Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Aug 1.
Published in final edited form as: Thromb Haemost. 2024 Jan 25;124(8):725–738. doi: 10.1055/a-2253-9359

ADAMTS13 or Caplacizumab Reduces the Accumulation of Neutrophil Extracellular Traps and Thrombus in Whole Blood of COVID-19 Patients under Flow

Noritaka Yada 1,*, Quan Zhang 1, Antonia Bignotti 1, Zhan Ye 1, X Long Zheng 1,2
PMCID: PMC11260255  NIHMSID: NIHMS1986574  PMID: 38272066

Abstract

Background

Neutrophil NETosis and neutrophil extracellular traps (NETs) play a critical role in pathogenesis of coronavirus disease 2019 (COVID-19)-associated thrombosis. However, the extents and reserve of NETosis, and potential of thrombus formation under shear in whole blood of patients with COVID-19 are not fully elucidated. Neither has the role of recombinant ADAMTS13 or caplacizumab on the accumulation of NETs and thrombus in COVID-19 patients’ whole blood under shear been investigated.

Methods

Flow cytometry and microfluidic assay, as well as immunoassays, were employed for the study.

Results

We demonstrated that the percentage of H3Cit + MPO+ neutrophils, indicative of NETosis, was dramatically increased in patients with severe but not critical COVID-19 compared with that in asymptomatic or mild disease controls. Upon stimulation with poly [I:C], a double strain DNA mimicking viral infection, or bacterial shigatoxin-2, the percentage of H3Cit + MPO+ neutrophils was not significantly increased in the whole blood of severe and critical COVID-19 patients compared with that of asymptomatic controls, suggesting the reduction in NETosis reserve in these patients. Microfluidic assay demonstrated that the accumulation of NETs and thrombus was significantly enhanced in the whole blood of severe/critical COVID-19 patients compared with that of asymptomatic controls. Like DNase I, recombinant ADAMTS13 or caplacizumab dramatically reduced the NETs accumulation and throm-bus formation under arterial shear.

Conclusion

Significantly increased neutrophil NETosis, reduced NETosis reserve, and enhanced thrombus formation under arterial shear may play a crucial role in the pathogenesis of COVID-19-associated coagulopathy. Recombinant ADAMTS13 or caplacizumab may be explored for the treatment of COVID-19-associated thrombosis.

Keywords: NETosis, neutrophil extracellular traps, ADAMTS13, von Willebrand factor, SARS-CoV-2, COVID-19, Thrombosis

Visual summary.

NETS and Potential Targets. (A) Underflow the released VWF captures platelets (PLT) and neutrophils (N), which undergo NETosis resulting in the release of histone-DNA or histone-MPO complexes (NETs) that bind VWF and activate platelets to enhance thrombus formation. (B) DNase I that cleaves extracellular DNA strings, or recombinant ADAMTS13 that cleaves VWF strings, or caplacizumab that blocks platelet-VWF interaction inhibits NET formation and thrombosis in patients with severe/critical COVID-19.

Introduction

Venous and arterial thrombosis is well recognized in patients with severe/critical COVID-19, resulting from infection of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2).1–3 Thrombotic events are associated with poor outcomes such as an increased intubation and mortality rate.4,5 More importantly, approximately 40% of thrombotic events still occurred in patients receiving low-molecular-weight heparin as a prophylaxis in hospital or home therapies.5 In the early pandemic, more than 70% of nonsurviving coronavirus disease 2019 (COVID-19) patients fulfilled the diagnostic criteria of disseminated intravascular coagulation with increased levels of plasma D-dimer.6 However, the molecular mechanism of coagulation activation, resulting in thrombus formation in patients with severe/critical COVID-19, is not fully understood.

It has been postulated that SARS-CoV-2 and damage-associated molecular patterns (DAMPs) released from injured host tissues may activate monocytes, triggering the production of inflammatory cytokines and chemokines that then stimulate neutrophils, lymphocytes, platelets, and vascular endothelial cells.3,7–9 Blood neutrophils may be activated to undergo a process known as NETosis, which releases cytoplasmic and intranuclear contents, including myeloperoxidase (MPO), cathepsin-G, human neutrophil peptides (HNPs), and histone–DNA complexes, as well as cell-free DNA (cfDNA), etc. These compounds may form a “web-like” structure namely the neutrophil extracellular traps (NETs). NETs play an important role in entrapping invaded microorganisms10–12 but may contribute to pathogenesis of acute respiratory distress syndrome (ARDS) through the formation of microvascular thrombosis in the lungs following SARS-CoV-2 infection.13 Increased NETosis and plasma levels of soluble NET fragments are observed in many other infectious diseases, including sepsis,14,15 systemic lupus erythematosus,16 deep venous thrombosis, and heparin-induced thrombocytopenia.17,18 To date, there has been no systemic investigation on potential of NETs and thrombus formation under flow in the whole blood from patients with COVID-19 with various disease severities.

Moreover, endotheliopathy is a hallmark of severe/critical COVID-19, which correlates with the mortality and other long-term complications.9,19,20 In an acute disease, vascular endothelium may be activated or directly injured by viruses, inflammatory cytokines, DAMPs, and activated complements, etc.21–24 This results in acute or chronic releases of endothelial ultra-large von Willebrand factor (VWF), which may spontaneously recruit platelets and leukocytes from circulation to the sites of vascular injury, a critical step for normal hemostasis.25–27 The size and activity of endothelium-derived VWF are regulated by a plasma metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, 13).28–30 Deficiency of plasma ADAMTS13 activity, resulting from ADAMTS13 mutations or autoantibodies against ADAMTS13, results in a potentially fatal blood disorder called thrombotic thrombocytopenic purpura (TTP).27,31,32

Several recent studies have demonstrated significantly elevated levels of plasma VWF with a concomitant but modest reduction of plasma ADAMTS13 activity in patients with COVID-19.33–36 An increased ratio of VWF to ADAMTS13 may result in further endothelial damage and is associated with an increased mortality rate.37,38 Thus, therapeutic plasma exchange that removes the excessive ultra-large VWF while supplementing plasma ADAMTS13 has been shown to reduce the mortality in patients with severe/critical COVID-19.39

VWF, platelets, and neutrophil interactions appear to be required for NETs and thrombus formation in several other inflammatory/thrombotic diseases.40–42 Thus, therapeutic targeting at VWF–platelet or platelet–neutrophil interaction may prevent NETs and thrombus formation. For instance, aspirin or clopidogrel that inhibits platelet function has been shown to be efficacious in treatment of COVID-19-associated thrombosis.43,44 With that, we hypothesize that either recombinant ADAMTS13 that cleaves ultra-large VWF or caplacizumab that blocks VWF–platelet interaction may inhibit the NET accumulation and thrombus formation in patients with severe/critical COVID-19.

To test this hypothesis, we first assessed the status of suicidal neutrophil NETosis in vivo and the reserve of neutrophil NETosis (or vital NETosis) that is inducible by an agonist using flow cytometry and a microfluidic system under arterial flow ex vivo in patients with SARS-CoV-2 infection and asymptomatic or healthy controls. We then determined the role of recombinant ADAMTS13 or caplacizumab (an anti-VWF nanobody) on NETs accumulation and thrombus formations under flow in these patient blood samples. Our results demonstrate significantly elevated levels of neutrophil NETosis in vivo and a reduced NETosis reserve (or vital NETosis) and enhanced NET and thrombus formation under arterial flow in patients with severe/critical COVID-19. Like DNase I, recombinant ADAMTS13 or caplacizumab prevents the NETs accumulation and thrombus formation under arterial flow, likely via the blockage of VWF–platelet–neutrophil interactions. Our findings suggest a potentially novel therapeutic strategy for macro/microvascular thrombosis associated with severe/critical COVID-19 or other inflammatory/thrombotic disorders.

