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Original research
An in vitro model for Extracellular DNA Traps (ETs)-rich Human Thrombus Analogs
  1. Yang Liu1,2,
  2. Daying Dai1,
  3. Mehdi Abbasi1,
  4. Oana Madalina Mereuta1,
  5. Scott I Gamb3,
  6. Ramanathan Kadirvel1,
  7. David F Kallmes1,
  8. Waleed Brinjikji1
  1. 1Radiology, Mayo Clinic, Rochester, Minnesota, USA
  2. 2Global Institute of Future Technology, Shanghai Jiao Tong University, Shanghai, China
  3. 3Electron Microscopy Core Facility, Mayo Clinic, Rochester, Minnesota, USA
  1. Correspondence to Dr Daying Dai, Radiology, Mayo Clinic, Rochester, Minnesota, USA; dai.daying{at}mayo.edu

Abstract

Background Extracellular DNA traps (ETs) have important implications in both thrombosis and thrombolysis. Thus, developing benchtop thrombus analogs that recapitulate clinical ETs is potentially of great value for preclinical development and testing of thrombolytic agents and thrombectomy devices. In this study, we aimed to develop ETs-rich thrombus analogs for preclinical testing.

Methods Red blood cell (RBC)-rich, fibrin-rich, and platelet-rich thrombus analogs were created using human whole blood, platelet-poor plasma, and platelet-rich plasma obtained from the blood bank following institutional approval. Peripheral blood mononuclear cells (9.9×106 cells/mL) isolated from human whole blood and lipopolysaccharide (1 µg/mL) were added to induce ETs. Histochemical, immunohistochemistry and immunofluorescence were used to identify thrombus components and ETs. Scanning electronic microscopy was used to investigate the ultrastructure of the thrombus analogs. The thrombus compositions, morphologic features of ETs and citrullinated histone H3 (H3Cit) expression were compared with those of thrombi retrieved from patients by thrombectomy.

Results ETs-rich thrombus analogs were more compacted th-an the ETs-poor thrombus analogs. ETs were identified in both ETs-rich thrombus analogs and patient thrombi showing morphologic features including nuclear lobulation, nuclear swelling, diffused chromatin within cytoplasm, DNA/chromatin extending intracellularly and extracellularly, and extracellular chromatin patches and bundles. In the ETs-poor thrombus analogs, ETs were not observed and H3Cit expression was absent to minimal. The compositions and H3Cit expression in the ETs-rich thrombus analogs fell in the range of patient thrombi.

Conclusions ETs-rich thrombus analogs can be consistently created in vitro and may benefit the preclinical development and testing of new thrombolytic agents and thrombectomy devices.

  • Thrombectomy
  • Thrombolysis
  • Stroke
  • Drug
  • Platelets

Data availability statement

All data relevant to the study are included in the article or uploaded as supplemental information.

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Data availability statement

All data relevant to the study are included in the article or uploaded as supplemental information.

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Footnotes

  • YL and DD contributed equally.

  • Contributors YL, DD, and WB conceived the concept and designed the study. YL and DD developed the thrombus model. DD, OMM, and MA performed histology. SIG performed SEM. YL and DD drafted the article. The article was critically revised by DFK and WB. RK, DFK, and WB provided administrative, technical, supervisory, or other support. All authors reviewed the submitted version, and YL approved it on behalf of all the authors.

  • Funding This work was supported by National Institutes of Health grant number NS105853.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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