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

Evaluation of FAK as a common signaling effector molecule regulating thromboxane generation downstream of integrins and G12/13 pathways.Non-aspirin-treated, washed human platelets were pre-treated with varying concentrations of TAE-226 for 5 minutes at 37°C (A) and murine platelets from WT and Pf4-Cre/Fak-Floxed mice (B) were stimulated with 2MeSADP (100 nM) for 3.5 minutes and TXB2 levels were analyzed as described for Figure 1. Aggregation tracings were measured from WT and Pf4-Cre/Fak-Floxed mice and representative tracings are shown (C). The values are representative of 3 independent experiments mean ± S.E.M (n = 3). The data were analyzed by ANOVA and student t-test, * P≤0.05 was considered significant.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3037367&req=5

pone-0016586-g005: Evaluation of FAK as a common signaling effector molecule regulating thromboxane generation downstream of integrins and G12/13 pathways.Non-aspirin-treated, washed human platelets were pre-treated with varying concentrations of TAE-226 for 5 minutes at 37°C (A) and murine platelets from WT and Pf4-Cre/Fak-Floxed mice (B) were stimulated with 2MeSADP (100 nM) for 3.5 minutes and TXB2 levels were analyzed as described for Figure 1. Aggregation tracings were measured from WT and Pf4-Cre/Fak-Floxed mice and representative tracings are shown (C). The values are representative of 3 independent experiments mean ± S.E.M (n = 3). The data were analyzed by ANOVA and student t-test, * P≤0.05 was considered significant.

Mentions: We next evaluated whether activated FAK played a role in ADP-induced TXA2 generation. TAE-226 has recently been identified as a selective inhibitor of FAK with an IC50 of 5.5 nM [36]. To determine the effect of FAK inhibition on ADP-induced thromboxane generation platelets were treated with varying concentrations of TAE-226. As demonstrated in Fig. 5A, TXA2 generation was significantly inhibited by TAE-226 at a higher concentration of 2 µM. Pharmacological inhibitors are often known to have off target and broad-spectrum effects. The specificity of TAE-226 was never evaluated in platelets and reports suggest that TAE-226 inhibits Pyk2 (a Focal Adhesion kinase family member) with an IC50 of 5 nM [37]. Hence, we studied thromboxane generation in WT and Pf4-Cre/FAK-floxed mice platelets. Murine platelets from WT and Pf4-Cre/FAK-floxed were stimulated with 100 nM of 2MeSADP and thromboxane levels were measured from WT and Pf4-Cre/FAK-floxed mice samples. As shown in Fig. 5B there was no significant difference observed thromboxane levels and aggregation tracings (Fig. 5C) between WT and Pf4-Cre/FAK-floxed. These results suggest that TAE-226 might exhibit some non-specific effects, and FAK is not the common signaling molecule regulating thromboxane generation downstream of integrins and G12/13 pathways.


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)

Evaluation of FAK as a common signaling effector molecule regulating thromboxane generation downstream of integrins and G12/13 pathways.Non-aspirin-treated, washed human platelets were pre-treated with varying concentrations of TAE-226 for 5 minutes at 37°C (A) and murine platelets from WT and Pf4-Cre/Fak-Floxed mice (B) were stimulated with 2MeSADP (100 nM) for 3.5 minutes and TXB2 levels were analyzed as described for Figure 1. Aggregation tracings were measured from WT and Pf4-Cre/Fak-Floxed mice and representative tracings are shown (C). The values are representative of 3 independent experiments mean ± S.E.M (n = 3). The data were analyzed by ANOVA and student t-test, * P≤0.05 was considered significant.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016586-g005: Evaluation of FAK as a common signaling effector molecule regulating thromboxane generation downstream of integrins and G12/13 pathways.Non-aspirin-treated, washed human platelets were pre-treated with varying concentrations of TAE-226 for 5 minutes at 37°C (A) and murine platelets from WT and Pf4-Cre/Fak-Floxed mice (B) were stimulated with 2MeSADP (100 nM) for 3.5 minutes and TXB2 levels were analyzed as described for Figure 1. Aggregation tracings were measured from WT and Pf4-Cre/Fak-Floxed mice and representative tracings are shown (C). The values are representative of 3 independent experiments mean ± S.E.M (n = 3). The data were analyzed by ANOVA and student t-test, * P≤0.05 was considered significant.
Mentions: We next evaluated whether activated FAK played a role in ADP-induced TXA2 generation. TAE-226 has recently been identified as a selective inhibitor of FAK with an IC50 of 5.5 nM [36]. To determine the effect of FAK inhibition on ADP-induced thromboxane generation platelets were treated with varying concentrations of TAE-226. As demonstrated in Fig. 5A, TXA2 generation was significantly inhibited by TAE-226 at a higher concentration of 2 µM. Pharmacological inhibitors are often known to have off target and broad-spectrum effects. The specificity of TAE-226 was never evaluated in platelets and reports suggest that TAE-226 inhibits Pyk2 (a Focal Adhesion kinase family member) with an IC50 of 5 nM [37]. Hence, we studied thromboxane generation in WT and Pf4-Cre/FAK-floxed mice platelets. Murine platelets from WT and Pf4-Cre/FAK-floxed were stimulated with 100 nM of 2MeSADP and thromboxane levels were measured from WT and Pf4-Cre/FAK-floxed mice samples. As shown in Fig. 5B there was no significant difference observed thromboxane levels and aggregation tracings (Fig. 5C) between WT and Pf4-Cre/FAK-floxed. These results suggest that TAE-226 might exhibit some non-specific effects, and FAK is not the common signaling molecule regulating thromboxane generation downstream of integrins and G12/13 pathways.

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