Protease-activated Receptor 4 causes Akt phosphorylation independently of PI3 kinase pathways.

Abstract

PI-3 Kinase plays an important role in platelet activation mainly through regulation of RASA3. Akt phosphorylation is an indicator for the activity of PI3 kinase. The aim of this study is to characterize the pathways leading to Akt phosphorylation in platelets. We performed concentration response curves of LY294002, a pan-PI3 kinase inhibitor, on platelet aggregation and Akt phosphorylation, in washed human and mouse platelets. At concentrations as low as 3.12 μM, LY294002 abolished Akt phosphorylation induced by 2MeSADP and SFLLRN, but not by AYPGKF. It required much higher concentrations of LY294002 (12.5-25 μM) to abolish AYPGKF-induced Akt phosphorylation, both in wild type and P2Y12 null mouse platelets. We propose that 3.12 μM LY294002 is sufficient to inhibit PI3 kinase isoforms in platelets and higher concentrations might inhibit other pathways regulating Akt phosphorylation by AYPGKF. We conclude that Protease-activated receptor 4 (PAR4) might cause Akt phosphorylation through pathways distinctly different from those of Protease-activated receptor 1 (PAR1).

Introduction

Platelets play a critical role in both homeostasis as well as hemostasis. An important signaling pathway in platelets involves phosphatidylinositol 3-kinases (PI3K) which are divided into three families – Classes I, II, and III. ^{(1, 2)} The Class I PI3KS are responsible for agonist-induced production of the second messengers phosphoinositide (PI) (3,4,5)P3 and PI(3,4)P2 which participate in a variety of platelet functions. The Class 1b isoform is typically activated by G-protein-coupled receptors.⁽³⁾ We have previously shown that GPCR-mediated Akt phosphorylation in platelets depends on the P2Y12 signaling,⁽⁴⁾ while Li and co-workers have shown that AYPGKF can cause Akt phosphorylation independently of the P2Y12 receptor in mice.⁽⁵⁾ Platelets contain all Class I PI3K isoforms with lower levels of p110δ. In order to evaluate the functional role of PI-3 kinases many investigators employ the pan PI3K inhibitor, LY294002 and measure agonist-induced aggregation and Akt phosphorylation. Most studies use 10 to 100 μM LY294002 without evaluating the minimum concentration required for maximum specificity (6, 7). In this study we evaluated the concentration requirements of LY294002 downstream of different GPCR agonists in both human and mouse platelets and concluded that 3.12 μM LY294002 is sufficient to specifically inhibit Akt phosphorylation in platelets. As we have used Akt as a read out for PI3 kinase we suggest that this inhibition of Akt phosphorylation is due to inhibition of PI3 kinase isoforms. In addition, we show that, unlike PAR-1, PAR-4 mediated Akt phosphorylation is not inhibited by 3.12 μM LY294002 or a structurally different PI3 kinase inhibitor PI103 (8).

Materials and methods

Materials

All materials were obtained from ThermoFisher Scientific unless otherwise noted. Apyrase and 2-MeSADP, were purchased from Sigma (St. Louis, MO). PAR 4 peptide AYPGKF-NH2 and PAR 1 peptide SLFLLRN-NH2 were synthesized by GenScript (Piscataway, NJ). Anti-Akt (Ser473) cat#4060 was obtained from Cell Signaling (Danvers, MA). Antibody to total Akt cat#TA504230 was purchased from Origene (Rockville, MD). Blocking buffer, infrared dye-labeled goat anti-mouse (926-68020) and anti-rabbit antibodies (926-32211) were purchased from LI-COR (Lincoln, NE). PI103 was purchased from Tocris Bioscience (Minneapoli, MN)

Mice

All work involving mice was done according to the Temple University Institution Animal Care and Use Committee (Approval #4864). All mice were housed in a pathogen-free environment of the Temple University Central Animal Facility. P2Y12-null mice were generated using standard techniques.⁽⁹⁾ In-house bred 129Sv × C57BL/6 F2 mice derived from the same 129 Sv parent ES cell line as the P2Y12-null mice that were used as controls. These wild-type mice do not differ genetically from P2Y12-null mice except at the P2Y12-targeted locus.

Isolation of Murine Platelets

Mouse blood was collected and processed as previously described(10). Briefly, blood from anesthetized mice was collected via cardiac puncture into one-tenth volume 3.8% sodium citrate and centrifuged at 100 × g for 10 minutes. The platelet-rich plasma (PRP) was removed and sodium citrate was added to the remaining packed cells prior to a second 100 × g spin. The resulting PRPs were combined and 1 μM PGE1 was added to the PRP before centrifugation at 400 × g for 10 minutes. The resulting platelet pellet was resuspended in Tyrodes buffer (138 mM NaCl, 2.7 mM KCl, 2 mM MgCl ₂ , 0.42 mM NaH ₂ PO ₄ , 10 mM HEPES, 0.1% glucose and 0.2 U/mL apyrase, pH 7.4), and platelets were counted using a Hemavet 950FS blood cell counter (Drew Scientific, Dallas, TX) and adjusted to a final concentration of 1.5 × 10 ⁸ cells/ml.

