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Thromb Haemost 2009; 102(03): 520-528
DOI: 10.1160/TH09-03-0143
Platelets and Blood Cells
Schattauer GmbH

Integrin-dependent translocation of LASP-1 to the cytoskeleton of activated platelets correlates with LASP-1 phosphorylation at tyrosine 171 by Src-kinase

Julia Traenka
1   Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Wuerzburg, Germany
,
Christof R Hauck
2   Institute of Cell Biology, University of Konstanz, Konstanz, Germany
,
Urs Lewandrowski
3   Department of Bioanalytics, ISAS Institute for Analytical Sciences, Bunsen, Dortmund, Germany
,
Albert Sickmann
3   Department of Bioanalytics, ISAS Institute for Analytical Sciences, Bunsen, Dortmund, Germany
,
Stepan Gambaryan
1   Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Wuerzburg, Germany
4   Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Science, St. Petersburg, Russia
,
Petra Thalheimer
1   Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Wuerzburg, Germany
,
Elke Butt
1   Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Wuerzburg, Germany
› Author AffiliationsFinancial support: This investigation was supported by research grants 107706 from Deutsche Krebshilfe to E.B. and the Ministerium für Innovation, Wissenschaft, Forschung and Technology NRW and the BMBF to A.S.
Further Information

Publication History

Received: 03 March 2009

Accepted after major revision: 21 May 2009

Publication Date:
22 November 2017 (online)

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Summary

During platelet adhesion, the complex cytoskeletal structure is rearranged resulting in the formation of F-actin-based filopodia and lamellipodia. Stimulatory platelet signalling pathways include binding of integrin αIIbβ3 to fibrinogen followed by activation of protein tyrosine kinases (PTK) and phosphorylation of downstream signalling proteins. In this study, we demonstrate that the scaffolding and F-actin binding protein LASP-1 undergoes tyrosine phosphorylation in thrombin-stimulated human platelets. By means of specific inhibitors we identified Src-kinase as the primary enzyme phosphorylating LASP-1 in intact cells. These data were confirmed in platelet model cells (A5-CHO cells), constitutively expressing integrin αIIbβ3. Fibrinogen-mediated cell stimulation resulted in a similar tyrosine phosphorylation of transiently transfected LASP-1. Site directed mutagenesis identified tyrosine 171 as the Src-kinase phosphorylation site. Immunofluorescence microscopic analysis of these cells revealed a relocation of LASP-1 to focal contacts and the leading edge of the membrane upon fibrinogen activation and tyrosine 171 phosphorylation. This translocation was also seen in adherent platelets. Concomitant with adhesion, LASP-1 translocated from the cytosol along the arms of the pseudopodia into the leading lamellae of the spreading platelets, indicating a crucial role of the protein in platelet cytoskeleton rearrangement.

Keywords

LASP-1 - Src-kinase - adhesion - tyrosine phosphorylation - platelets
 

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