Methods

Patients

The Institutional Review Board at the University of Kansas Medical Center has approved the study protocol (#00148313). From January to March 2022, 124 consecutive patients admitted to our hospital with a positive SARS-CoV-2 test were prospectively enrolled into the study. The residual blood samples following complete blood count (ethylenediaminetetraacetic acid [EDTA]-anticoagulated) and/or coagulation tests (sodium citrate-anticoagulated) were collected for this study. Of those, 76 buffy coat samples (primarily leukocytes and platelets) were processed for the analysis of NETosis in vivo by flow cytometry, and 112 plasma samples were collected and cryopreserved (at −80 °C) for assessing plasma levels of NETs markers (e.g., histone–DNA complexes and cfDNA). Additionally, whole blood samples (sodium citrate-anticoagulated) (n = 20) were processed for assessing the formation of NETs and thrombosis under flow using a microfluidic assay.

All patients enrolled into the study were classified according to the World Health Organization (WHO) guidelines into: (1) asymptomatic, those who were hospitalized for reasons other than COVID-19 but happened to have a positive SARS-CoV-2 PCR test (patient controls); (2) mild to moderate, those who did not require oxygenation; (3) severe, those who required oxygenation, and (4) critical, those who exhibited multiple organ failure and/or required mechanical ventilation (https://www.mhcluster.org/WHO-2019-nCoV-therapeutics-2021.2-eng.pdf).

Patient Sampling and Data Collection

On the days of admission (Day 1) and 3 days (Day 4) following admission with a standard of care treatment, the residual blood samples were collected and processed for various experiments as described in the previous section. All samples were collected during Omicron outbreak between January 2022 and March 2022. Patients’ demographic, clinical, and routine laboratory data were extracted from electronic medical records. All patients were followed for up to 60 days to assess their long-term outcomes. The patients’ information includes age, gender, comorbidities (e.g., hypertension, hyperlipidemia, diabetes, cardiovascular disease, history of malignancy, history of thrombosis, etc.), thrombosis, ventilator usage, and mortality. Laboratory data include white blood cell count, neutrophil count, lymphocyte count, platelet count, albumin, C-reactive protein, lactate dehydrogenase, prothrombin time, activated thromboplastin time, D-dimer, and creatinine, etc.

Recombinant ADAMTS13

Recombinant human full-length ADAMTS13 protein was expressed in a stably transfected HEK-293 cell line and purified with a combination of ion exchange and affinity Ni-NTA chromatography as described previously.45 The sodium dodecyl sulfate–polyacrylamide gel electrophoresis plus Coomassie blue staining determined the purity and integrity of the purified recombinant human ADAMTS13 protein.

Caplacizumab

An anti-VWF nanobody, caplacizumab, is approved by the Food and Drug Administration for treatment of immune TTP,46,47 a potentially fatal hematological disorder, resulting from severe ADAMTS13 deficiency. Caplacizumab was pur-chased from Sanofi (Bridgewater, New Jersey, United States) through our hospital pharmacy department.

NETosis and Its Reserve by Flow Cytometry

EDTA-anticoagulated whole blood was centrifuged at 500× g for 10 minutes to obtain the buffy coat containing leukocytes and platelets. Following wash with a phosphate-buffered saline (PBS) containing 2% bovine serum albumin, the cells were either stimulated or not with a polyinosinic polycytidylic acid (poly [I:C]) at 5 μg/mL (Invitrogen, Waltham, Massachusetts, United States) for 3 hours or with bacterial Shigatoxin-2 at 100 ng/mL for 15 minutes to trigger further NETosis (i.e., the NETosis reserve or vital NETosis). The cells were then fixed with 2% paraformaldehyde in PBS and stained with anti-CD15-eFluor450 (Invitrogen, Waltham, Massachusetts, United States) for neutrophils, a rabbit anti-citrullinated histone H3 (Abcam, Boston, Massachusetts, United States) and anti-MPO-PE antibody (Novus Biologicals, Centennial, Colorado, United States) followed by Alexa488 goat anti-rabbit antibody (Invitrogen)18. Samples were analyzed with a BD LSR II flow cytometer (Becton Dickinson, Franklin Lakes, New Jersey, United States).

Soluble NETs or NETs Fragments by ELISA

Plasma levels of NETs markers in patients were determined by an ELISA-based assay. Plasma levels of histone–DNA complexes were determined using the cell death detection Kit (Sigma-Aldrich, St. Louis, Missouri, United States).48 Plasma levels of cfDNA were assessed using Quanti-iT PicoGreen dsDNA assay kit (AAT Bioquest, Pleasanton, California). Plasma VWF antigen levels were determined using our ELISA-based assay.49

Microfluidic Shear-Based Assay

Sodium citrate anticoagulated whole blood from patients with severe and critical COVID-19 were collected for these experiments. The anticoagulated whole bloods were perfused at 100 dyne/cm2 through microfluidic channels coated with type I fibrillar collagen (100 μg/mL) (Chrono-Log, Havertown, Pennsylvania, United States) in 0.01 M hydrochloride acid (HCl) as previously described.50,51 Platelets and neutrophils were first stained for 15 minutes with rhodamine 6G (40 μg/mL) (Sigma-Aldrich, St. Louis, Missouri, United States). The samples were treated without or with DNase I (200 IU/mL) (Genentech, South San Francisco, California, United States), or recombinant ADAMTS13 (6 μg/mL), or caplacizumab (3 μg/mL) (Sanofi, Paris, France) prior to perfusion. The accumulation of platelets and neutrophils (fluorescence signal) over time was observed in a real time and quantified surface area coverage using Montage software. The cells were further fixed with 2% paraformaldehyde in PBS, then extracellular DNA was stained with SytoxGreen (0.3 × 10−6 mol/L) (Invitrogen, Waltham, Massachusetts, United States), nuclei with Hoechst 33342 (10 μg/mL) (ThermoFisher Scientific, Waltham, Massachusetts, United States), and platelets with anti-CD41 (5 μg/mL) (BD Biosciences, Franklin Lakes, New Jersey, United States) conjugated with APC. The microfluidic channels were imaged under a Nikon A1R confocal microscope and analyzed with the NIS-Elements Viewer 5.21 software (Melville, New York, United States).

Statistical Analysis

Student’s t-tests were used for differences between two groups for normally distributed data, and Mann–Whitney U tests were used for two group comparison of nonparametric data. One-way ANOVA tests were used for differences among three or more groups with normally distributed data and Kruskal–Wallis ANOVA tests were used for these groups with nonparametric data. Paired t-tests were used for changes in the same groups for normally distributed data, and Wilcoxon signed-rank tests were used for nonparametric data. Chi-square tests were used for differences in frequency. Statistical significance was set at p < 0.05. Data analysis and graphs were performed using GraphPad Prism 9 software (Boston, Massachusetts, United States). When the distribution of the data was not known, nonparametric statistical analysis was performed to determine the significance.

