Limits...
G(12/13) signaling pathways substitute for integrin αIIbβ3-signaling for thromboxane generation in platelets.

Bhavaraju K, Lakhani PR, Dorsam RT, Jin J, Hitchcock IS, Sanjay A, Kunapuli SP - PLoS ONE (2011)

Bottom Line: This observation suggested an existence of a common signaling effector downstream of integrins and G(12/13) pathways.Selective activation of G(12/13) pathways resulted in the activation of FAK, in the absence of integrin signaling.Therefore, we conclude that differential activation of FAK occurs downstream of Integrins and G(12/13) pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: We have previously shown that ADP-induced TXA(2) generation requires signaling from αIIbβ3 integrin in platelets. Here we observed that, unlike ADP, protease-activated receptor (PAR)-mediated TXA(2) generation occurs independently of αIIbβ3. PAR agonists, but not ADP, activate G(12/13) signaling pathways. Hence, we evaluated the role of these pathways in TXA(2) generation.

Principal findings: Inhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by co-stimulation of G(12/13) pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G(12/13) pathways. Hence, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. Selective activation of G(12/13) pathways resulted in the activation of FAK, in the absence of integrin signaling. Interestingly, αIIbβ3-mediated FAK activation occurred in a Src family kinase (SFK)-independent manner whereas G(12/13) pathway caused FAK activation in a SFK and RhoA-dependent manner. A FAK selective inhibitor TAE-226, blocked TXA(2) generation. However, in comparison to WT mice, Pf4-Cre/Fak-Floxed mice did not show any difference in platelet TXA(2) generation.

Conclusions: Therefore, we conclude that differential activation of FAK occurs downstream of Integrins and G(12/13) pathways. However, the common effector molecule, possibly a tyrosine kinase downstream of integrins and G(12/13) pathways contributing to TXA(2) generation in platelets remains elusive.

Show MeSH

Related in: MedlinePlus

Focal Adhesion Kinase is activated downstream of integrins and G12/13 pathways.Aspirin treated, washed platelets were stimulated with 2MeSADP (100 nM) in presence or absence of reagents (as indicated) for 60 seconds under stirring conditions at 37°C (A). The lysates were then subjected to western blotting analysis and probed with anti- phospho- FAK (Y-397) and total FAK antibodies as lane loading control. The data are representative of mean ± S.E.M (n = 3). The data was analyzed by ANOVA and * P≤0.05 was considered significant (B). Aspirin-treated washed platelets were stimulated with AYPGKF (500 µM) in presence or absence of YM254890 (150 nM) (C), The lysates were then subjected to western blotting analysis and probed with anti- phospho- FAK (Y-397) and total FAK antibodies as lane loading control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3037367&req=5

pone-0016586-g003: Focal Adhesion Kinase is activated downstream of integrins and G12/13 pathways.Aspirin treated, washed platelets were stimulated with 2MeSADP (100 nM) in presence or absence of reagents (as indicated) for 60 seconds under stirring conditions at 37°C (A). The lysates were then subjected to western blotting analysis and probed with anti- phospho- FAK (Y-397) and total FAK antibodies as lane loading control. The data are representative of mean ± S.E.M (n = 3). The data was analyzed by ANOVA and * P≤0.05 was considered significant (B). Aspirin-treated washed platelets were stimulated with AYPGKF (500 µM) in presence or absence of YM254890 (150 nM) (C), The lysates were then subjected to western blotting analysis and probed with anti- phospho- FAK (Y-397) and total FAK antibodies as lane loading control.

Mentions: We next evaluated the activation of FAK by ADP receptors, using Y397 phosphorylation as an activation marker. FAK contains multiple tyrosine phosphorylation sites and the sequential tyrosine phosphorylations of these sites causes complete FAK activation beginning with autophosphorylation Y397 phosphorylation [34]. As shown in Figs. 3A & B, FAK is activated downstream of ADP receptors and this activation is blocked by a fibrinogen receptor antagonist but not by a pan SFK inhibitor PP2. These results indicate FAK activation by ADP occurs in an integrin-clustering-dependent manner, independent of SFKs.


