo

G

Salvatore

S

Harp

J

Baxter-Stoltzfus

A

Laurence

J

2020

Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: a report of five cases

Translational Research220113

10.1016/j.trsl.2020.04.007

32299776

Marceau

F

Bachelard

H

Bouthillier

J

Fortin

JP

Morissette

G

Bawolak

MT

Charest-Morin

X

Gera

L

2020

Bradykinin receptors: agonists, antagonists, expression, signaling, and adaptation to sustained stimulation

International Immunopharmacology82

106305

10.1016/j.intimp.2020.106305

32106060

McCarthy

DJ

Chen

Y

Smyth

GK

2012

Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation

Nucleic Acids Research4042884297

10.1093/nar/gks042

22287627

Mestres

G

Puigmacià

R

Blanco

C

Yugueros

X

Esturrica

M

Riambau

V

2020

Risk of peripheral arterial thrombosis in COVID-19

Journal of Vascular Surgery72756757

10.1016/j.jvs.2020.04.477

32417015

Michalovich

D

Rodriguez-Perez

N

Smolinska

S

Pirozynski

M

Mayhew

D

Uddin

S

Van Horn

S

Sokolowska

M

Altunbulakli

C

Eljaszewicz

A

Pugin

B

Barcik

W

Kurnik-Lucka

M

Saunders

KA

Simpson

KD

Schmid-Grendelmeier

P

Ferstl

R

Frei

R

Sievi

N

Kohler

M

Gajdanowicz

P

Graversen

KB

Lindholm Bøgh

K

Jutel

M

Brown

JR

Akdis

CA

Hessel

EM

O'Mahony

L

2019

Obesity and disease severity magnify disturbed microbiome-immune interactions in asthma patients

Nature Communications10

5711

10.1038/s41467-019-13751-9

31836714

Middeldorp

S

Coppens

M

van Haaps

TF

Foppen

M

Vlaar

AP

Müller

MCA

Bouman

CCS

Beenen

LFM

Kootte

RS

Heijmans

J

Smits

LP

Bonta

PI

van Es

N

2020

Incidence of venous thromboembolism in hospitalized patients with COVID-19

Journal of Thrombosis and Haemostasis1819952002

10.1111/jth.14888

32369666

Mohammed

BM

Matafonov

A

Ivanov

I

Sun

MF

Cheng

Q

Dickeson

SK

Li

C

Sun

D

Verhamme

IM

Emsley

J

Gailani

D

2018

An update on factor XI structure and function

Thrombosis Research16194105

10.1016/j.thromres.2017.10.008

29223926

Panigada

M

Bottino

N

Tagliabue

P

Grasselli

G

Novembrino

C

Chantarangkul

V

Pesenti

A

Peyvandi

F

Tripodi

A

2020

Hypercoagulability of COVID-19 patients in intensive care unit: a report of thromboelastography findings and other parameters of hemostasis

Journal of Thrombosis and Haemostasis1817381742

10.1111/jth.14850

32302438

Pieters

M

Wolberg

AS

2019

Fibrinogen and fibrin: an illustrated review

Research and Practice in Thrombosis and Haemostasis3161172

10.1002/rth2.12191

31011700

Prada

AE

Zahedi

K

Davis

AE

1998

Regulation of C1 inhibitor synthesis

Immunobiology199377388

10.1016/S0171-2985(98)80042-9

9777421

Preis

M

Hirsch

J

Kotler

A

Zoabi

A

Stein

N

Rennert

G

Saliba

W

2017

Factor XI deficiency is associated with lower risk for cardiovascular and venous thromboembolism events

Blood12912101215

10.1182/blood-2016-09-742262

28039189

Revenko

AS

Gao

D

Crosby

JR

Bhattacharjee

G

Zhao

C

May

C

Gailani

D

Monia

BP

MacLeod

AR

2011

Selective depletion of plasma prekallikrein or coagulation factor XII inhibits thrombosis in mice without increased risk of bleeding