Results

Clinical Characteristics and Laboratory Findings of Patients with COVID-19 and Asymptomatic Controls

A total of 76 patients with a positive polymerase chain reaction (PCR) test for SARS-CoV-2 were assessed by flow cytometry for NETosis. These included 11 asymptomatic controls, 28 mild to moderate, 22 severe, and 15 critical patients. The demographic, clinical, and laboratory data of these patients on admission are shown in ►Table 1. As shown, no statistically significant difference was observed in age and gender among all four groups. No significant difference in the incident rate of cardiovascular diseases, malignancy, and history of thrombotic events, but significant differences between the critical group and other groups in diabetes mellitus, hypertension, and hyperlipidemia were detected. The use of ventilator and the mortality rate were significantly higher in critical (46.7%) than in other groups (0%). No patient was treated with extracorporeal membrane oxygenation. The rate of thrombosis in patients with critical disease (20%) was also significantly higher than that in patients with asymptomatic controls, or mild to moderate disease (0%).

Table 1.

The demographic, clinical, and laboratory characteristics of 76 patients with SARS-CoV-2 infections

Asymptomatic control (n = 11) Mild–moderate (n = 28) Severe (n = 22) Critical (n = 15) p-Value
Age, y, median (IQR) 61.0 (48.0–65.0) 64.5 (50.5–73.5) 62.0 (57.0–69.8) 71.0 (65.0–74.0) 0.124
Gender, male, n (%) 7 (63.6) 12 (42.9) 13 (59.1) 7 (46.7) 0.565
Comorbidities
 Hypertension, n (%) 1 (9.1) 14 (50.0)** 8 (36.4) 11 (73.3)** 0.008
 Hyperlipidemia, n (%) 0 (0) 8 (28.6) 4 (18.2) 7 (46.7)* 0.037
 Diabetes, n (%) 2 (18.2) 5 (17.9) 3 (13.6) 8 (53.3)* 0.041
 Cardiovascular disease, n (%) 2 (18.2) 4 (14.3) 1 (4.5) 3 (20.0) 0.455
 History of malignancy, n (%) 1 (9.1) 7 (25.0) 11 (50.0) 3 (20.0) 0.068
 History of thrombosis, n (%) 1 (9.1) 5 (17.9) 1 (4.5) 2 (13.3) 0.591
Thrombosis, n (%) 0 (0) 0 (0) 2 (9.1) 3 (20.0)* 0.047
Ventilator, n (%) 0 (0) 0 (0) 0 (0) 7 (46.7)**** <0.001
Mortality, n (%) 0 (0) 0 (0) 0 (0) 5 (33.3)**** <0.001
White blood cell, ×109/L 5.70 (4.85–11.15) 6.00 (3.89–9.55) 6.10 (3.73–10.95) 7.80 (5.40–8.70) 0.719
Neutrophil, ×109/L (4.1–7.7) 4.33 (2.91–10.32) 4.01 (2.70–7.06) 4.72 (2.70–6.95) 6.66 (4.66–7.52) 0.306
Lymphocyte, 109/L (1.0–4.8) 1.13 (0.80–1.37) 1.10 (0.81–1.43) 0.47 (0.33–0.76)**** 0.40 (0.29–0.72)**** <0.001
Platelet, 109/L 223 (178–271) 224 (178–266) 178 (88–278) 226 (179–295) 0.508
Albumin, g/dL 4.2 (3.8–4.4) 4.0 (3.4–4.2) 3.3 (3.0–3.6)*** 3.5 (3.0–3.8)**** 0.003
CRP, mg/dL (<1.0) NA 2.33 (1.17–18.70) 8.20 (4.75–21.19) 5.59 (3.59–38.81) 0.339
LDH, U/L (100–200) 160.5 (147.8–173.3) 233.5 (181.0–359.5) 363.0 (237.0–462.0) 348.5 (319.5–410.5) 0.080
PT, s (11.0–13.5) 13.1 (11.5–14.8) 14.3 (13.4–17.0) 15.0 (13.2–21.4) 16.8 (15.2–22.8) 0.283
APTT, s (20–37.5) 27.2 (27.2–27.2) 32.0 (31.0–33.5) 33.4 (31.8–41.0) 32.7 (30.9–37.8) 0.178
D-Dimer, μg/L (<500) NA 946 (456–1,363) 1,596 (1,091–3,129) 2,086 (1,258–13,841)** 0.008
Creatinine, mg/dL (0.46–1.09) 0.83 (0.67–1.05) 1.12 (0.90–2.46) 1.15 (0.83–1.69) 1.17 (0.70–1.70) 0.495
VWF:Ag, % (50–200) 140.5 (113.3–217.0) 135.0 (91.0–205.5) 273.0 (168.7–538.0)*** 420.0 (116.0–592.8)**** <0.001
Open in a new tab

Abbreviations: APTT, activated partial thromboplastin time; CRP, C-reactive protein; LDH, lactate dehydrogenase; n, number of patients; PT, prothrombin time; VWF:Ag, von Willebrand factor antigen.

Note: All data are expressed as median (interquartile range, IQR) unless specified otherwise.

*, **, ***, and ****

indicate p-value <0.05, <0.01, <0.005, and <0.001, compared with the asymptomatic controls. Normal range is included for each biomarker in parenthesis.

Laboratory findings on admission showed no significant difference in white blood cell counts, neutrophil counts, and platelet counts among all four groups, although lymphocyte counts were significantly lower in the critical group than in other groups. The levels of C-reactive protein, lactate dehydrogenase, prothrombin time, and activated thromboplastin time, and creatinine were not statistically different among all groups. Plasma levels of VWF antigen and plasma D-dimers were significantly higher in patients with the critical disease than those with mild-to-moderate disease or asymptomatic controls.

NETosis and Its Reserve in Patients with COVID-19 and Controls

Neutrophils undergo NETosis following SARS-CoV-2 infection.52–54 However, the extent of NETosis and its reserve (or vital NETosis) in patients with COVID-19 of different disease severities are not fully understood. Utilizing flow cytometric analysis, we were able to determine the percentage (%) of neutrophils undergoing NETosis on admission and its reserve or vital NETosis in response to external stimuli. All gating strategy, negative, and positive controls for detecting NETosis are shown in ►Supplementary Fig. S1 (available in the online version). Our results showed that very low percentage (median 2.8%, interquartile range [IQR]: 1.0–4.8%) of neutrophils was double positive for H3Cit and MPO, indicative of NETosis,18 in the whole blood from asymptomatic control patients (►Fig. 1A). The percentage of H3Cit + MPO+ neutrophils (median, IQR) was significantly increased in patients with severe disease (13.4%, 2.8–26.5%) (►Fig. 1B) (p < 0.05), but not significantly increased in patients with the critical disease (1.7%, 1.0–8.9%) compared with those in asymptomatic (2.8%, 1.0–4.8%) or mild-to-moderate disease (5.9%, 1.7–13.6%) (all p > 0.05) (►Fig. 1C). Following a challenge with Shigatoxin-2 (100 ng/mL) for 15 minutes, the percentage of H3Cit + MPO+ neutrophils (median, IQR) was significantly increased (23%, 10.1–42.6%) from the baseline in whole blood samples of the asymptomatic/mild-to-moderate patients (2.8%, 1.0–4.8%) (p < 0.01). However, only minimal to modest increase in the percentage of H3Cit + MPO+ neutrophils following Shiga-toxin-2 was observed in the whole blood from patients with severe (16.2%, 11.4–36.3%) (p > 0.05) or critical (11.2%, 8.8– 20.5%) disease (p > 0.05) (►Fig. 1D–F, and ►Fig. 1J). Similarly, an incubation of the whole blood with a poly [I:C] (5 μg/mL) for 3 hours resulted in a significantly increased percentage of H3Cit + MPO+ neutrophils in control patients (p < 0.05), but only minimally changed the percentage of H3Cit + MPO+ neutrophils in severe (p > 0.05) and critical (p > 0.05) patient samples (►Fig. 1G, I, and K). Longitudinal assessment showed that there was a trend toward reduction in NETosis in mild-to-severe disease, but paradoxically increased in the critical group, although the difference was not statistically significant (►Fig. 1L). Together, the results support that neutrophil NETosis is significantly increased in patients with severe COVID-19, and the neutrophils in patients with severe and critical diseases appear to show a significantly reduced NETosis reserve.

