Biochemical Journal

Research article

Role of phosphoinositide 3-kinase β in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways

Analia Garcia, Soochong Kim, Kamala Bhavaraju, Simone M. Schoenwaelder, Satya P. Kunapuli

Abstract

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms α, β, γ and δ in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kβ-selective inhibitor, but not by PIK75 (a PI3Kα inhibitor), AS252424 (a PI3Kγ inhibitor) or IC87114 (a PI3Kδ inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1−/− mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kβ in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kβ plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kβ mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.

  • ADP
  • Akt
  • extracellular-signal-regulated kinase (ERK)
  • phosphoinositide 3-kinase (PI3K)
  • platelet
  • thromboxane A2 (TXA2)

INTRODUCTION

Platelets are key cellular components of haemostasis [1]. Upon vessel injury, platelets in circulation adhere to the exposed collagen and become activated. Platelet activation leads to release of ADP from dense granules and TX (thromboxane) A2 generation from the phospholipids [2]. The secreted ADP and generated TXA2 thus act as positive-feedback mediators and activate more platelets amplifying initial platelet responses and aiding in formation of a stable haemostatic plug [3].

ADP is an important platelet agonist that causes platelet shape change, aggregation and TXA2 generation [4]. ADP activates platelets through two purinergic receptors, P2Y1 and P2Y12, which couple to Gq and Gi respectively. Concomitant signalling through Gq-coupled P2Y1 and Gi-coupled P2Y12 is necessary and sufficient for the fibrinogen receptor activation [57]. The importance of these two ADP receptors was uncovered by studies on P2Y1−/− and P2Y12−/− mice respectively. The P2Y1 receptor stimulation results in an increase in intracellular calcium, PKC (protein kinase C) activation and causes platelet shape change [5,6]. The P2Y12 receptor stimulation results in Gi-mediated inhibition of stimulated adenylate cyclase and Gβγ-mediated activation of PI3K (phosphoinositide 3-kinase) [8], Akt [9] and Rap1b [10,11]. P2Y12-coupled Gi signalling is also critical for potentiating dense granule secretion [12], TXA2 generation and irreversible aggregation [13].

Platelet PI3Ks catalyse the phosphorylation of phosphoinositides at position 3 of the inositol ring to produce the second messenger PtdIns(3,4)P2 and PtdIns(3,4,5)P3. PI3Ks are divided into three classes based on their primary structure, mode of regulation and substrate specificity. PI3K class I family members have a p110 catalytic subunit, and those p110 subunits associate with regulatory subunits. There are three p110 catalytic subunits of class IA PI3Ks: p110α, p110β and p110δ. Intriguingly, besides phosphorylating lipids, all p110 catalytic subunits possess intrinsic serine protease activity, the function of which is still not clear [14]. The only member of the PI3K class IB family is PI3Kγ. This enzyme is present only in mammals and is preferentially expressed in leucocytes [15]. The catalytic subunit p110γ of this enzyme interacts with a different regulatory subunit termed p101. This enzyme is distinct from the class IA PI3Ks in that it is not activated by tyrosine-phosphorylated proteins, but rather by Gβγ subunits of heterotrimeric G-proteins. Previous studies have dissected the functional importance of these PI3K isoforms in various cell types, including platelets, using pharmacological inhibitors specific to each isoforms: PIK75 for PI3Kα, TGX-221 for PI3Kβ, IC87114 for PI3Kδ and AS252424 for PI3Kγ [1625]. These studies indicated that PI3Kα plays a role in cancer, cell growth and division, PI3Kβ in thrombosis and stenosis reduction, PI3Kδ in B- and T-cell signalling, and PI3Kγ in inflammation, leucocyte chemotaxis and neutrophil activation.

Although it has been shown that PI3K pathways downstream of Gi are important for ADP-induced platelet aggregation and Akt activation [9,26], there is controversy in the role of different PI3K isoforms in Gi signalling. Although it has been shown that PI3Kβ is the dominant PI3K isoform regulating Gi-dependent integrin αIIbβ3 activation, through the catalytic modulation of Rap1b and/or Akt in ADP-stimulated platelets [27], a recent study has indicated that ADP-induced Akt phosphorylation is regulated by both PI3Kβ and PI3Kγ, whereas ADP-induced Rap1b activation and aggregation are only partially regulated by those two isoforms [28]. Furthermore, previous studies have shown that co-stimulation of the Gi and Gz pathways caused enhanced TXA2 generation [29]. We have also shown previously that ADP-induced TXA2 generation occurs through the regulation of ERK (extracellular-signal-regulated kinase) activation [30]. However, the role of PI3Ks in ADP-induced ERK activation and TXA2 generation was not completely understood.

