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Front. Cardiovasc. Med. | doi: 10.3389/fcvm.2019.00099

Comparative Analysis of Microfluidics Thrombus Formation in Multiple Genetically Modified Mice: Link to Thrombosis and Hemostasis

 Magdolna Nagy1, Johanna P. van Geffen1,  David Stegner2, 3, David J. Adams4, Attila Braun2, 3, Susanne M. de Witt1,  Margitta Elvers5,  Mitchell J. Geer6,  Marijke J. Kuijpers1, Karl Kunzelmann7,  Jun Mori6,  Cécile Oury8,  Joachim Pircher9, 10, Irina Pleines2, 3,  Alastair W. Poole11, Yotis A. Senis6, Remco Verdoold1,  Christian Weber1, 12, Bernhard Nieswandt2, 3, Johan W. Heemskerk1* and  Constance C. Baaten1, 13*
  • 1Cardiovascular Research Institute Maastricht, Maastricht University, Netherlands
  • 2Institute of Experimental Biomedicine, University Hospital of Würzburg, Germany
  • 3Rudolf Virchow Zentrum für experimentelle Biomedizin, Universität Würzburg, Germany
  • 4Wellcome Trust Sanger Institute (WT), United Kingdom
  • 5Heinrich Heine University of Düsseldorf, Germany
  • 6Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, United Kingdom
  • 7Institute of Physiology, University of Regensburg, Germany
  • 8GIGA Cardiovascular Sciences, University of Liège Hospital, Belgium
  • 9Medizinische Klinik und Poliklinik I, Klinikum der Universität München, Germany
  • 10Partner site Munich Heart Alliance,DZHK, (German Centre for Cardiovascular Research), Germany
  • 11School of Physiology, Pharmacology and Neuroscience, Faculty of Biomedical Sciences, University of Bristol, United Kingdom
  • 12Institute for Cardiovascular Prevention, Hospital of the University of Munich, Germany
  • 13Institut für Molekulare Herz-Kreislaufforschung (IMCAR), Germany

Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin 61 pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time.
In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis.

Keywords: Arterial thrombus formation, bleeding, Collagen, Glycoprotein VI, platelets, Microfluidics

Received: 06 May 2019; Accepted: 03 Jul 2019.

Edited by:

Paul Jurasz, University of Alberta, Canada

Reviewed by:

Daniel Duerschmied, Department of Cardiology and Angiology I, Heart Center, University of Freiburg, Germany
Plinio Cirillo, University of Naples Federico II, Italy  

Copyright: © 2019 Nagy, van Geffen, Stegner, Adams, Braun, de Witt, Elvers, Geer, Kuijpers, Kunzelmann, Mori, Oury, Pircher, Pleines, Poole, Senis, Verdoold, Weber, Nieswandt, Heemskerk and Baaten. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Prof. Johan W. Heemskerk, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, Netherlands,
PhD. Constance C. Baaten, Institut für Molekulare Herz-Kreislaufforschung (IMCAR), Aachen, Germany,