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Role of Focal Adhesion Tyrosine Kinases in GPVI-Dependent Platelet Activation and Reactive Oxygen Species Formation
BACKGROUND: We have previously shown the presence of a TRAF4/p47(phox)/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2,...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240642/ https://www.ncbi.nlm.nih.gov/pubmed/25415317 http://dx.doi.org/10.1371/journal.pone.0113679 |
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author | Carrim, Naadiya Walsh, Tony G. Consonni, Alessandra Torti, Mauro Berndt, Michael C. Metharom, Pat |
author_facet | Carrim, Naadiya Walsh, Tony G. Consonni, Alessandra Torti, Mauro Berndt, Michael C. Metharom, Pat |
author_sort | Carrim, Naadiya |
collection | PubMed |
description | BACKGROUND: We have previously shown the presence of a TRAF4/p47(phox)/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI. AIMS: To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway. METHODS AND RESULTS: Human and mouse washed platelets (from WT or Pyk2 KO mice) were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively) and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P) surface expression) and integrin α(IIb)β(3) activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk), PI3-K and Bruton's tyrosine kinase (Btk) and upstream of Rac1, PLCγ2, Ca(2+) release, PKC, Hic-5, NOX1 and α(IIb)β(3) activation. CONCLUSION: Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway. |
format | Online Article Text |
id | pubmed-4240642 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-42406422014-11-26 Role of Focal Adhesion Tyrosine Kinases in GPVI-Dependent Platelet Activation and Reactive Oxygen Species Formation Carrim, Naadiya Walsh, Tony G. Consonni, Alessandra Torti, Mauro Berndt, Michael C. Metharom, Pat PLoS One Research Article BACKGROUND: We have previously shown the presence of a TRAF4/p47(phox)/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI. AIMS: To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway. METHODS AND RESULTS: Human and mouse washed platelets (from WT or Pyk2 KO mice) were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively) and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P) surface expression) and integrin α(IIb)β(3) activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk), PI3-K and Bruton's tyrosine kinase (Btk) and upstream of Rac1, PLCγ2, Ca(2+) release, PKC, Hic-5, NOX1 and α(IIb)β(3) activation. CONCLUSION: Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway. Public Library of Science 2014-11-21 /pmc/articles/PMC4240642/ /pubmed/25415317 http://dx.doi.org/10.1371/journal.pone.0113679 Text en © 2014 Carrim et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Carrim, Naadiya Walsh, Tony G. Consonni, Alessandra Torti, Mauro Berndt, Michael C. Metharom, Pat Role of Focal Adhesion Tyrosine Kinases in GPVI-Dependent Platelet Activation and Reactive Oxygen Species Formation |
title | Role of Focal Adhesion Tyrosine Kinases in GPVI-Dependent Platelet Activation and Reactive Oxygen Species Formation |
title_full | Role of Focal Adhesion Tyrosine Kinases in GPVI-Dependent Platelet Activation and Reactive Oxygen Species Formation |
title_fullStr | Role of Focal Adhesion Tyrosine Kinases in GPVI-Dependent Platelet Activation and Reactive Oxygen Species Formation |
title_full_unstemmed | Role of Focal Adhesion Tyrosine Kinases in GPVI-Dependent Platelet Activation and Reactive Oxygen Species Formation |
title_short | Role of Focal Adhesion Tyrosine Kinases in GPVI-Dependent Platelet Activation and Reactive Oxygen Species Formation |
title_sort | role of focal adhesion tyrosine kinases in gpvi-dependent platelet activation and reactive oxygen species formation |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240642/ https://www.ncbi.nlm.nih.gov/pubmed/25415317 http://dx.doi.org/10.1371/journal.pone.0113679 |
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