The results from the present study show that PI3Kβ plays an important role in ADP-induced platelet aggregation, Akt and ERK phosphorylation, and TXA2 generation. We also show that PI3Kβ mediates platelet aggregation, ERK phosphorylation and TXA2 generation induced by Gi plus Gz pathways. Hence, we conclude that PI3Kβ, but not α, γ or δ, plays a critical role in ADP-induced platelet responses.

EXPERIMENTAL

Materials

Apyrase (Type VII), BSA (fraction V), acetylsalicylic acid, 2MeSADP (2-methyl-thio-ADP), apyrase (type V), and MRS-2179 were from Sigma–Aldrich. Anti-phospho-ERK and anti-(total ERK) antibodies were purchased from Cell Signaling Technology. HRP (horseradish peroxidase)-labelled secondary antibody was from Santa Cruz Biotechnology. Wortmannin was from Biomol Research Laboratories. TGX-221 was purchased from Cayman Chemical. PIK75, AS252424 and IC87114 were provided by Dr Shaun Jackson (Monash University, Victoria, Australia).

Isolation of human platelets

Approval for this study was obtained from the Institutional Review Board of Temple University (Philadelphia, PA, U.S.A.). Whole blood was drawn from healthy consenting human volunteers into tubes containing a one-sixth volume of ACD solution (2.5 g of sodium citrate, 1.5 g of citric acid and 2 g of glucose in 100 ml of deionized water). Blood was centrifuged (Eppendorf 5810R centrifuge) at 230 g for 20 min at room temperature (23 °C) to obtain PRP (platelet-rich plasma). PRP was incubated with 1 mM acetylsalicylic acid (aspirin) for 30 min at 37 °C. The PRP was then centrifuged at 980 g for 10 min at room temperature to pellet the platelets. Platelets were resuspended in Tyrode's buffer (10 mM Hepes, pH 7.4, 138 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 3 mM NaH2PO4, 5 mM glucose and 0.2% BSA) containing 0.1 unit/ml apyrase. Cells were counted using the Coulter Z1 Particle Counter and the concentration of cells was adjusted to 2×108 platelets/ml. All experiments using washed platelets were performed in the absence of extracellular calcium unless otherwise indicated. In the experiments with TXB2 measurements, the treatment of PRP with acetylsalicylic acid was omitted.

Aggregometry

Aggregation of 0.5 ml of washed platelets was analysed using a PICA lumiaggregometer (Chrono-log). Aggregation was measured using light transmission under stirring conditions (900 rev./min) at 37 °C. Agonists were added simultaneously for platelet stimulation; however, platelets were pre-incubated with each inhibitor (where indicated) at 37 °C. Each sample was allowed to aggregate for at least 3 min. The chart recorder (Kipp and Zonen) was set for 0.2 mm/s. All samples contained exogenously added human fibrinogen (1 mg/ml) without added calcium. Aggregation tracings are representative of results obtained from three separate experiments for three different donors.

Western blot analysis

Platelets were stimulated with agonists in the presence or absence of inhibitors for the appropriate time, and the reaction was stopped by the addition of 3× SDS sample buffer. Platelet samples were boiled for 10 min and proteins were separated by SDS/PAGE (10% gels) and transferred on to PVDF membranes. Non-specific binding sites were blocked by incubation in TBST (Tris-buffered saline/Tween 20; 20 mM Tris/HCl, 140 mM NaCl and 0.1% Tween 20) containing 2% (w/v) BSA for 30 min at room temperature, and membranes were incubated overnight at 4 °C with the primary antibody [1:1000 dilution in TBST with 2% (w/v) BSA] with gentle agitation. After three washes for 5 min each with TBST, the membranes were probed with a HRP-labelled secondary antibody [1:10000 dilution in TBST with 2% (w/v) BSA] for 1 h at room temperature. After additional washing steps, membranes were then incubated with chemiluminescent HRP substrate (Millipore) for 3 min at room temperature and immunoreactivity was detected using a FujiFilm Luminescent Image Analyzer (LAS-3000 CH).