Fig. 1.

Fig. 1

Open in a new tab

Flow cytometric detection of NETosis in the whole blood from patients with COVID-19. (A–C) The cell gating counting profiles and percentage of H3Cit + MPO+ neutrophils (Q2) in unstimulated samples from asymptomatic controls, patients with severe COVID-19, and patients with critical COVID-19, respectively. (D–F) The gating profile and the percentage of H3Cit + MPO+ neutrophils (Q2) in Shiga toxin 2-treated blood sample from asymptomatic controls, patients with severe, and critical COVID-19, respectively. (G–I) The gating profile and the percentage of H3Cit + MPO+ neutrophils in poly [I:C]-treated blood samples from control, severe, and critical patients, respectively. (J, K) The effects of shigatoxin-2 and poly [I:C], respectively, on the percentage of H3Cit + MPO+ neutrophils in COVID-19 patients of various disease severities. (L) Longitudinal change (Day 1 vs. Day 4) of the percentage of H3Cit + MPO+ neutrophils in COVID-19 patients of various disease severities. Kruskal–Willis test was performed to determine the difference of values on Day 1 among four different groups, but a Wilcoxon signed-rank test was performed to determine difference in the values of two different days or two groups of the same samples before and after treatment with shigatoxin-2 (Stx2) or Poly [I:C]. The data shown are individual values, median (bar), and interquartile range (IQR). Here, Con, asymptomatic control; MM, mild–moderate; MPO, myeloperoxidase; n, the number of samples. Here, n.s., *, and ** indicate a p value >0.05, <0.05, and <0.01, respectively.

Plasma Soluble Nucleosomes in COVID-19 Patients and Controls

NETosis results in the release of neutrophil contents including histone–DNA complexes and cfDNA into plasma. 8,48,53 To confirm the NETosis did occur in vivo, we determined the soluble plasma levels of nucleosomes or NET fragments using ELISA-based methods as described.48,55 We showed that the admission plasma levels of histone–DNA complexes were markedly elevated in patients with a critical disease, but less so in those with moderate to severe diseases when compared with those in the asymptomatic controls or those with a mild-to-moderate disease (►Fig. 2A). Similarly, plasma levels of cfDNA were dramatically elevated in patients with critical disease, but to a lesser degree in patients with mild and moderate diseases, compared with the levels in the asymptomatic controls (►Fig. 2B). Longitudinal studies demonstrated that the plasma levels of histone/DNA complexes (►Fig. 2C–E) or cfDNA (►Fig. 2F–H) on 3 days following therapy were not dramatically altered in all patient groups compared with the levels on admission, suggesting that current standard of care treatment does not appear to halt neutrophil NETosis, tissue damage, and eliminate the release of soluble nucleosomes or NET fragments.

Fig. 2.

Fig. 2

Open in a new tab

Plasma levels of NETs in patients with COVID-19 of various disease severities and with or without treatment. (A, B) Plasma levels of histone–DNA complexes and cell-free DNA, respectively, in patients with various disease severities on admission (Day 1) and 3 days following therapy (Day 4). (C–E) Longitudinal changes of plasma levels of histone/DNA complexes in each individual patient with mild to moderate, severe, and critical disease, respectively, from admission Day 1 to day 3 following therapy (Day 4). (F–H) Longitudinal changes of plasma levels of cell-free DNA in each individual patient with mild to moderate, severe, and critical disease, respectively, from admission Day 1 to day 3 following therapy (Day 4). The data in A and B are shown as individual values, median (bar) and interquartile range (IQR). Panels C, D, E, F, G, and H are shown as the individual levels on admission Day 1 and 3 days after therapy (Day 4) with the line indicating the trend of change. Kruskal–Wallis one-way test was determined to assess the significance of the differences among multiple groups in panels A and B, but a paired Student’s t-test was used to determine the difference in longitudinal change of the same patient. Here, MM, mild to moderate; n.s., not significant; n, number of samples tested, *, **, ***, and **** indicate p < 0.05, <0.01, <0.005, and <0.001, respectively.

Recombinant ADAMTS13 or Caplacizumab Also Inhibits NETs Accumulation in COVID-19 Patient’s Blood under Arterial Flow

NETs may play a crucial role in venous and arterial thromboses,56–60 thus, eliminating NETs is expected to inhibit thrombosis in patients with severe/critical COVID-19. To test this hypothesis, we developed a microfluidic shear assay for assessing ex vivo NETs and thrombus formation under arterial shear as previously described.17,51 We showed that when a whole blood sample from patients with severe/critical COVID-19 was incubated with DNase I (200 IU/mL) and then perfused over a fibrillar collagen-coated surface at arterial shear (100 dyne/cm2), the rate of thrombus formation as indicated by the accumulation of rhodamine 6G-labeled platelets and leukocytes was significantly reduced as a function of time compared with that in a buffer-treated control (►Supplementary Fig. S2A–C (available in the online version), and ►Supplementary Video S1). At the completion of the real-time imaging, the channels were washed and fixed with paraformaldehyde and stained with anti-CD41 and SytoxGreen for platelets and extracellular DNA, respectively. Under a confocal fluorescence microscope, we observed a significantly reduced number of extracellular DNA strings, surface coverage of platelets, and leukocytes in the presence of DNase I compared with those in the buffer-treated controls (►Supplementary Fig. S2D–F, available in the online version). These results indicate that the removal of NETs by DNase I alone may be sufficient to inhibit the thrombus formation under arterial flow in the whole blood from patients with severe and critical COVID-19.

VWF and platelet interaction are also shown to play an important role in NETs formation.40–42 Additionally, the released NETs may bind to VWF and provide a scaffold for adhesion of activated platelets and red blood cells (RBCs).60 To determine if recombinant ADAMTS13 that cleaves ultra-large VWF or caplacizumab that blocks platelet–VWF inter-action would inhibit the accumulation of NETs under flow, we incubated a buffer (control) or a recombinant ADAMTS13 (6 μg/mL) or caplacizumab (3 μg/mL) with a citrate-anti-coagulated whole blood obtained from severe/critical COVID-19 patients for 15 minutes and then perfused under arterial shear over a fibrillar collagen-coated surface at arterial shear (100 dyne/cm2). The results showed that like DNase I, either recombinant ADAMTS13 (►Fig. 3A, B, and ►Supplementary Video S2) or caplacizumab (►Fig. 4A, B, and ►Supplementary Video S3) could dramatically reduce the thrombus formation under flow; following a fixation, the platelets, extracellular DNA, and nuclei of neutrophils were stained with APC-conjugated antiplatelet CD41, SytoxGreen, and Hoechst 33342, respectively, and visualized under a confocal microscope. The platelet/neutrophil aggregates and extracellular DNA strings were only observed in the channels that were treated with a buffer, but not with recombinant ADAMTS13 (►Fig. 3C–E) or caplacizumab (►Fig. 4C–E). These results indicate that recombinant ADAMTS13 or caplacizumab, as efficacious as the DNase I, may be therapeutic for COVID19-associated thrombosis by eliminating excessive thrombus formation and accumulation of NETs under (patho)physiological conditions.

