PB0379 - Functional expression of GFP-tagged integrin αIIb in a transgenic (knockin) mouse line

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Charly Kusch

PhD Student
University Hospital Würzburg, Germany
Würzburg, Bayern, Germany

Co-Author(s)

Denise Johnson, MSc

PhD student
Institute of Experimental Biomedicine I, University Hospital Würzburg, Germany and Rudolf Virchow Center for Integrative and Translational Biomaging University of Würzburg, Germany
Würzburg, Bayern, Germany
Philipp Burkard, MSc

PhD student
Institute of Experimental Biomedicine - Chair I, University Hospital Würzburg and Rudolf Virchow Center for Integrative and Translational Biomaging, University of Würzburg, Germany
Würzburg, Bayern, Germany
Patricia Öftering, MSc

PhD student
University Hospital Würzburg and Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Germany
Würzburg, Bayern, Germany
David Stegner, PhD

Professor
Institute of Experimental Biomedicine - Chair I, University Hospital and Rudolf Virchow Center for Integrative and Translational Bioimaging, Würzburg, Germany
Würzburg, Bayern, Germany
Sarah Beck, PhD

Postdoc
Institute of Experimental Biomedicine - Chair I, University Hospital and Rudolf Virchow Center for Integrative and Translational Bioimaging, Würzburg, Germany
Würzburg, Bayern, Germany
BN

Bernhard Nieswandt

Professor
Institute of Experimental Biomedicine - Chair I, University Hospital Würzburg and Rudolf Virchow Center for Integrative and Translational Biomaging, University of Würzburg, Germany
Würzburg, Bayern, Germany

Background: As heterodimeric adhesion molecules, integrins facilitate cell-cell and cell-matrix interactions. The dominant platelet integrin αIIbβ3 is a paradigmatic model to study integrin biology and is central in thrombosis, haemostasis, and inflammation. Dynamic visualisation of αIIbβ3 in vivo as well as in vitro is difficult as its ligation, e.g. by antibodies, rapidly alters its localisation (clustering and capping) and function.

Aims: To generate and analyze a mouse line expressing eGFP-tagged integrin αIIbβ3.

Methods: Generation and characterization of a mouse line expressing eGFP-tagged integrin αIIb (Itga2bGFP) allowing the dynamic visualization of αIIbβ3 in the absence of antibodies.

Results: It has previously been shown in heterologous systems (CHO cells) that tagging of the integrin αIIb-subunit with GFP is possible without impairing the integrin’s functionality (Buensuceso et al., JBC 2003). Following this strategy, we generated a mutant mouse line expressing eGFP fused to the intracellular tail of αIIb (Itga2bGFP). Flow cytometric analyses of Itga2bGFP platelets confirmed the expression of the αIIb-eGFP fusion protein. Notably, αIIbβ3 surface abundance was slightly reduced in platelets from homozygous mutant mice but unaltered in heterozygous platelets compared to wild-type controls. Surface expression of major platelet glycoproteins, as well as αIIbβ3 activation and P-selectin exposure in response to classical platelet agonists were indistinguishable between Itga2bGFP and control platelets. This was confirmed in aggregometry studies and by normal tail bleeding times of Itga2bGFP mice. Fluorescence microscopy confirmed that the eGFP signal allows to monitor αIIbβ3 in live platelets during spreading on fibrinogen in vitro and in intravital microscopy-based disease models.

Conclusion(s): Itga2bGFP mice express functional eGFP-tagged integrin αIIbβ3 and now enable studies on αIIbβ3 dynamics in platelets and megakaryocytes in vitro and in vivo.

This work was supported by TR240 grant with project number 374031971 and BE5084/5-1.