Measurement of TXA2 generation

Washed, human platelets without aspirin-treatment were prepared as above to a concentration of 2×108 platelets/ml. Stimulations were performed in a platelet aggregometer under stirring conditions (900 rev./min) at 37 °C without added calcium. The PI3K inhibitors and the vehicle, as indicated in the Figure legends, were added 5 min prior to addition of the agonist. Stimulations were performed for 3.5 min and the reaction was stopped by snap-freezing. The samples were stored at −80 °C until TXB2 analysis was performed (as TXA2, which is unstable, is rapidly converted into a stable non-enzymatic hydration product TXB2, TXB2 levels are measured to determine TXA2 generation). Levels of TXB2 were determined in duplicate using a Correlate-EIA TXB2 enzyme immunoassay kit (Assay Designs), according to the manufacturer's instructions.

Results represent the normalized values obtained from at least three donors and are given as means±S.E.M.

Preparation of washed mouse platelets

Blood was collected from the vena cava of anesthetized P2Y1−/− mice into syringes containing one-tenth of blood volume of 3.8% (w/v) sodium citrate, as an anticoagulant. Red blood cells were removed by centrifugation at 100 g for 10 min. PRP was recovered and platelets were pelleted at 400 g for 10 min. The platelet pellet was resuspended in Tyrode's buffer (pH 7.4) containing 0.05 unit/ml apyrase.

Statistical analysis

Statistical significance was determined by one-way ANOVA. All statistical tests were carried out using Prism software (version 3.0). Results are presented as means±S.E.M.

RESULTS

Role of PI3K isoforms in 2MeSADP-induced platelet aggregation

PI3Ks play an important role in ADP-induced platelet aggregation. In order to identify the role of the specific PI3K isoform that plays an important role in ADP-induced platelet aggregation, we evaluated the effect of different PI3K isoform selective inhibitors [1618,20,22,23]. As shown in Figure 1 (upper panel), 2MeSADP-induced aggregation in washed non-aspirin-treated human platelets was blocked in the presence of the PI3K inhibitor wortmannin. 2MeSADP-induced platelet aggregation was significantly inhibited by the PI3Kβ inhibitor TGX-221, whereas the PI3Kα inhibitor PIK75, the PI3Kγ inhibitor AS252424 and the PI3Kδ inhibitor IC87114 showed no effect on 2MeSADP-induced platelet aggregation. In order to rule out the possible secondary effects of TXA2 on 2MeSADP-induced platelet aggregation, we evaluated the effect of PI3K isoform inhibitors on aspirin-treated platelets. Similar results were obtained with washed aspirin-treated platelets as shown in Figure 1 (lower panel), indicating that the PI3Kβ isoform plays an important role in 2MeSADP-induced platelet aggregation.

Figure 1 Effect of PI3K isoform inhibitors on 2MeSADP-induced platelet aggregation

Washed human non-aspirin-treated (upper panel) and aspirin-treated (lower panel) platelets were pre-treated with PI3K-isoform-selective inhibitors PIK75 (100 nM), TGX-221 (200 nM), AS252424 (2 μM), IC87114 (1 μM) and wortmannin (100 nM) at 37 °C for 5 min followed by stimulation with 100 nM 2MeSADP for 5 min under stirring conditions (addition indcated by arrow). Platelet aggregations were measured by aggregometry. Tracings are representative of experiments performed using platelets from at least three different donors.

Effect of PI3K isoform inhibitors on 2MeSADP-induced ERK phosphorylation and TXA2 generation

The inhibitory effect of wortmannin and the PI3Kβ inhibitor TGX-221 on 2MeSADP-induced aggregation in non-aspirin-treated platelets (Figure 1) suggests a role for PI3Ks in ADP-mediated TXA2 generation. We have shown previously that ADP-induced TXA2 generation occurs through the regulation of ERK activation [30]. Hence, we evaluated the role of specific PI3K isoforms in 2MeSADP-induced TXA2 generation and ERK activation. Washed non-aspirin-treated platelets were stimulated with 2MeSADP in the presence and absence of PI3K isoform inhibitors. As shown in Figure 2(A), 2MeSADP-induced TXA2 generation was significantly inhibited in the presence of wortmannin and the PI3Kβ inhibitor TGX-221, indicating the critical role for PI3Kβ in 2MeSADP-induced TXA2 generation. Consistent with the result in Figure 2(A), we observed that 2MeSADP-induced ERK phosphorylation was markedly inhibited by wortmannin and TGX-221 and partially inhibited by the PI3Kα inhibitor PIK75, whereas the PI3Kγ inhibitor AS252424 and the PI3Kδ inhibitor IC87114 showed little or no effect on 2MeSADP-induced ERK phosphorylation (Figures 2B and 2C), thereby demonstrating the key role of PI3Kβ and a less critical role of PI3Kα in 2MeSADP-induced ERK phosphorylation. Time course analysis of 2MeSADP-induced ERK phosphorylation in the absence and presence of TGX-221 showed that there was a significant inhibition of ERK phosphorylation in the presence of TGX-221 (Figure 2D), consistent with the result in Figure 2(B). As our previous studies have shown that ERK mediates TXA2 generation [30], these studies suggest that PI3Kβ plays a key role in 2MeSADP-induced TXA2 generation through the regulation of ERK phosphorylation in platelets.