Blood11853025311

10.1182/blood-2011-05-355248

21821705

Riewald

M

Petrovan

RJ

Donner

A

Mueller

BM

Ruf

W

2002

Activation of endothelial cell protease activated receptor 1 by the protein C pathway

Science29618801882

10.1126/science.1071699

12052963

Robinson

MD

McCarthy

DJ

Smyth

GK

2010

edgeR: a bioconductor package for differential expression analysis of digital gene expression data

Bioinformatics26139140

10.1093/bioinformatics/btp616

19910308

Salomon

O

Steinberg

DM

Koren-Morag

N

Tanne

D

Seligsohn

U

2008

Reduced incidence of ischemic stroke in patients with severe factor XI deficiency

Blood11141134117

10.1182/blood-2007-10-120139

18268095

Salomon

O

Steinberg

DM

Zucker

M

Varon

D

Zivelin

A

Seligsohn

U

2011

Patients with severe factor XI deficiency have a reduced incidence of deep-vein thrombosis

Thrombosis and Haemostasis105269273

10.1160/TH10-05-0307

21057700

Schmaier

AH

2016

The contact activation and kallikrein/kinin systems: pathophysiologic and physiologic activities

Journal of Thrombosis and Haemostasis142839

10.1111/jth.13194

26565070

Schmaier

AH

Emsley

J

Feener

EP

Gailani

D

Govers-Riemslag

JWP

Kaplan

AP

Maas

C

Morrissey

JH

Renné

T

Sidelmann

JJ

Meijers

JCM

2019

Nomenclature of factor XI and the contact system

Journal of Thrombosis and Haemostasis : JTH1722162219

10.1111/jth.14595

31410964

Sengupta

S

Tang

SY

Devine

JC

Anderson

ST

Nayak

S

Zhang

SL

Valenzuela

A

Fisher

DG

Grant

GR

López

CB

FitzGerald

GA

2019

Circadian control of lung inflammation in influenza infection

Nature Communications10

4107

10.1038/s41467-019-11400-9

31511530

Shetty

S

Padijnayayveetil

J

Tucker

T

Stankowska

D

Idell

S

2008

The fibrinolytic system and the regulation of lung epithelial cell proteolysis, signaling, and cellular viability

American Journal of Physiology-Lung Cellular and Molecular Physiology295L967L975

10.1152/ajplung.90349.2008

Siegerink

B

Govers-Riemslag

JW

Rosendaal

FR

Ten Cate

H

Algra

A

2010

Intrinsic coagulation activation and the risk of arterial thrombosis in young women: results from the risk of arterial thrombosis in relation to oral contraceptives (RATIO) case-control study

Circulation12218541861

10.1161/CIRCULATIONAHA.110.943738

20956210

Simpson-Haidaris

PJ

Courtney

MA

Wright

TW

Goss

R

Harmsen

A

Gigliotti

F

1998

Induction of fibrinogen expression in the lung epithelium during Pneumocystis carinii pneumonia

Infection and Immunity6644314439

10.1128/IAI.66.9.4431-4439.1998

9712798

Stavrou

EX

2018

Factor XII in inflammation and wound healing

Current Opinion in Hematology25403409

10.1097/MOH.0000000000000450

29994899

Su

H

Yang

M

Wan

C

Yi

LX

Tang

F

Zhu

HY

Yi

F

Yang

HC

Fogo

AB

Nie

X

Zhang

C

2020

Renal histopathological analysis of 26 postmortem findings of patients with COVID-19 in China

Kidney International98219227

10.1016/j.kint.2020.04.003

32327202

Sun

Q

Fang

L

Roth

M

Tang

X

Papakonstantinou

E

Zhai

W

Louis

R

Heinen

V

Schleich

FN

Lu

S

Savic

S

Tamm

M

Stolz

D

2019

Bronchial thermoplasty decreases airway remodelling by blocking epithelium-derived heat shock protein-60 secretion and protein arginine methyltransferase-1 in fibroblasts

