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

Frontiers in Cardiovascular Medicine

Christian Weber¹  Constance C. F. M. J. Baaten²  Susanne M. de Witt³  Marijke J. E. Kuijpers³  Magdolna Nagy³  Johan W. M. Heemskerk³  Johanna P. van Geffen³  Remco Verdoold³  Alastair W. Poole⁴  Margitta Elvers⁵  Cécile Oury⁶  Yotis A. Senis⁷  Mitchell J. Geer⁷  Jun Mori⁷  Karl Kunzelmann⁸  Joachim Pircher⁹  Attila Braun^1,10  Irina Pleines^1,10  Bernhard Nieswandt^1,10  David Stegner^1,10  David J. Adams^1,11 

[1] 0Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-Universität München, Munich, Germany;1Institute for Molecular Cardiovascular Research (IMCAR), University Hospital Aachen, Aachen, Germany;Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, Netherlands;Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom;Department of Vascular Surgery, Experimental Vascular Medicine, Heinrich Heine University, Düsseldorf, Germany;GIGA-Cardiovascular Sciences, University of Liège, Liège, Belgium;Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom;Institute of Physiology, University of Regensburg, Regensburg, Germany;Medizinische Klinik und Poliklinik I, Klinikum der Universität München, Ludwig-Maximilians-University, and DZHK (German Center for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany;Rudolf Virchow Center, Institute of Experimental Biomedicine, University Hospital Würzburg, University of Würzburg, Würzburg, Germany;Wellcome Trust Sanger Institute, Cambridge, United Kingdom;

关键词: arterial thrombus formation;    bleeding;    collagen;    glycoprotein VI;    platelets;    microfluidics;   

DOI  :  10.3389/fcvm.2019.00099

来源: DOAJ

【 摘 要 】

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 α6β1 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.

【 授权许可】

Unknown