Figure 2 Effect of PI3K isoform inhibitors on 2MeSADP-induced ERK phosphorylation and TX generation

Washed human (A) non-aspirin-treated and (B) aspirin-treated platelets were pre-treated with PI3K isoform selective inhibitors PIK75 (100 nM), TGX-221 (200 nM), AS252424 (2 μM), IC87114 (1 μM) or wortmannin (100 nM) at 37 °C for 5 min followed by stimulation with 2MeSADP (100 nM) for 60 s (ERK phosphorylation) and for 3.5 min (TX) under stirring conditions. (A) TXA2 generation and (B) ERK phosphorylation (P-ERK) were measured as described in the Experimental section. (C) Densitometric measurement of phospho-ERK, expressed as a percentage of the agonist control. (D) Platelets were stimulated with 2MeSADP (100 nM) for the indicated time in the presence and absence of TGX-221 (200 nM) or 100 ng/ml convulxin (CVX, positive control). ERK phosphorylation was measured. Results are representative of experiments performed using platelets from at least three different donors. Results are means±S.E.M. (n=3); *P<0.05 compared with agonist.

Role of PI3Kβ in Gi plus Gz-induced platelet aggregation

We have shown previously that co-stimulation of the Gi and Gz pathways in the absence of Gq signalling leads to platelet aggregation and TXA2 generation [29]. Hence, we evaluated the role of PI3K isoforms in this event. We first evaluated the effect of PI3K-selective inhibitors on platelet aggregation induced by Gi plus Gz stimulation with 2MeSADP plus adrenaline (epinephrine) in the presence of P2Y1 receptor antagonist MRS-2179. Gi plus Gz-mediated platelet aggregation was blocked by wortmannin and the PI3Kβ inhibitor TGX-221 in both washed non-aspirin-treated (Figure 3, upper panel) and aspirin-treated (Figure 3, lower panel) platelets, whereas other PI3K-selective inhibitors showed no effect, indicating that PI3Kβ is an essential PI3K isoform in Gi plus Gz-mediated platelet aggregation.

Figure 3 Effect of PI3K isoform inhibitors on Gi plus Gz-induced platelet aggregation

Washed human (A) non-aspirin-treated and (B) aspirin-treated platelets were pre-treated with MRS 2179, a P2Y1 receptor antagonist, and PI3K isoform selective inhibitors PIK75 (100 nM), TGX-221 (200 nM), AS252424 (2 μM), IC87114 (1 μM) or wortmannin (100 nM) at 37 °C for 5 min followed by stimulation with 2MeSADP (100 nM) plus adrenaline (10 μM) for 5 min under stirring conditions (addition indcated by arrow). Platelet aggregations were measured by aggregometry. Tracings are representative of three independent experiments.

Role of PI3Kβ in Gi plus Gz-mediated ERK phosphorylation and TX generation

As we have shown that co-stimulation of Gi and Gz pathways mediate TXA2 generation [29] through the regulation of ERK phosphorylation [30], we studied the role of PI3K isoforms in Gi plus Gz-mediated TXA2 generation and ERK phosphorylation. We stimulated washed non-aspirin-treated human platelets with 2MeSADP, adrenaline and MRS2179 in the presence and absence of PI3K-selective inhibitors. Consistent with the effect of TGX-221 on Gi plus Gz-mediated platelet aggregation, the PI3Kβ inhibitor TGX-221 significantly inhibited Gi plus Gz-mediated TXA2 generation (Figure 4A) and ERK phosphorylation (Figures 4B and 4C), indicating that PI3Kβ mediates Gi plus Gz-induced TXA2 generation through the regulation of ERK phosphorylation. The other PI3K isoform inhibitors showed no significant effect.