European Respiratory Journal54

1900300

10.1183/13993003.00300-2019

31467116

Suri

MFK

Yamagishi

K

Aleksic

N

Hannan

PJ

Folsom

AR

2010

Novel hemostatic factor levels and risk of ischemic stroke: the atherosclerosis risk in communities (ARIC) Study

Cerebrovascular Diseases29497502

10.1159/000297966

Tang

N

Li

D

Wang

X

Sun

Z

2020

Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia

Journal of Thrombosis and Haemostasis18844847

10.1111/jth.14768

32073213

van de Veerdonk

FL

Kouijzer

IJE

de Nooijer

AH

van der Hoeven

HG

Maas

C

Netea

MG

Brüggemann

RJM

2020

Outcomes associated with use of a kinin B2 receptor antagonist among patients with COVID-19

JAMA Network Open3

e2017708

10.1001/jamanetworkopen.2020.17708

32789513

Wang

Y

Ivanov

I

Smith

SA

Gailani

D

Morrissey

JH

2019

Polyphosphate, Zn²⁺ and high molecular weight kininogen modulate individual reactions of the contact pathway of blood clotting

Journal of Thrombosis and Haemostasis1721312140

10.1111/jth.14612

31420909

Wang

J

Hajizadeh

N

Moore

EE

McIntyre

RC

Moore

PK

Veress

LA

Yaffe

MB

Moore

HB

Barrett

CD

2020

Tissue plasminogen activator (tPA) treatment for COVID-19 associated acute respiratory distress syndrome (ARDS): A case series

Journal of Thrombosis and Haemostasis1817521755

10.1111/jth.14828

32267998

Weigt

SS

Wang

X

Palchevskiy

V

Li

X

Patel

N

Ross

DJ

Reynolds

J

Shah

PD

Danziger-Isakov

LA

Sweet

SC

Singer

LG

Budev

M

Palmer

S

Belperio

JA

2019

Usefulness of gene expression profiling of bronchoalveolar lavage cells in acute lung allograft rejection

The Journal of Heart and Lung Transplantation38845855

10.1016/j.healun.2019.05.001

31122726

WHO Rapid Evidence Appraisal for COVID-19 Therapies (REACT) Working GroupSterne

JAC

Murthy

S

Diaz

JV

Slutsky

AS

Villar

J

Angus

DC

Annane

D

Azevedo

LCP

Berwanger

O

Cavalcanti

AB

Dequin

PF

Du

B

Emberson

J

Fisher

D

Giraudeau

B

Gordon

AC

Granholm

A

Green

C

Haynes

R

Heming

N

Higgins

JPT

Horby

P

Jüni

P

Landray

MJ

Le Gouge

A

Leclerc

M

Lim

WS

Machado

FR

McArthur

C

Meziani

F

Møller

MH

Perner

A

Petersen

MW

Savovic

J

Tomazini

B

Veiga

VC

Webb

S

Marshall

JC

2020

Association between administration of systemic corticosteroids and mortality among critically ill patients with COVID-19: a Meta-analysis

Jama32413301341

10.1001/jama.2020.17023

32876694

Wood

JP

Ellery

PE

Maroney

SA

Mast

AE

2014

Biology of tissue factor pathway inhibitor

Blood12329342943

10.1182/blood-2013-11-512764

24620349

Xu

Z

Shi

L

Wang

Y

Zhang

J

Huang

L

Zhang

C

Liu

S

Zhao

P

Liu

H

Zhu

L

Tai

Y

Bai

C

Gao

T

Song

J

Xia

P

Dong

J

Zhao

J

Wang

FS

2020

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

The Lancet Respiratory Medicine8420422

10.1016/S2213-2600(20)30076-X

32085846

Zhang

HH

Li

CX

Tang

LF

2019

The differential expression profiles of miRNA-let 7a, 7b, and 7c in bronchoalveolar lavage fluid from infants with asthma and airway foreign bodies