G(12/13) signaling pathways substitute for integrin αIIbβ3-signaling for thromboxane generation in platelets.

Bhavaraju K, Lakhani PR, Dorsam RT, Jin J, Hitchcock IS, Sanjay A, Kunapuli SP - PLoS ONE (2011)

Focal Adhesion Kinase is activated downstream of integrins and G12/13 pathways.Aspirin treated, washed platelets were stimulated with 2MeSADP (100 nM) in presence or absence of reagents (as indicated) for 60 seconds under stirring conditions at 37°C (A). The lysates were then subjected to western blotting analysis and probed with anti- phospho- FAK (Y-397) and total FAK antibodies as lane loading control. The data are representative of mean ± S.E.M (n = 3). The data was analyzed by ANOVA and * P≤0.05 was considered significant (B). Aspirin-treated washed platelets were stimulated with AYPGKF (500 µM) in presence or absence of YM254890 (150 nM) (C), The lysates were then subjected to western blotting analysis and probed with anti- phospho- FAK (Y-397) and total FAK antibodies as lane loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037367&req=5

pone-0016586-g003: Focal Adhesion Kinase is activated downstream of integrins and G12/13 pathways.Aspirin treated, washed platelets were stimulated with 2MeSADP (100 nM) in presence or absence of reagents (as indicated) for 60 seconds under stirring conditions at 37°C (A). The lysates were then subjected to western blotting analysis and probed with anti- phospho- FAK (Y-397) and total FAK antibodies as lane loading control. The data are representative of mean ± S.E.M (n = 3). The data was analyzed by ANOVA and * P≤0.05 was considered significant (B). Aspirin-treated washed platelets were stimulated with AYPGKF (500 µM) in presence or absence of YM254890 (150 nM) (C), The lysates were then subjected to western blotting analysis and probed with anti- phospho- FAK (Y-397) and total FAK antibodies as lane loading control.
Mentions: We next evaluated the activation of FAK by ADP receptors, using Y397 phosphorylation as an activation marker. FAK contains multiple tyrosine phosphorylation sites and the sequential tyrosine phosphorylations of these sites causes complete FAK activation beginning with autophosphorylation Y397 phosphorylation [34]. As shown in Figs. 3A & B, FAK is activated downstream of ADP receptors and this activation is blocked by a fibrinogen receptor antagonist but not by a pan SFK inhibitor PP2. These results indicate FAK activation by ADP occurs in an integrin-clustering-dependent manner, independent of SFKs.

Bottom Line: This observation suggested an existence of a common signaling effector downstream of integrins and G(12/13) pathways.Selective activation of G(12/13) pathways resulted in the activation of FAK, in the absence of integrin signaling.Therefore, we conclude that differential activation of FAK occurs downstream of Integrins and G(12/13) pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: We have previously shown that ADP-induced TXA(2) generation requires signaling from αIIbβ3 integrin in platelets. Here we observed that, unlike ADP, protease-activated receptor (PAR)-mediated TXA(2) generation occurs independently of αIIbβ3. PAR agonists, but not ADP, activate G(12/13) signaling pathways. Hence, we evaluated the role of these pathways in TXA(2) generation.

Principal findings: Inhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by co-stimulation of G(12/13) pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G(12/13) pathways. Hence, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. Selective activation of G(12/13) pathways resulted in the activation of FAK, in the absence of integrin signaling. Interestingly, αIIbβ3-mediated FAK activation occurred in a Src family kinase (SFK)-independent manner whereas G(12/13) pathway caused FAK activation in a SFK and RhoA-dependent manner. A FAK selective inhibitor TAE-226, blocked TXA(2) generation. However, in comparison to WT mice, Pf4-Cre/Fak-Floxed mice did not show any difference in platelet TXA(2) generation.

Conclusions: Therefore, we conclude that differential activation of FAK occurs downstream of Integrins and G(12/13) pathways. However, the common effector molecule, possibly a tyrosine kinase downstream of integrins and G(12/13) pathways contributing to TXA(2) generation in platelets remains elusive.

Show MeSH
Related in: MedlinePlus