Fig. 3.

Fig. 3

Open in a new tab

Recombinant ADAMTS13 eliminates the accumulation of NETs and thrombus formation under arterial flow. (A, B) Representative images of final platelet coverage and the rate of platelet adhesion and aggregation (mean and standard error of the mean, n = 3) onto the collagen-coated surfaces, respectively, under arterial shear (100 dynes/cm2) following perfusion of a whole blood from a patient with severe COVID-19 with or without recombinant ADAMTS13 (rA13). (C) Confocal fluorescence images of final platelets (purple), neutrophils (green), and extracellular DNA strings (elongated green) on the collagen-coated surface at the end of perfusion of whole blood samples with or without rA13 following fixation and re-staining. (D) 5× enlarged images illustrating aggregated platelets (purple), neutrophils (green), and extracellular DNA strings. (E) The number of extracellular DNA strings formed on the collagen surface shown in panel C. The data shown are mean and standard error of the mean (n = 4). Paired Student’s t-test determined the differences between two groups. Here, *p < 0.05.

Fig. 4.

Fig. 4

Open in a new tab

Caplacizumab also eliminates the accumulation of NETs and thrombus formation under arterial flow. (A, B) Representative images of final platelet coverage and the rate of platelet adhesion and aggregation (mean and standard error of the mean, n = 3) onto the collagen-coated surfaces, respectively, under arterial shear (100 dynes/cm2) following perfusion of a whole blood from a patient with severe COVID-19 with or without caplacizumab. (C) Confocal fluorescence images demonstrate the final coverage of platelets (purple), neutrophils (green), and extracellular DNA strings (elongated green) on the collagen-coated surface at the end of perfusion of a whole blood sample with or without rA13, following fixation and re-staining. (D) 5× enlarged images illustrate aggregated platelets (purple), neutrophils (green), and extracellular DNA strings. (E) The number of extracellular DNA strings formed on the collagen surface shown in panel C. The data shown are mean and standard error of the mean (n = 4). Paired Student’s t-test determined the differences between the two groups. Here, *p < 0.05.

Discussion

The present study demonstrates that in vivo neutrophil NETo-sis is significantly increased in patients with severe and critical COVID-19. Consequently, the NETosis reserve or inducible NETosis in the remaining neutrophils is significantly reduced in these patients. Most importantly, like DNase I, recombinant ADAMTS13 or caplacizumab appears to be highly efficacious in eliminating the excessive accumulation of NETs, platelet–leu-kocyte aggregation, and thrombus under arterial flow ex vivo in the whole blood of patient with severe/critical COVID-19. These results demonstrate the critical role of endothelial VWF and platelet–VWF interaction in the accumulation of NETs under flow, which provides scientific basis for further exploring the potential use of recombinant ADAMTS13 or caplacizumab in the management of COVID-19 or other infection-associated thrombosis to reduce mortality and long-term complications.

Neutrophils are the first line of defense of human innate immunity. They migrate to sites of infection from circulation to engulf and kill invasive pathogens like bacteria, fungi, and viruses.61 In response to an infection, neutrophils undergo a process known as NETosis, which releases their cytoplasmic and nuclear contents including HNPs,62,63 MPO,64–66 citrul-linated histone H3,15,18,49 and cfDNA,49,67 etc. Nucleosomes or soluble NET fragments are not only released upon an enhanced shear, but also directly contribute to the increase of shear, leading to stretching of VWF that enhances platelet adhesion and aggregation.68 NETs may act as a scaffold to form platelet and RBC thrombi, thus limiting the infectious agents by encapsulation.69,70 However, NETs may also result in potential tissue damage if excessive.58,59

We show that a significant number of neutrophils in patients with severe/critical COVID-19 patients undergo NETosis with many neutrophils being somewhat consumed. The remaining neutrophils in the whole blood lack the response toward an external challenge, such as shigatoxin-2 that activates the nitrogen oxide (NOX)-dependent pathway71,72 or poly [I:C] that activates the toll-like receptor (TLR)-3 pathway.73–75 A similar phenomenon of reduced NETosis reserve or inducible NETosis was reported in patients with sepsis in whom the neutrophils did not respond to phorbol 12-myristate 13-acetate stimulation15 and in patients with immune TTP in whom the inducible NETosis is significantly diminished during acute episode and restored following disease remission.76 Support for this novel hypothesis also comes from significantly elevated plasma levels of soluble NET fragments (e.g., histone–DNA complexes and cfDNA) in patients with severe and critical COVID-19, although cfDNA is found to be a less specific marker for NETs.

Please be mindful that there are some technical limitations measuring NETosis in vivo indicated by the double positivity of MPO and citrullinated histone by flow cytometry, because NETs are fragile and appear to exhibit three-dimensional structures with heterogenous compositions and complex structures. The plasma histone–DNA complexes may also derive from other cells other than neutrophils. However, when comparing flow cytometric data with ELISA data, the formation of DNA strings in microfluidic channels from stimulated neutrophils can be rapidly removed by DNase I, suggesting that flow cytometric assay is suitable to quantify the extend of NETosis in vivo.

NETs may not only provide a scaffold to recruit RBC, platelets, and leukocytes, but also bind plasma proteins such as VWF, fibronectin, and fibrinogen60,77 to encapsulate the invaded microorganisms or cause thrombosis and result in vascular endothelial injury if excessive.17,78 NETs are found in thrombi of patients with venous or arterial thrombosis by immunohistochemistry.56 NETs can be rapidly degraded by plasma DNase I, which is found to be significantly reduced in many patients with immune inflammatory disorders, such as COVID-19, immune TTP, and other autoimmune diseases.79–82 Using the microfluidic assay, we found that the accumulation of NETs and thrombus formation are dramatically increased in patients’ whole blood of severe/critical COVID-19. As expected, an addition of exogenous DNase I to the whole blood eliminates the NETs accumulation and dramatically reduces adhesion and aggregation of platelets and leukocytes. Animal studies showed that DNase I decreases detectable plasma levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice.83 Additionally, an intravenous infusion of recombinant DNase I mitigates peri-bronchiolar, perivascular, and interstitial inflammation, ARDS, induced by poly [I:C].79 A recent pilot clinical trial has also demonstrated its therapeutic efficacy of long-acting nanoparticle DNase I in COVID-19 patients, leading to reduction of plasma levels of cfDNA and neutrophil activities.84

VWF and platelets are shown to play a critical role in neutrophil recruitment and activation, as well as NETs formation. We therefore tested if recombinant ADAMTS13 that cleaves ultra-large VWF under shear27 or caplacizumab that blocks VWF–platelet interaction46,47 would reduce an accumulation of NETs and thrombus formation in COVID-19 blood samples. As efficacious as DNase I, recombinant ADAMTS13 or caplacizumab dramatically reduces the accumulation of NETs and thrombus formation on collagen surface under arterial flow. It is known that neutrophil adhesion to endothelial surface requires the participation of VWF and platelets. In the absence of platelets, no neutrophil would adhere to endothelial surface and generate NETs. The nucleosome (DNA/histone complexes) may also directly interact with VWF strings, which further enhances platelet adhesion and aggregation and thrombus formation (►Fig. 5). Our findings support the potential therapeutic usage of recombinant ADAMTS13 or caplacizumab for the treatment of macro/micro-vascular thrombosis associated with severe/critical COVID-19 and perhaps other inflammatory thrombotic disorders.

Fig. 5.