Isolation of Human Platelets

Blood was drawn from informed healthy volunteers according to a protocol approved by the Institutional Review Board of Temple University (Approval #23688) in accordance with the Declaration of Helsinki into one-sixth volume of acid-citrate-dextrose (85 mM sodium citrate, 111 mM glucose, 71.4 mM citric acid). Platelet-rich plasma (PRP) was isolated by centrifugation at 230 × g for 20 min. Platelets were obtained by centrifugation of the PRP for 10 min at 980 × g and resuspended in Tyrode’s buffer as described above. Platelet counts were adjusted to 2 × 10⁸ platelets/ml.

Platelet Aggregation

All aggregation experiments were performed using a lumi-aggregometer (Chrono-log) at 37°C under stirring conditions. Platelets (250 μl for mouse platelets and 500 μl for human platelets) were stimulated with AYPGKF, SFLLRN or 2-MeSADP and aggregation was measured via light transmission.

Platelet Sample Preparation and Western Blotting Analysis

Platelets were stimulated with agonists for 3.5 minutes under stirring conditions at 37 °C, and the proteins were precipitated by the addition of HClO₄ to a final concentration of 0.6 N. The protein pellets were washed one time with water and solubilized in sample buffer containing 0.1 M Tris base, 2% SDS, 1%(v/v) glycerol, 0.1% bromphenol blue, and 100 mM DTT and boiled for 10 min. Proteins were resolved using SDS-PAGE and transferred to nitrocellulose using an iBlot 2 (Invitrogen). Membranes were blocked with the Odyssey blocking buffer for 1 h at room temperature, incubated overnight at 4 °C with the desired primary antibody, and washed four times with Tris-buffered saline-Tween (TBS-T; 25 mM Tris, pH 7.4, 137 mM NaCl, 0.1% Tween 20) before incubation with appropriate infrared dye-labeled secondary antibody for 1 h at room temperature. Washed membranes were examined with a LI-COR Odyssey infrared imaging system.

Statistical analysis

All statistical analyses were carried out using Kaleidagraph software. Data are presented as means +/− S.E. Statistical significance was determined by Student’s t test and analysis of variance. p<0.05 was considered statistically significant.

Results and Discussion

Concentration of LY294002 required for inhibition of PI3 kinase in platelets

The pan PI3 kinase inhibitor LY294002 has been used in several platelet studies to block PI3 kinase.^(11-15) In all these studies the concentration of LY294002 used ranged from 10-100 μM and phosphorylation of Akt was used as an indicator of PI3 kinase activity.⁽¹⁶⁾ Indeed, our own laboratory has standardly used 25 μM LY294002 to inhibit PI3 kinase activity.^{(4, 17, 18)} Concerned about any non-specific inhibitory effects of LY294002, we performed a concentration response curve of the inhibitor on agonist-induced aggregation and Akt phosphorylation in human and mouse platelets, as a proxy for PI3 kinase activity. In human platelets LY294002 caused a reversal of aggregation induced by 2MeSADP or SFLLRN at a minimum concentration of 1.56 μM, while AYPGKF-induced aggregation was untouched at 25 μM LY294002 (Figure 1A). Similarly, 2MeSADP- or SFLLRN-induced Akt phosphorylation was abolished at 3.12 μM LY294002 but it required a minimum concentration of 12.5-25 μM LY294002 to inhibit Akt phosphorylation induced by AYPGKF (Figure 1B). These data are statistically significant (Figure 1C), clearly shows a right ward shift of the concentration response curve by AYPGKF compared to SFLLRN (insert in Fig. 1C). Similar results were obtained in mouse platelets for 2MeSADP and AYPGKF (Fig. 2A-C) although AYPGKF-induced Akt phosphorylation was abolished by 6.25 μM LY294002. These results indicate that LY294002 can inhibit Akt phosphorylation possibly through complete inhibition of PI3 kinase at a minimum concentration of 3.12 μM. At higher concentration of the inhibitor AYPGKF-induced Akt phosphorylation is abolished possibly through the non-specific inhibitory actions of LY294002. Previously, Resendiz et al (19) have shown that SFLLRN causes Akt phosphorylation in platelets through PI3 kinase pathways whereas AYPGKF-induced Akt phosphorylation occurs independently of PI3 kinase pathways. These investigators used higher (10 μM) concentration of LY294002 (19) than our study.

Figure 1:

Isolated human platelets were pre-treated with either vehicle control (DMSO) or various concentrations of LY294002 for 5 min at 37 °C. Platelets were stimulated with indicated agonists for 3.5 minutes at 37 °C with stirring: 2-MeSADP (100 nM), SFLLRN (25 μM), or AYPGKF (500 μM). A. Representative aggregometer tracings and B. Representative Akt phosphorylation C. Percent inhibition of Akt phosphorylation induced by each agonist was calculated from 4 independent experiments and graphed as mean +/− SEM. Akt inhibition induced by individual concentrations of LY294002 when platelets were activated with 2MeSADP or SFLLRN were compared to the inhibition when activated with AYPGKF (*, p<0.05; NS, not significant). Insert showing expanded x-axis with low concentrations.