Figure 4 Effect of PI3K isoform inhibitors on Gi plus Gz-induced ERK phosphorylation and TX generation

Washed human (A) non-aspirin-treated and (B) aspirin-treated platelets were pre-treated with MRS 2179, a P2Y1 receptor antagonist, and PI3K isoform selective inhibitors PIK75 (100 nM), TGX-221 (200 nM), AS252424 (2 μM), IC87114 (1 μM) or wortmannin (100 nM) at 37 °C for 5 min following stimulation with 2MeSADP (100 nM) plus adrenaline (10 μM) for 60 s (ERK phosphorylation) and for 3.5 min (TX) under stirring conditions. (A) TXA2 generation (B) and ERK phosphorylation (P-ERK) were measured as described in the Experimental section. (C) Densitometric measurement of phospho-ERK, expressed as a percentage of the agonist control. Results are representative of at least three independent experiments and are means±S.E.M. (n=3); *P<0.05 compared with agonist.

In order to verify the role of PI3Kβ in Gi plus Gz-induced platelet responses in mouse, as well as human platelets, we stimulated platelets from P2Y1−/− mice with 2MeSADP and adrenaline in the presence and absence of PI3K inhibitors. Consistent with the results shown in Figure 4, TGX-221 significantly inhibited Gi plus Gz-mediated platelet aggregation (Figure 5A), TXA2 generation (Figure 5B) and ERK phosphorylation (Figures 5C and 5D). Hence, our results confirm that PI3Kβ plays a key role in regulating platelet aggregation, TXA2 generation and ERK phosphorylation mediated by Gi plus Gz pathways in both human and mouse platelets.

Figure 5 Role of PI3Kβ in Gi plus Gz-mediated aggregation, ERK phosphorylation and TX generation in murine platelets

(A and B) Non-aspirin-treated platelets or (C) aspirin-treated platelets from P2Y1−/− mice were pre-treated with PI3K isoform selective inhibitors PIK75 (100 nM), TGX-221 (200 nM), AS252424 (2 μM), IC87114 (1 μM) or wortmannin (100 nM) at 37 °C for 5 min followed by stimulation with 2MeSADP (100 nM) plus adrenaline (10 μM) under stirring conditions (addition indcated by arrow). (A) Platelet aggregation, (B) TXA2 generation and (C) ERK phosphorylation (P-ERK) were measured. (D) Densitometric measurement of phospho-ERK, expressed as a percentage of the agonist control. Results are representative of at least three independent experiments and are means±S.E.M. (n=3); *P<0.05 compared with agonist.

Role of PI3Kβ in 2MeSADP-induced Akt phosphorylation

It is known that PI3K is the main upstream regulator of Akt, and we have shown that PI3K is a key element in Akt phosphorylation induced by various agonists including ADP [9]. However, the role of the PI3K isoforms in ADP-induced Akt phosphorylation has not been identified. Thus we evaluated the contribution of PI3K isoforms to 2MeSADP-induced Akt phosphorylation by using PI3K isoform inhibitors. As shown in Figure 6, 2MeSADP-induced Akt phosphorylation was completely inhibited by TGX-221, and partly inhibited by PIK75, but was not affected by AS252424 and IC87114, indicating an essential role for PI3Kβ in ADP-mediated Akt phosphorylation.

Figure 6 Role of PI3Kβ in 2MeSADP-induced Akt phosphorylation

(A) Washed human aspirin-treated platelets pre-treated with PI3K-isoform-selective inhibitors PIK75 (100 nM), TGX-221 (200 nM), AS252424 (2 μM), IC87114 (1 μM) or wortmannin (100 nM) were stimulated at 37 °C for 3 min with 2MeSADP (100 nM). Samples were separated by SDS/PAGE and probed with an anti-phospho-Akt (P-Akt; Ser473) or anti-Akt (loading control) antibody. (B) Densitometric measurement of phospho-Akt expressed as percentage of the agonist control. The Western blots are representative of three independent experiments. Results are means±S.E.M. (n=3); *P<0.05 compared with agonist.

DISCUSSION

It has been known that PI3K plays an important role in ADP-induced platelet aggregation [26], and our results using the PI3K inhibitor wortmannin show that PI3K is important for ADP-induced ERK phosphorylation, TXA2 generation and platelet aggregation. However, it was not clear which subtype of PI3K (α, β, δ or γ) was involved in this process. Therefore we have used several PI3K inhibitors to selectively inhibit different isoforms of PI3K to define the role of individual PI3K isoforms in regulating ADP-induced platelet responses.