Journal of Evidence-Based Integrative Medicine24

2515690X18821906

10.1177/2515690X18821906

30789054

Zhou

F

Yu

T

Du

R

Fan

G

Liu

Y

Liu

Z

Xiang

J

Wang

Y

Song

B

Gu

X

Guan

L

Wei

Y

Li

H

Wu

X

Xu

J

Tu

S

Zhang

Y

Chen

H

Cao

B

2020a

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

The Lancet39510541062

10.1016/S0140-6736(20)30566-3

32171076

Zhou

P

Yang

XL

Wang

XG

Hu

B

Zhang

L

Zhang

W

Si

HR

Zhu

Y

Li

B

Huang

CL

Chen

HD

Chen

J

Luo

Y

Guo

H

Jiang

RD

Liu

MQ

Chen

Y

Shen

XR

Wang

X

Zheng

XS

Zhao

K

Chen

QJ

Deng

F

Liu

LL

Yan

B

Zhan

FX

Wang

YY

Xiao

GF

Shi

ZL

2020b

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Nature579270273

10.1038/s41586-020-2012-7

32015507

10.7554/eLife.64330.sa1

Decision letter

Emoto

Noriaki

Reviewing EditorKobe Pharmaceutical UniversityJapan

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This manuscript captivated the connection between COVID-19 and cardiovascular disease. Following their previous studies, the authors further investigate the dysregulation of the coagulation and fibrinolytic pathways on COVID-19 patients. The authors' conclusions are well supported, and the authors draw on deep expertise in the field to make exquisite use of gene expression data to obtain deep insights into COVID-19 pathogenesis.

Decision letter after peer review:

Thank you for submitting your article "SARS-CoV-2 Suppresses Anticoagulant and Fibrinolytic Gene Expression in the Lung" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Jos van der Meer as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Summary:

This manuscript extends the authors previous work using transcriptomics data to formulate their "bradykinin storm" hypothesis. Their previous work was extremely high-impact, as it challenged the dogma that a cytokine storm was the principle mechanisms underlying lung pathology in COVID-19. This manuscript further extends their prior work, linking bradykinin signaling to the accumulation of fibrin deposits within pulmonary vessels and the alveolar space, inferred from gene expression data from COVID-19 patients. The authors' conclusions are well supported, and the authors draw on deep expertise in the field to make exquisite use of gene expression data to obtain deep insights into COVID-19 pathogenesis.

Essential Revisions:

1) The reviewer acknowledges the extensive expertise of the impressive author group and congratulates the authors for an interesting investigation. That being said, the reviewer does not think your Abstract reflects the importance or insight of your findings, as your conclusion in the Abstract states "analysis documents increased vascular permeability, enhanced coagulation, and reduced fibrinolytic activity in the lungs of patients with COVID-19". The potential for acute lung injury, hypercoagulability, and microvascular thrombotic sequela of COVID 19 are well known. the reviewer thinks you need to include some of your important aspects of the investigation's findings in the Abstract rather than generalized conclusions.

2) The route of viral entry into the body to the respiratory tract initially triggers the local inflammatory response in the lung, with a host defense mechanism of microvascular thrombotic sequela due to microcirculatory injury, but as the disease progresses without any potential immunity, then multiple other organs may be involved. Again, with the initial insult to the lung and subsequent systemic responses that seem to be the sort of chronology of the infection, as a result, it would be helpful to include some time course of the disease progression that you describe. For example, the skin and other organ involvement is a later, not initial event in most patients. the reviewer thinks your Introduction could be shortened with the extensive literature that exists already about COVID 19 coagulopathy, but also, it would be helpful to provide some sense of the time course of the disease progression. The bronchial alveolar lavage specimens you have our impatience intubated and ventilated most likely, and clearly in an advance disease state. The reviewer thinks that needs to be specifically explained as well.