Figure 2:

Isolated mouse platelets were pre-treated with either vehicle control (DMSO) or various concentrations of LY294002 for 5 min at 37 °C. Platelets were stimulated with indicated agonists for 3.5 minutes at 37 °C with stirring: 2-MeSADP (100 nM) or AYPGKF (500 μM). A. Representative aggregometer tracings and B. Representative Akt phosphorylation C. Percent inhibition of Akt phosphorylation induced by each agonist was calculated from 4 independent experiments and graphed as mean +/− SEM. Akt inhibition induced by individual concentrations of LY294002 when platelets were activated with 2MeSADP or SFLLRN were compared to the inhibition when activated with of AYPGKF. (*, p<0.05; NS, not significant). Insert showing expanded x-axis with low concentrations.

Previous studies in other cells have shown that LY294002 can inhibit other kinases non-specifically (6, 7). In order to evaluate whether this residual phosphorylation of Akt, downstream of AYPGKF in the presence of 3.12 μM LY294002, was due to another isoform of PI3 kinase or some other nonspecific kinase, we used another PI3 kinase inhibitor, PI103 that is structurally distinct from LY294002 (8). As shown in Fig. 3 A-C, in the presence of 100 nM PI103, 2MeSADP-induced Akt phosphorylation, but not AYPGKF-induced Akt phosphorylation, was abolished suggesting that this AYPGKF-induced residual phosphorylation is due to non PI3 kinase pathways.

Figure 3.

Isolated mouse platelets were pre-treated with either vehicle control (DMSO) or various concentrations of PI103 for 5 min at 37 °C. Platelets were stimulated with indicated agonists for 3.5 minutes at 37 °C with stirring: 2-MeSADP (100 nM), or AYPGKF (500 μM). A. Representative aggregometer tracings and B. Representative Akt phosphorylation C. Percent inhibition of Akt phosphorylation induced by each agonist was calculated from 4 independent experiments and graphed as mean +/− SEM. Akt inhibition induced by individual concentrations of PI103 when platelets were activated with 2MeSADP was compared to the inhibition when activated with of AYPGKF. Insert showing expanded x-axis with low concentrations.

Our previous studies have shown that GPCR agonist-induced Akt phosphorylation occurs though secreted ADP activating the P2Y12-Gi-PI3 kinase pathways.⁽⁴⁾ Li and co-workers have shown that AYPGKF causes Akt phosphorylation independently of the P2Y12 receptor.⁽⁵⁾ However, in these studies, the authors did not take into account the possibility that other secreted agents from platelets, such as SDF-1, could weakly activate Gi pathways and hence cause PI3 kinase activation and Akt phosphorylation.⁽¹⁴⁾ Our current study reconciles these studies and provides evidence for Akt phosphorylation independently of PI3 kinase pathways. Li and coworkers also employed 20 μM LY294002 to inhibit AYPGKF-induced Akt phosphorylation in P2Y12 ^−/− platelets.⁽⁵⁾ We therefore compared AYPGKF-induced Akt phosphorylation in WT and P2Y12 ^−/− platelets pre-incubated with either 3.12 μM or 25 μM LY294002. Figure 4 demonstrates that, even in the absence of P2Y12, AYPGKF is capable of phosphorylating Akt in the presence of 3.12 μM LY294002. Our earlier study has also shown that Akt translocates to the membrane independently of the P2Y12 and PI3 kinase pathways.⁽²⁰⁾ Interestingly this translocation occurs whether the platelets are activated with SFLLRN or AYPGKF. However, in the current study there is a remarkable difference by SFLLRN vs AYPGKF as 3.12 μM LY294002 abolished SFLLRN-induced, but not AYPGKF-induced, Akt phosphorylation. This highlights the remarkable signaling difference between the two PAR receptors, PAR1 and PAR4. Earlier studies by Kuliopulos and co-workers have shown that PAR1 mediated intracellular calcium increases are transient while those of PAR4 are sustained for a longer period.⁽²¹⁾ These studies indicate that although both PAR1 and PAR4 activate Gq and G12/13 pathways, there is an inherent difference in the way these two receptors signal and these differences in signaling need to be further explored.

Figure 4.

Isolated WT or P2Y12 KO platelets were pre-incubated with either vehicle (DMSO), 3.1 μM or 25 μM LY294002 for 5 min at 37 °C and activated with 500 μM AYPGKF for 3.5 minutes at 37 °C with stirring. A. Representative Akt phosphorylation and B. Percent inhibition of Akt phosphorylation was calculated from 4 independent experiments and graphed as mean +/− SEM (*, p<0.05).

In conclusion, we provide evidence that 3.12 μM LY294002 is sufficient to inhibit PI3 kinase in platelets and at higher concentrations it might elicit non-specific effects to inhibit Akt phosphorylation. Our study also clearly demonstrates that unlike SFLLRN, AYPGKF causes Akt phosphorylation independently of PI3 kinase pathways.