Fig. 5

Open in a new tab

A proposed model of action for DNase I, recombinant ADAMTS13, and caplacizumab to inhibit the accumulation of NETs and thrombus formation. (A) Under flow, when ultra-large VWF multimers are released from activated vascular endothelial cells (EC), they are anchored on the endothelial surface and capable of capturing circulating platelets (PLT) and neutrophils (N). The neutrophils undergo NETosis, resulting in the release of histone–DNA and histone–MPO complexes (NETs). These NETs can bind to VWF multimers and activate platelets to enhance thrombus formation. (B) DNase I that cleaves extracellular DNA strings, recombinant ADAMTS13 that cleaves VWF strings, and caplacizumab that blocks platelet–VWF interaction can all efficiently inhibit the accumulation of NETs and thrombus formation under flow. This results in reduction of acute inflammation and thrombosis in patients with severe/critical COVID-19.

We conclude that flow cytometry plus shigatoxin-2 or poly [I:C] stimulation is a useful tool for assessing the extent and reserve of neutrophil NETosis in vivo in patients with COVID-19. The increased NETosis with a concomitant decrease of NETosis reserve or inducible NETosis by an external stimulator is associated with COVID-19 disease severity. Most importantly, recombinant ADAMTS13 or caplacizumab, as efficacious as DNase I, may be therapeutic for COVID-19-associated coagulopathy by eliminating excessive accumulation of NETs on an injured vascular endothelial surface, thus reducing macro/micro-vascular thrombosis in these patients.

Supplementary Material

Figure S1
Figure S2
Video S1

Supplementary Video S1 DNase I (top) dramatically reduces the rate of platelet and neutrophil adhesion and aggregation on type 1 fibrillar collagen surface under arterial flow (100 dyne/cm2) compared with the buffer control (bottom).

Download video file (6.6MB, mov)
Video S2

Supplementary Video S2 Recombinant ADAMTS13 (top) also dramatically reduces the rate of platelet and neutrophil adhesion and aggregation on type 1 fibrillar collagen surface under arterial flow (100 dyne/cm2) compared with the buffer control (bottom).

Download video file (6.5MB, mov)
Video S3

Supplementary Video S3 Caplacizumab (top) also dramatically reduces the rate of platelet and neutrophil adhesion and aggregation on type 1 fibrillar collagen surface under arterial flow(100 dyne/cm2) compared with the buffer control (bottom).

Download video file (6.5MB, mov)

What is known about this topic?

  • Neutrophil NETosis is involved in pathogenesis of COVID-19-associated coagulopathy.

  • However, the extents and reserve of NETosis in vivo and thrombus formation under shear in patients with SARS-CoV-2 infection are not fully understood.

  • Neither has the role of recombinant ADAMTS13 or caplacizumab in the NET accumulation under flow been investigated.

What does this paper add?

  • In vivo NETosis and ex vivo NET accumulation under floware significantly increased in patients with severe and critical COVID-19.

  • Like DNase I, recombinant ADAMTS13 or caplacizumab effectively eliminates the accumulation of NETs formed in the whole blood of COVID-19 patients under flow.

  • Our findings may help design a potential therapeutic strategy for severe COVID-19-associated coagulopathy.

Funding

The study was supported in part by grants from NHLBI (HL144552 and HL157975-01A1) to X.L.Z.

Footnotes

Conflict of Interest

X.L.Z. is a consultant for Alexion, Sanofi, Takeda, Apollo, GC Biopharma, and Stago. X.L.Z. is also the co-founder of Clotsolution. All other authors have declared no relevant conflict.