3) In the subsection “Conclusions”, your first three sentences, including the goal of the present study, need to be incorporated in your Introduction. The reviewer would start your conclusion section with the sentence "We detected pronounced changes in mRNA levels encoding proteins involved in regulation of coagulation, fibrinolysis, and inflammation in the lungs of COVID-19 patients.".

4) Gene and protein naming conventions: when specific proteins or hormones are listed in the text, it would be helpful to also list their formal names, e.g., the reviewer assumes that "tissue factor" in the Abstract refers to human protein CD142 – though later the informal name is given as "extravascular tissue factor (TF)". The informal name, however, is confusing since TF is also the formal symbol for the gene "Homo sapiens transferrin (TF)", RefSeq NM_001354704. It would be particularly helpful to list both gene and protein names parenthetically at each such mention. While this is less valuable for those who study the human system directly, it is very important for biologists specializing in model organisms who may be less familiar with medical parlance and informal gene and protein names. It is also extremely useful for AI/ML algorithms that parse Abstracts in search of gene names.

5) Introductory material in the Results section: The Results sections begin with lengthy introductions to the biomedical literature surrounding specific pathways, forcing the reader to scan for novel material. It would be better either to move this material to the Introduction with call-outs to specific Results sections, or, alternatively, to label such introductory paragraphs explicitly so that readers already familiar with the literature can quickly scan past them. As is, it took me a few reads to find the novel result within the first paragraph labeled, "Changes in transcripts encoding proteins in the Kallikrein-Kinin/Bradykinin System".

6) Speculative discussion in the Results section: The final sentence of the first section of Results is speculative, and would fit better in the Discussion:

(6a) "Consistent with our hypothesis that COVID-19 triggers a Bradykinin Storm, the net effect of increased BK production, reduced BK degradation, and increased local expression of BK receptors, may be to enhance vasodilation and vascular permeability that expose blood components to extravascular proteins that promote fibrin formation and contribute to tissue damage."

Or, alternatively, re-word to posit as a conclusion supported by a combination of data in this study and prior literature, perhaps as follows:

"Our observations of increased BK production, reduced BK degradation, and increased local expression of BK receptors, are expected in a system undergoing enhanced vasodilation and vascular permeability, where blood components are exposed to extravascular proteins that promote fibrin formation and contribute to tissue damage. Further, these findings are broadly consistent with our hypothesis that COVID-19 triggers a Bradykinin Storm."

or something like that – at least get rid of the "…may be to…".

(6b) Similarly, we have, "On balance, these data suggest that pulmonary fibrin deposition does not stem from enhanced local TF production and that counterintuitively, COVID-19 may dampen TF-dependent mechanisms in the lungs."

It would be better to use "indicate" than "suggest", or to rephrase something like:

"On balance, these data are inconsistent pulmonary fibrin deposition from enhanced local TF production – but are consistent, if perhaps counterintuitively, with the hypothesis that COVID-19 dampens TF-dependent mechanisms in the lungs."

(6c) The paragraph that begins, "Increased thrombin generation can also be initiated by the plasma protease factor XIIa (FXIIa).(50, 51)…" Appears to be entirely "Discussion" and should probably be removed from Results. Did I miss a new result there?

(6d) The last two sentences of the Results section are also quite speculative:

"The combination of increased fibrinogen expression, reduced production of uPA and uPAR, and a modest increase in PAI-1 suggests a loss of local fibrinolytic capacity. This change may permit the accumulation of fibrin deposits within pulmonary vessels and the alveolar space."

How about the following instead?:

"The combination of increased fibrinogen expression, reduced production of uPA and uPAR, and a modest increase in PAI-1 indicates a loss of local fibrinolytic capacity – consistent with a lung environment permissive of the accumulation of fibrin deposits within pulmonary vessels and the alveolar space (CITE)." and of course a citation should be added where indicated above.

7) Comments on Figures 1 and 2: please increase the resolution of the figure to at least 300dpi and ensure that all fonts are at least size 9. Providing a color scale would make the data more readable for the reader.

