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

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 thrombus 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 - thrombosisAuthors' Contribution
N.Y., Q.Z., and X.L.Z. designed the study, analyzed the results, and wrote the manuscript. N.Y., A.B., and Q.Z. performed experiments and data analysis. All authors approved the final version of the manuscript for submission.
* Current affiliation: Department of General Medicine, Nara Medical University, Nara, Japan.
Publication History
Received: 01 September 2023
Accepted: 23 January 2024
Accepted Manuscript online:
25 January 2024
Article published online:
05 March 2024
© 2024. Thieme. All rights reserved.
Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany
-
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
- 2 Porfidia A, Pola R. Venous thromboembolism in COVID-19 patients. J Thromb Haemost 2020; 18 (06) 1516-1517
- 3 Iba T, Levy JH, Levi M, Thachil J. Coagulopathy in COVID-19. J Thromb Haemost 2020; 18 (09) 2103-2109
- 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
- 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
- 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
- 7 Kaur S, Bansal R, Kollimuttathuillam S. et al. The looming storm: blood and cytokines in COVID-19. Blood Rev 2021; 46: 100743
- 8 Zhu Y, Chen X, Liu X. NETosis and neutrophil extracellular traps in COVID-19: immunothrombosis and beyond. Front Immunol 2022; 13: 838011
- 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
- 10 Branzk N, Papayannopoulos V. Molecular mechanisms regulating NETosis in infection and disease. Semin Immunopathol 2013; 35 (04) 513-530
- 11 Zawrotniak M, Rapala-Kozik M. Neutrophil extracellular traps (NETs) - formation and implications. Acta Biochim Pol 2013; 60 (03) 277-284
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 27 Zheng XL. ADAMTS13 and von Willebrand factor in thrombotic thrombocytopenic purpura. Annu Rev Med 2015; 66: 211-225
- 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
- 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
- 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
- 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
- 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
- 33 Delrue M, Siguret V, Neuwirth M. et al. von Willebrand factor/ADAMTS13 axis and venous thromboembolism in moderate-to-severe COVID-19 patients. Br J Haematol 2021; 192 (06) 1097-1100
- 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. 01) 129-136
- 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
- 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
- 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
- 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
- 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
- 40 Andrews RK, Arthur JF, Gardiner EE. Neutrophil extracellular traps (NETs) and the role of platelets in infection. Thromb Haemost 2014; 112: 659-665
- 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
- 42 Ma AC, Kubes P. Platelets, neutrophils, and neutrophil extracellular traps (NETs) in sepsis. J Thromb Haemost 2008; 6 (03) 415-420
- 43 Lal A, Gajic O. Response to aspirin therapy in COVID-19: prevention of NETosis. Arch Bronconeumol 2023; 59 (02) 130
- 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
- 45 Xiao J, Jin SY, Xue J, Sorvillo N, Voorberg J, Zheng XL. Essential domains of a disintegrin and metalloprotease with thrombospondin type 1 repeats-13 metalloprotease required for modulation of arterial thrombosis. Arterioscler Thromb Vasc Biol 2011; 31 (10) 2261-2269
- 46 Peyvandi F, Callewaert F. Caplacizumab for acquired thrombotic thrombocytopenic purpura. N Engl J Med 2016; 374 (25) 2497-2498
- 47 Duggan S. Caplacizumab: first global approval. Drugs 2018; 78 (15) 1639-1642
- 48 Sui J, Lu R, Halkidis K. et al. Plasma levels of S100A8/A9, histone/DNA complexes, and cell-free DNA predict adverse outcomes of immune thrombotic thrombocytopenic purpura. J Thromb Haemost 2021; 19 (02) 370-379
- 49 Kumar M, Cao W, McDaniel JK. et al. Plasma ADAMTS13 activity and von Willebrand factor antigen and activity in patients with subarachnoid haemorrhage. Thromb Haemost 2017; 117 (04) 691-699
- 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
- 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
- 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
- 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
- 54 Behzadifard M, Soleimani M. NETosis and SARS-COV-2 infection related thrombosis: a narrative review. Thromb J 2022; 20 (01) 13
- 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
- 56 Laridan E, Martinod K, De Meyer SF. Neutrophil extracellular traps in arterial and venous thrombosis. Semin Thromb Hemost 2019; 45 (01) 86-93
- 57 Martinod K, Wagner DD. Thrombosis: tangled up in NETs. Blood 2014; 123 (18) 2768-2776
- 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
- 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
- 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
- 61 Segal AW. How neutrophils kill microbes. Annu Rev Immunol 2005; 23: 197-223
- 62 Ganz T, Selsted ME, Szklarek D. et al. Defensins. Natural peptide antibiotics of human neutrophils. J Clin Invest 1985; 76 (04) 1427-1435
- 63 Lehrer RI, Lu W. α-Defensins in human innate immunity. Immunol Rev 2012; 245 (01) 84-112
- 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
- 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
- 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
- 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
- 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
- 69 Ito T. PAMPs and DAMPs as triggers for DIC. J Intensive Care 2014; 2 (01) 67
- 70 Engelmann B, Massberg S. Thrombosis as an intravascular effector of innate immunity. Nat Rev Immunol 2013; 13 (01) 34-45
- 71 Feitz WJC, Suntharalingham S, Khan M. et al. Shiga toxin 2a induces NETosis via NOX-dependent pathway. Biomedicines 2021; 9 (12) 9
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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 Haemost 2015; 13 (05) 732-742
- 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
- 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
- 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