References

  • 1.Middeldorp S, Coppens M, van Haaps TF, et al. Incidence of venous thromboembolism in hospitalized patients with COVID-19. J Thromb Haemost 2020;18(08):1995–2002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Porfidia A, Pola R. Venous thromboembolism in COVID-19 patients. J Thromb Haemost 2020;18(06):1516–1517 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Iba T, Levy JH, Levi M, Thachil J. Coagulopathy in COVID-19. J Thromb Haemost 2020;18(09):2103–2109 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hanif A, Khan S, Mantri N, et al. Thrombotic complications and anticoagulation in COVID-19 pneumonia: a New York City hospital experience. Ann Hematol 2020;99(10):2323–2328 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Tamayo-Velasco Á, Bombín-Canal C, Cebeira MJ, et al. Full characterization of thrombotic events in all hospitalized COVID-19 patients in a Spanish tertiary hospital during the first 18 months of the pandemic. J Clin Med 2022;11(12):11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Tang N, Li D, Wang X, Sun Z. Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia. J Thromb Haemost 2020;18(04):844–847 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Kaur S, Bansal R, Kollimuttathuillam S, et al. The looming storm: blood and cytokines in COVID-19. Blood Rev 2021;46:100743 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Zhu Y, Chen X, Liu X. NETosis and neutrophil extracellular traps in COVID-19: immunothrombosis and beyond. Front Immunol 2022;13:838011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Wibowo A, Pranata R, Lim MA, Akbara MR, Martha JW. Endotheliopathy marked by high von Willebrand factor (vWF) antigen in COVID-19 is associated with poor outcome: a systematic review and meta-analysis. Int J Infect Dis 2022;117:267–273 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Branzk N, Papayannopoulos V. Molecular mechanisms regulating NETosis in infection and disease. Semin Immunopathol 2013;35 (04):513–530 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Zawrotniak M, Rapala-Kozik M. Neutrophil extracellular traps (NETs) - formation and implications. Acta Biochim Pol 2013;60 (03):277–284 [PubMed] [Google Scholar]
  • 12.Almyroudis NG, Grimm MJ, Davidson BA, Röhm M, Urban CF, Segal BH. NETosis and NADPH oxidase: at the intersection of host defense, inflammation, and injury. Front Immunol 2013;4:45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Middleton EA, He XY, Denorme F, et al. Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome. Blood 2020;136(10):1169–1179 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Gavillet M, Martinod K, Renella R, et al. Flow cytometric assay for direct quantification of neutrophil extracellular traps in blood samples. Am J Hematol 2015;90(12):1155–1158 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Lee KH, Cavanaugh L, Leung H, et al. Quantification of NETs-associated markers by flow cytometry and serum assays in patients with thrombosis and sepsis. Int J Lab Hematol 2018;40 (04):392–399 [DOI] [PubMed] [Google Scholar]
  • 16.Zharkova O, Tay SH, Lee HY, et al. A flow cytometry-based assay for high-throughput detection and quantification of neutrophil extracellular traps in mixed cell populations. Cytometry A 2019; 95(03):268–278 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Gollomp K, Kim M, Johnston I, et al. Neutrophil accumulation and NET release contribute to thrombosis in HIT. JCI Insight 2018;3 (18):3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Perdomo J, Leung HHL, Ahmadi Z, et al. Neutrophil activation and NETosis are the major drivers of thrombosis in heparin-induced thrombocytopenia. Nat Commun 2019;10(01):1322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Fogarty H, Townsend L, Morrin H, et al. ; Irish COVID-19 Vasculopathy Study (iCVS) investigators. Persistent endotheliopathy in the pathogenesis of long COVID syndrome. J Thromb Haemost 2021;19(10):2546–2553 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Fogarty H, Ward SE, Townsend L, et al. ; Irish COVID-19 Vasculopathy Study (iCVS) Investigators. Sustained VWF-ADAMTS-13 axis imbalance and endotheliopathy in long COVID syndrome is related to immune dysfunction. J Thromb Haemost 2022;20 (10):2429–2438 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Ladikou EE, Sivaloganathan H, Milne KM, et al. Von Willebrand factor (vWF): marker of endothelial damage and thrombotic risk in COVID-19? Clin Med (Lond) 2020;20(05):e178–e182 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Rambaldi A, Gritti G, Micò MC, et al. Endothelial injury and thrombotic microangiopathy in COVID-19: Treatment with the lectin-pathway inhibitor narsoplimab. Immunobiology 2020;225 (06):152001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Turecek PL, Peck RC, Rangarajan S, et al. Recombinant ADAMTS13 reduces abnormally up-regulated von Willebrand factor in plasma from patients with severe COVID-19. Thromb Res 2021; 201:100–112 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Zhang D, Li L, Chen Y, et al. Syndecan-1, an indicator of endothelial glycocalyx degradation, predicts outcome of patients admitted to an ICU with COVID-19. Mol Med 2021;27(01):151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Fernández-Pérez MP, Águila S, Reguilón-Gallego L, et al. Neutrophil extracellular traps and von Willebrand factor are allies that negatively influence COVID-19 outcomes. Clin Transl Med 2021; 11(01):e268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Yang J, Wu Z, Long Q, et al. Insights into immunothrombosis: the interplay among neutrophil extracellular trap, von Willebrand factor, and ADAMTS13. Front Immunol 2020;11:610696 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Zheng XL. ADAMTS13 and von Willebrand factor in thrombotic thrombocytopenic purpura. Annu Rev Med 2015;66:211–225 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Zheng X, Chung D, Takayama TK, Majerus EM, Sadler JE, Fujikawa K. Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura. J Biol Chem 2001;276(44):41059–41063 [DOI] [PubMed] [Google Scholar]
  • 29.Levy GG, Nichols WC, Lian EC, et al. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 2001;413(6855):488–494 [DOI] [PubMed] [Google Scholar]
  • 30.Dong JF, Moake JL, Nolasco L, et al. ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions. Blood 2002;100 (12):4033–4039 [DOI] [PubMed] [Google Scholar]
  • 31.Furlan M, Robles R, Galbusera M, et al. von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome. N Engl J Med 1998;339(22): 1578–1584 [DOI] [PubMed] [Google Scholar]
  • 32.Tsai HM, Lian EC. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med 1998;339(22):1585–1594 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Delrue M, Siguret V, Neuwirth M, et al. von Willebrand factor/ADAMTS13 axis and venous thromboembolism in moder-ate-to-severe COVID-19 patients. Br J Haematol 2021;192(06): 1097–1100 [DOI] [PubMed] [Google Scholar]
  • 34.Henry BM, Benoit SW, de Oliveira MHS, et al. ADAMTS13 activity to von Willebrand factor antigen ratio predicts acute kidney injury in patients with COVID-19: Evidence of SARS-CoV-2 induced secondary thrombotic microangiopathy. Int J Lab Hematol 2021;43(Suppl 1):129–136 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Rodríguez Rodríguez M, Castro Quismondo N, Zafra Torres D, Gil Alos D, Ayala R, Martinez-Lopez J. Increased von Willebrand factor antigen and low ADAMTS13 activity are related to poor prognosis in covid-19 patients. Int J Lab Hematol 2021;43(04): O152–O155 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Sweeney JM, Barouqa M, Krause GJ, Gonzalez-Lugo JD, Rahman S, Gil MR. Low ADAMTS13 activity correlates with increased mortality in COVID-19 patients. TH Open 2021;5(01):e89–e103 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Cotter AH, Yang ST, Shafi H, Cotter TM, Palmer-Toy DE. Elevated von Willebrand factor antigen is an early predictor of mortality and prolonged length of stay for coronavirus disease 2019 (COVID-19) inpatients. Arch Pathol Lab Med 2022;146(01):34–37 [DOI] [PubMed] [Google Scholar]
  • 38.Joly BS, Darmon M, Dekimpe C, et al. Imbalance of von Willebrand factor and ADAMTS13 axis is rather a biomarker of strong inflammation and endothelial damage than a cause of thrombotic process in critically ill COVID-19 patients. J Thromb Haemost 2021;19(09):2193–2198 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Ward SE, Fogarty H, Karampini E, et al. ; Irish COVID-19 Vasculopathy Study (iCVS) investigators. ADAMTS13 regulation of VWF multimer distribution in severe COVID-19. J Thromb Haemost 2021;19(08):1914–1921 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Andrews RK, Arthur JF, Gardiner EE. Neutrophil extracellular traps (NETs) and the role of platelets in infection. Thromb Haemost 2014;112:659–665 [DOI] [PubMed] [Google Scholar]
  • 41.von Brühl ML, Stark K, Steinhart A, et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med 2012;209(04):819–835 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Ma AC, Kubes P. Platelets, neutrophils, and neutrophil extracellular traps (NETs) in sepsis. J Thromb Haemost 2008;6(03):415–420 [DOI] [PubMed] [Google Scholar]