8) Table 1: Please provide the log2 data for the observed gene. Adding statistical analysis and providing the statistical significance would make the data more convincing.

9) Materials and methods: In this paper, the authors use 9 COVID-19 patient's BALF samples and 40 control. The reviewer found that in a previous paper (Garvin et al., 2020), the author's group also use the same number of COVID-19 cases and controls. Are these the same samples?

10.7554/eLife.64330.sa2

Author response

Essential Revisions:

1) The reviewer acknowledges the extensive expertise of the impressive author group and congratulates the authors for an interesting investigation. That being said, the reviewer does not think your Abstract reflects the importance or insight of your findings, as your conclusion in the Abstract states "analysis documents increased vascular permeability, enhanced coagulation, and reduced fibrinolytic activity in the lungs of patients with COVID-19". The potential for acute lung injury, hypercoagulability, and microvascular thrombotic sequela of COVID 19 are well known. the reviewer thinks you need to include some of your important aspects of the investigation's findings in the Abstract rather than generalized conclusions.

We removed general background information from the Abstract and recapitulated the most important results to make the impact of the work clear.

2) The route of viral entry into the body to the respiratory tract initially triggers the local inflammatory response in the lung, with a host defense mechanism of microvascular thrombotic sequela due to microcirculatory injury, but as the disease progresses without any potential immunity, then multiple other organs may be involved. Again, with the initial insult to the lung and subsequent systemic responses that seem to be the sort of chronology of the infection, as a result, it would be helpful to include some time course of the disease progression that you describe. For example, the skin and other organ involvement is a later, not initial event in most patients. the reviewer thinks your Introduction could be shortened with the extensive literature that exists already about COVID 19 coagulopathy, but also, it would be helpful to provide some sense of the time course of the disease progression. The bronchial alveolar lavage specimens you have our impatience intubated and ventilated most likely, and clearly in an advance disease state. The reviewer thinks that needs to be specifically explained as well.

We made major changes to the Introduction. Overall we shortened the Introduction. Some portions of the first paragraph were moved to the "Overview of pathways examined" sections in the Results and Discussion, while some text from the “Conclusions” subsection was incorporated into this section.

We added text relating to disease progression to help put our model into context (e.g., Introduction, second paragraph, second sentence). The Results and Discussion have been changed extensively to make our model more clear.

We added Table 2 with the clinical characteristics of the patients from which the BALF were taken. All were admitted to the ICU, which we note in the new document.

We added text in the Materials and methods section "Study participants" regarding the severity of illness and the condition of the patients at the time of sampling.

3) In the subsection “Conclusions”, your first three sentences, including the goal of the present study, need to be incorporated in your Introduction. The reviewer would start your conclusion section with the sentence "We detected pronounced changes in mRNA levels encoding proteins involved in regulation of coagulation, fibrinolysis, and inflammation in the lungs of COVID-19 patients.".

The first 3 sentences from the subsection “Conclusions” were moved to the Introduction.

4) Gene and protein naming conventions: when specific proteins or hormones are listed in the text, it would be helpful to also list their formal names, e.g., the reviewer assumes that "tissue factor" in the Abstract refers to human protein CD142 – though later the informal name is given as "extravascular tissue factor (TF)". The informal name, however, is confusing since TF is also the formal symbol for the gene "Homo sapiens transferrin (TF)", RefSeq NM_001354704. It would be particularly helpful to list both gene and protein names parenthetically at each such mention. While this is less valuable for those who study the human system directly, it is very important for biologists specializing in model organisms who may be less familiar with medical parlance and informal gene and protein names. It is also extremely useful for AI/ML algorithms that parse Abstracts in search of gene names.

We changed gene and protein names throughout the text to use the formal names and include gene and/or protein symbols parenthetically where appropriate.