The activation of PI3K downstream of Gi has been implicated in stabilization of platelet aggregation [31]. It has also been shown that PI3K activity is required for P2Y12 receptor-mediated platelet aggregation [26] and P2Y12-dependent prolongation of the Ca2+ response relies on PI3Kβ [32]. PI3K activity is necessary for ADP-induced Akt phosphorylation, which depends on the Gi-coupled P2Y12 receptor [9,33]. An early study has shown decreased Akt phosphorylation in response to ADP in PI3Kγ-deficient platelets [34], but more recent studies have shown that the contribution of PI3Kγ to Gi-mediated Akt or Rap1b activation is minimal [20,27,35]. Moreover, it has been shown recently that both PI3Kβ and PI3Kγ are required for ADP-induced Akt phosphorylation whereas ADP-induced Rap1b activation and platelet aggregation occurs independently of both isoforms [28]. In addition, other studies have shown that Gi-dependent Rap1b and Akt activation is inhibited by TGX-221 indicating that PI3Kβ has an important role in P2Y12/Gi signalling in platelets [20,27]. A further recent study has reported an important role for PI3Kβ in regulating the avidity of high-affinity integrin αIIbβ3 receptors [36]. It has also been suggested that PI3Kβ plays a key role in PI3K signalling downstream of GPCRs (G-protein-coupled receptors) and GPVI (glycoprotein VI) [20,24,25,27,37,38]. Consistent with previous findings, the results from the present study show that 2MeSADP-induced platelet aggregation is significantly inhibited by the PI3Kβ inhibitor TGX-221, whereas the PI3Kα inhibitor PIK75, the PI3Kγ inhibitor AS252424 and the PI3Kδ inhibitor IC87114 have little or no effect on platelet aggregation, indicating an important role for PI3Kβ in the ADP-induced platelet aggregation. However, our results do not exclude the contribution of other PI3K isoforms, because some degree of platelet aggregation and TXA2 generation occurs in the presence of TGX-221 when compared with wortmannin.

We have shown previously that ERK is activated downstream of P2Y receptors and ERK plays an important role in ADP-mediated TXA2 generation [30]. In addition, we, and others, have also shown that P2Y12 receptor potentiates PAR (protease-activated receptor)-mediated TXA2 generation through the regulation of ERK phosphorylation [39,40]. Therefore, on the basis of these findings, we evaluated the role of PI3K downstream of P2Y12/Gi signalling in 2MeSADP-induced TXA2 generation and ERK phosphorylation. We showed that there was a significant inhibition of 2MeSADP-induced TXA2 generation in the presence of TGX-221 or wortmannin, suggesting an important role for PI3Kβ in ADP-induced TXA2 generation. Similarly, 2MeSADP-induced ERK phosphorylation was significantly inhibited in the presence of TGX-221. A partial inhibition of ERK phosphorylation was observed in the presence of the PI3Kα inhibitor PIK75 suggesting there is contribution from PI3Kα, but TGX-221 had a similar extent of inhibition on TXA2 generation and ERK phosphorylation compared with wortmannin, which indicates that the function of PI3Kβ is less redundant than PI3Kα. Taken together, our results show that PI3Kβ is the main PI3K isoform in ADP-mediated TXA2 generation and that it functions by regulating ERK phosphorylation. The mechanism of ERK phosphorylation is quite complicated and could be regulated by the Ras/Raf pathway, via PKCs or by other mechanisms, and the present study is unable to provide the mechanism by which PI3Kβ regulates ERK phosphorylation.

