Immune versus thrombotic stimulation of platelets differentially regulates signalling pathways, intracellular protein-protein interactions, and α-granule release

Sybille Rex; Lea M. Beaulieu; David H. Perlman; Olga Vitseva; Price S. Blair; Mark E. McComb; Catherine E. Costello; Jane E. Freedman

doi:10.1160/TH08-08-0513

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2009; 102(01): 97-110
DOI: 10.1160/TH08-08-0513

Platelets and Blood Cells

Schattauer GmbH

Immune versus thrombotic stimulation of platelets differentially regulates signalling pathways, intracellular protein-protein interactions, and α-granule release

Sybille Rex

¹Whitaker Cardiovascular Institute, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

²Cardiovascular Proteomics Center, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

,

Lea M. Beaulieu

¹Whitaker Cardiovascular Institute, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

,

David H. Perlman

²Cardiovascular Proteomics Center, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

,

Olga Vitseva

¹Whitaker Cardiovascular Institute, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

,

Price S. Blair

¹Whitaker Cardiovascular Institute, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

,

Mark E. McComb

²Cardiovascular Proteomics Center, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

,

Catherine E. Costello

²Cardiovascular Proteomics Center, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

³Mass Spectrometry Resource and Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts, USA

,

Jane E. Freedman

¹Whitaker Cardiovascular Institute, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

²Cardiovascular Proteomics Center, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

› Author Affiliations

Abstract

Summary

In addition to haemostasis, platelets mediate inflammation and clearance of bacteria from the bloodstream. As with platelet-platelet interactions, platelet-bacteria interactions involve cytoskeletal rearrangements and release of granular content. Stimulation of the immune Toll-like receptor 2 (TLR2) on the platelet surface, activates phosphoinositide-3-kinase (PI3K) and causes platelet activation and plateletdependent thrombosis. It remains unknown if platelet activation by immune versus thrombotic pathways leads to the differential regulation of signal transduction, protein-protein interactions, and α-granule release, and the physiological relevance of these potential differences. We investigated these processes after immune versus thrombotic platelet stimulation. We examined selected signalling pathways and found that phosphorylation kinetics of Akt, ERK1/2 and p38 differed dramatically between agonists. Next, we investigated platelet protein-protein interactions by mass spectrometry (MS)-based proteomics specifically targeting cytosolic factor XIIIa (FXIIIa) because of its function as a cytoskeleton-crosslinking protein whose binding partners have limited characterisation. Four FXIIIa-binding proteins were identified, two of which are novel interactions: FXIIIa-focal adhesion kinase (FAK) and FXIIIa-gelsolin. The binding of FAK to FXIIIa was found to be altered differentially by immune versus thrombotic stimulation. Lastly, we studied the effect of thrombin versus Pam₃CSK₄ stimulation on α-granule release and observed differential release patterns for selected granule proteins and decreased fibrin clot formation compared with thrombin. The inhibition of PI3K caused a decrease in protein release after Pam₃CSK₄ ^-but not after thrombin-stimulation. In summary, stimulation of platelets by either thrombotic or immune receptors leads to markedly different signalling responses and granular protein release consistent with differential contribution to coagulation and thrombosis.

Keywords

Toll-like receptor 2 - thrombin - signal transduction - mass spectrometry-based proteomics - factor XIIIA

Full Text

References

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