5) Introductory material in the Results section: The Results sections begin with lengthy introductions to the biomedical literature surrounding specific pathways, forcing the reader to scan for novel material. It would be better either to move this material to the Introduction with call-outs to specific Results sections, or, alternatively, to label such introductory paragraphs explicitly so that readers already familiar with the literature can quickly scan past them. As is, it took me a few reads to find the novel result within the first paragraph labeled, "Changes in transcripts encoding proteins in the Kallikrein-Kinin/Bradykinin System".

We separated these sections into subsections for "Overview of pathways examined" and

"Study findings."

6) Speculative discussion in the Results section: The final sentence of the first section of Results is speculative, and would fit better in the Discussion:

(6a) "Consistent with our hypothesis that COVID-19 triggers a Bradykinin Storm, the net effect of increased BK production, reduced BK degradation, and increased local expression of BK receptors, may be to enhance vasodilation and vascular permeability that expose blood components to extravascular proteins that promote fibrin formation and contribute to tissue damage."

Or, alternatively, re-word to posit as a conclusion supported by a combination of data in this study and prior literature, perhaps as follows:

"Our observations of increased BK production, reduced BK degradation, and increased local expression of BK receptors, are expected in a system undergoing enhanced vasodilation and vascular permeability, where blood components are exposed to extravascular proteins that promote fibrin formation and contribute to tissue damage. Further, these findings are broadly consistent with our hypothesis that COVID-19 triggers a Bradykinin Storm."

or something like that – at least get rid of the "…may be to…".

We moved this discussion point to the end of the “Conclusions” subsection.

(6b) Similarly, we have, "On balance, these data suggest that pulmonary fibrin deposition does not stem from enhanced local TF production and that counterintuitively, COVID-19 may dampen TF-dependent mechanisms in the lungs."

It would be better to use "indicate" than "suggest", or to rephrase something like:

"On balance, these data are inconsistent pulmonary fibrin deposition from enhanced local TF production – but are consistent, if perhaps counterintuitively, with the hypothesis that COVID-19 dampens TF-dependent mechanisms in the lungs."

We changed the text to "indicate" rather than "suggest."

(6c) The paragraph that begins, "Increased thrombin generation can also be initiated by the plasma protease factor XIIa (FXIIa).(50, 51)…" Appears to be entirely "Discussion" and should probably be removed from Results. Did I miss a new result there?

We added text to make the results clear; see text beginning with "Transcripts encoding FXII were slightly decreased (-4-fold)…", which is found in the section "Changes in transcripts encoding intrinsic pathway proteins that initiate thrombin generation" > "Study findings."

(6d) The last two sentences of the Results section are also quite speculative:

"The combination of increased fibrinogen expression, reduced production of uPA and uPAR, and a modest increase in PAI-1 suggests a loss of local fibrinolytic capacity. This change may permit the accumulation of fibrin deposits within pulmonary vessels and the alveolar space."

How about the following instead?:

"The combination of increased fibrinogen expression, reduced production of uPA and uPAR, and a modest increase in PAI-1 indicates a loss of local fibrinolytic capacity – consistent with a lung environment permissive of the accumulation of fibrin deposits within pulmonary vessels and the alveolar space (CITE)." and of course a citation should be added where indicated above.

We changed the text as suggested by the reviewer.

7) Comments on Figures 1 and 2: please increase the resolution of the figure to at least 300dpi and ensure that all fonts are at least size 9. Providing a color scale would make the data more readable for the reader.

Fixed resolution and added color scale.

8) Table 1: Please provide the log2 data for the observed gene. Adding statistical analysis and providing the statistical significance would make the data more convincing.

We added the log2-fold-change values and p-values, as well as the name of the protein for each gene.

9) Materials and methods: In this paper, the authors use 9 COVID-19 patient's BALF samples and 40 control. The reviewer found that in a previous paper (Garvin et al., 2020), the author's group also use the same number of COVID-19 cases and controls. Are these the same samples?

We added a table of the sample IDs (data available from NCBI) to help clarify that these are the same samples. This is listed as Table 1.