Gz signalling triggered by adrenaline through the α2A adrenergic receptor can supplement the Gi signalling [41], and it has been shown that combined stimulation of Gi signalling, through the P2Y12 receptor, and Gz signalling, through the α2A adrenergic receptor, causes platelet aggregation and TXA2 generation [26,29]. In addition, inhibition of MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] and subsequent ERK phosphorylation has been shown to block Gi plus Gz-mediated TXA2 generation [30], indicating that ERK also plays an important role in Gi plus Gz-mediated TXA2 generation. As we have shown previously that PI3K inhibition completely blocks platelet aggregation induced by Gi plus Gz signalling [29], we have examined the effect of PI3K isoforms on platelet aggregation, TXA2 generation and ERK phosphorylation caused by the simultaneous stimulation of the Gi and Gz pathways. Our results illustrate that inhibition of PI3Kβ significantly inhibited Gi plus Gz-mediated platelet aggregation, TXA2 generation and ERK phosphorylation. This suggests that PI3Kβ plays an important role in Gi plus Gz-mediated platelet aggregation and TXA2 generation through the regulation of ERK phosphorylation. It is known that the α2A adrenergic receptor agonist adrenaline mimics P2Y12 receptor-mediated Gi signalling events, and PI3Kβ plays an important role downstream of P2Y12-mediated Gi signalling. Similarly, PI3Kβ plays an important role when P2Y12 receptor stimulation was blocked and replaced by stimulation with adrenaline (Gq plus Gz) [20], which is consistent with our study (Gi plus Gz) and supports further an important role for PI3Kβ in Gi/Gz signalling processes.

We, and others, have reported that the response to ADP depends on Gi-coupled P2Y12 receptor for Akt activation in platelets, and PI3K activity is necessary for Akt activation in platelets [9,33]. In order to determine which PI3K isoforms are involved in ADP-induced Akt activation in platelets, we have used PI3K inhibitors. Consistent with the effect of isoform-specific PI3K inhibitors on ADP-induced ERK phosphorylation, we have found that the PI3Kβ inhibitor TGX-221 completely inhibited 2MeSADP-induced Akt phosphorylation, confirming an important role for PI3Kβ in ADP-mediated platelet responses. However, a partial inhibition of Akt phosphorylation was observed in the presence of the PI3Kα inhibitor PIK75 suggesting the contribution of PI3Kα in this event, whereas AS252424 or IC87114 had little or no effect on Akt phosphorylation. TGX-221 has the same extent of inhibition on Akt phosphorylation compared with the pan-PI3K inhibitor wortmannin, and it appears that the function of PI3Kβ is less redundant than PI3Kα. We conclude that PI3Kβ plays a major role in ADP-induced Akt phosphorylation although PI3Kα may have a minor role in this event. These results suggest that ADP depends on PI3Kβ to induce Akt phosphorylation.

ERK plays an important role in ADP-induced TXA2 generation, but the inhibition of ERK has no effect on ADP-induced platelet aggregation in aspirin-treated platelets [30]. In the present study, we found that wortmannin or the PI3Kβ inhibitor TGX-221 inhibits 2MeSADP- and Gi plus Gz-mediated platelet aggregation in both non-aspirin- and aspirin-treated platelets. Thus this suggests that PI3Kβ mediates the inhibitory effect on 2MeSADP- and Gi plus Gz-mediated platelet aggregation in non-aspirin-treated platelets through the regulation of ERK phosphorylation and subsequent TXA2 generation, whereas the inhibitory effect of PI3Kβ in aspirin-treated platelets occurs through the regulation of PI3K downstream signalling molecules other than ERK. Akt is one of the central downstream effectors of PI3K, and it is known that Akt plays an important role in platelet aggregation [33,42]. As the present study identified an important role for PI3Kβ in ADP-induced Akt phosphorylation, it is possible that PI3Kβ regulates ADP- and Gi plus Gz-induced platelet aggregation in aspirin-treated platelets through the regulation of Akt phosphorylation.

In conclusion, we have demonstrated that that the PI3Kβ inhibitor TGX-221 and PI3K inhibitor wortmannin blocked 2MeSADP-mediated platelet aggregation, TXA2 generation, and Akt and ERK phosphorylation, confirming that the PI3Kβ isoform plays an essential role in ADP-mediated platelet responses.

AUTHOR CONTRIBUTION

Analia Garcia and Soochong Kim equally contributed to the experimental design and performed research, analysed data and wrote the manuscript; Kamala Bhavaraju measured thromboxane generation; Simone Schoenwaelder helped with the experiments and Satya Kunapuli directed the research, interpreted experiments and edited the manuscript prior to acceptance.

FUNDING

This work was supported by National Institutes of Health [grant numbers HL60683, HL80444 (to S. P. K.) and K01 HL092586. (to A. G.)].

Abbreviations: ERK, extracellular-signal-regulated kinase; 2MeSADP, 2-methyl-thio-ADP; HRP, horseradish peroxidase; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PRP, platelet-rich plasma; TX, thromboxane; TBST, Tris-buffered saline/Tween 20

References

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