  • 43.Lal A, Gajic O. Response to aspirin therapy in COVID-19: prevention of NETosis. Arch Bronconeumol 2023;59(02):130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Arefizadeh R, Moosavi SH, Towfiqie S, Mohsenizadeh SA, Pishgahi M. Effect of ticagrelor compared to clopidogrel on short-term outcomes of COVID-19 patients with acute coronary syndrome undergoing percutaneous coronary intervention; a randomized clinical trial. Arch Acad Emerg Med 2023;11(01):e14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Xiao J, Jin SY, Xue J, Sorvillo N, Voorberg J, Zheng XL. Essential domains of a disintegrin and metalloprotease with thrombospon-din type 1 repeats-13 metalloprotease required for modulation of arterial thrombosis. Arterioscler Thromb Vasc Biol 2011;31(10): 2261–2269 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Peyvandi F, Callewaert F. Caplacizumab for acquired thrombotic thrombocytopenic purpura. N Engl J Med 2016;374(25):2497–2498 [DOI] [PubMed] [Google Scholar]
  • 47.Duggan S. Caplacizumab: first global approval. Drugs 2018;78 (15):1639–1642 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Sui J, Lu R, Halkidis K, et al. Plasma levels of S100A8/A9, histone/DNA complexes, and cell-free DNA predict adverse out-comes of immune thrombotic thrombocytopenic purpura. J Thromb Haemost 2021;19(02):370–379 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Kumar M, Cao W, McDaniel JK, et al. Plasma ADAMTS13 activity and von Willebrand factor antigen and activity in patients with sub-arachnoid haemorrhage. Thromb Haemost 2017;117(04):691–699 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Abdelgawwad MS, Cao W, Zheng L, Kocher NK, Williams LA, Zheng XL. Transfusion of platelets loaded with recombinant ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) is efficacious for inhibiting arterial thrombosis associated with thrombotic thrombocytopenic purpura. Arterioscler Thromb Vasc Biol 2018;38(11):2731–2743 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.McDaniel JK, Abdelgawwad MS, Hargett A, et al. Human neutrophil peptide-1 inhibits thrombus formation under arterial flow via its terminal free cysteine thiols. J Thromb Haemost 2019;17 (04):596–606 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Gillot C, Favresse J, Mullier F, Lecompte T, Dogné JM, Douxfils J. NETosis and the immune system in COVID-19: mechanisms and potential treatments. Front Pharmacol 2021;12:708302 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Morimont L, Dechamps M, David C, et al. NETosis and nucleosome biomarkers in septic shock and critical COVID-19 patients: an observational study. Biomolecules 2022;12(08):12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Behzadifard M, Soleimani M. NETosis and SARS-COV-2 infection related thrombosis: a narrative review. Thromb J 2022;20(01):13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.de Buhr N, von Köckritz-Blickwede M. Detection, visualization, and quantification of neutrophil extracellular traps (NETs) and NET markers. Methods Mol Biol 2020;2087:425–442 [DOI] [PubMed] [Google Scholar]
  • 56.Laridan E, Martinod K, De Meyer SF. Neutrophil extracellular traps in arterial and venous thrombosis. Semin Thromb Hemost 2019; 45(01):86–93 [DOI] [PubMed] [Google Scholar]
  • 57.Martinod K, Wagner DD. Thrombosis: tangled up in NETs. Blood 2014;123(18):2768–2776 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Brill A, Fuchs TA, Savchenko AS, et al. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost 2012;10(01):136–144 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Fuchs TA, Brill A, Wagner DD. Neutrophil extracellular trap (NET) impact on deep vein thrombosis. Arterioscler Thromb Vasc Biol 2012;32(08):1777–1783 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Fuchs TA, Brill A, Duerschmied D, et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A 2010;107(36): 15880–15885 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Segal AW. How neutrophils kill microbes. Annu Rev Immunol 2005;23:197–223 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Ganz T, Selsted ME, Szklarek D, et al. Defensins. Natural peptide antibiotics of human neutrophils. J Clin Invest 1985;76(04):1427–1435 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Lehrer RI, Lu W. α-Defensins in human innate immunity. Immunol Rev 2012;245(01):84–112 [DOI] [PubMed] [Google Scholar]
  • 64.Fuchs TA, Kremer Hovinga JA, Schatzberg D, Wagner DD, Lämmle B. Circulating DNA and myeloperoxidase indicate disease activity in patients with thrombotic microangiopathies. Blood 2012;120 (06):1157–1164 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Papayannopoulos V, Metzler KD, Hakkim A, Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J Cell Biol 2010;191(03):677–691 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Stapleton PP, Redmond HP, Bouchier-Hayes DJ. Myeloperoxidase (MPO) may mediate neutrophil adherence to the endothelium through upregulation of CD11B expression–an effect downregulated by taurine. Adv Exp Med Biol 1998;442:183–192 [DOI] [PubMed] [Google Scholar]
  • 67.Yalavarthi S, Gould TJ, Rao AN, et al. Release of neutrophil extracellular traps by neutrophils stimulated with antiphospholipid antibodies: a newly identified mechanism of thrombosis in the antiphospholipid syndrome. Arthritis Rheumatol 2015;67(11):2990–3003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Burmeister A, Vidal-Y-Sy S, Liu X, et al. Impact of neutrophil extracellular traps on fluid properties, blood flow and complement activation. Front Immunol 2022;13:1078891 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Ito T. PAMPs and DAMPs as triggers for DIC. J Intensive Care 2014;2 (01):67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Engelmann B, Massberg S. Thrombosis as an intravascular effector of innate immunity. Nat Rev Immunol 2013;13(01):34–45 [DOI] [PubMed] [Google Scholar]
  • 71.Feitz WJC, Suntharalingham S, Khan M, et al. Shiga toxin 2a induces NETosis via NOX-dependent pathway. Biomedicines 2021;9(12):9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72.Ramos MV, Mejias MP, Sabbione F, et al. Induction of neutrophil extracellular traps in shiga toxin-associated hemolytic uremic syndrome. J Innate Immun 2016;8(04):400–411 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Bunting RA, Duffy KE, Lamb RJ, et al. Novel antagonist antibody to TLR3 blocks poly(I:C)-induced inflammation in vivo and in vitro. Cell Immunol 2011;267(01):9–16 [DOI] [PubMed] [Google Scholar]
  • 74.Kannaki TR, Priyanka E, Abhilash M, Haunshi S. Co-administration of toll-like receptor (TLR)-3 agonist Poly I:C with different infectious bursal disease (IBD) vaccines improves IBD specific immune response in chicken. Vet Res Commun 2021;45(04): 285–292 [DOI] [PubMed] [Google Scholar]
  • 75.Stowell NC, Seideman J, Raymond HA, et al. Long-term activation of TLR3 by poly(I:C) induces inflammation and impairs lung function in mice. Respir Res 2009;10(01):43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 76.Yada N, Sui J, Zheng L, Zheng XL. Neutrophil extracellular traps (NETs) contribute to the formation of microvascular thrombosis in immune thrombotic thrombocytopenic purpura. Blood 2021; 138(Suppl. 1):1020 [Google Scholar]
  • 77.Gould TJ, Vu TT, Swystun LL, et al. Neutrophil extracellular traps promote thrombin generation through platelet-dependent and platelet-independent mechanisms. Arterioscler Thromb Vasc Biol 2014;34(09):1977–1984 [DOI] [PubMed] [Google Scholar]
  • 78.Schauer C, Janko C, Munoz LE, et al. Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines. Nat Med 2014;20(05):511–517 [DOI] [PubMed] [Google Scholar]
  • 79.Jiménez-Alcázar M, Rangaswamy C, Panda R, et al. Host DNases prevent vascular occlusion by neutrophil extracellular traps. Science 2017;358(6367):1202–1206 [DOI] [PubMed] [Google Scholar]
  • 80.Leffler J, Ciacma K, Gullstrand B, Bengtsson AA, Martin M, Blom AM. A subset of patients with systemic lupus erythematosus fails to degrade DNA from multiple clinically relevant sources. Arthritis Res Ther 2015;17(01):205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 81.Jiménez-Alcázar M, Napirei M, Panda R, et al. Impaired DNase1-mediated degradation of neutrophil extracellular traps is associated with acute thrombotic microangiopathies. J Thromb Hae-most 2015;13(05):732–742 [DOI] [PubMed] [Google Scholar]
  • 82.Katkar GD, Sundaram MS, NaveenKumar SK, et al. NETosis and lack of DNase activity are key factors in Echis carinatus venom-induced tissue destruction. Nat Commun 2016;7:11361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 83.Veras FP, Gomes GF, Silva BMS, et al. Targeting neutrophils extracellular traps (NETs) reduces multiple organ injury in a COVID-19 mouse model. Respir Res 2023;24(01):66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 84.Lee YY, Park HH, Park W, et al. Long-acting nanoparticulate DNase-1 for effective suppression of SARS-CoV-2-mediated neutrophil activities and cytokine storm. Biomaterials 2021; 267:120389 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1
NIHMS1986574-supplement-Figure_S1.png (2.8MB, png)
Figure S2
NIHMS1986574-supplement-Figure_S2.png (8.8MB, png)
Video S1

Supplementary Video S1 DNase I (top) dramatically reduces the rate of platelet and neutrophil adhesion and aggregation on type 1 fibrillar collagen surface under arterial flow (100 dyne/cm2) compared with the buffer control (bottom).

Download video file (6.6MB, mov)
Video S2

Supplementary Video S2 Recombinant ADAMTS13 (top) also dramatically reduces the rate of platelet and neutrophil adhesion and aggregation on type 1 fibrillar collagen surface under arterial flow (100 dyne/cm2) compared with the buffer control (bottom).

Download video file (6.5MB, mov)
Video S3

Supplementary Video S3 Caplacizumab (top) also dramatically reduces the rate of platelet and neutrophil adhesion and aggregation on type 1 fibrillar collagen surface under arterial flow(100 dyne/cm2) compared with the buffer control (bottom).

Download video file (6.5MB, mov)

RESOURCES