Insulin-like growth factor-1 regulates platelet activation through PI3-Kα isoform

At the site of vascular injury, platelets aggregate and release growth factors such as insulin-like growth factors (IGFs), platelet-derived growth factor, epidermal growth factor, endothelial cell growth factor, and transforming growth factor-β.^1,,,–5  IGF-1 plays an important role in wound healing and platelets release IGF-1 from α granules upon activation.⁶  ADP can mediate α-granule release in washed human platelets.⁷  Receptors for IGF-1 have been demonstrated on platelet surface membranes.⁸  A previous study has reported that IGF-1 can potentiate ADP-, collagen-, and thrombin-induced platelet aggregation.⁹  Recent study has shown that P2Y₁₂ receptor–mediated Akt activation requires IGF-1 receptor in C6 glioma cells.¹⁰  However, the functional role of IGF-1 and its signaling mechanisms in platelet activation are unclear

IGF-1 mediates the biological function by binding to its transmembrane receptor (IGF-1 receptor), and the biological actions of IGF-1 are modulated by a family of at least 6 IGF-binding proteins.¹¹  IGF-1 receptor contains a 30-residue signal peptide rich in polar amino acids, followed by α and β subunits.¹²  IGF-1 receptor is similar to the insulin receptor in structural features and consists of an α₂β₂ configuration, where 2 αβ subunits are linked by secondary disulfide bonds. Binding of IGF-1 to its receptor induces receptor autophosphorylation of 3 tyrosine residues in the intracellular kinase domain of the β subunit and results in activation of the tyrosine kinase activity of the IGF-1 receptor. The activated IGF-1 receptor phosphorylates intracellular substrate insulin receptor substrate-1 (IRS-1) and SH2-containing protein Shc (src homology domain–containing protein). Phosphorylated IRS-1, in turn, binds to certain SH2 domain–containing proteins such as phosphatidylinositol 3-kinase (PI3-K),¹³  Grb-2, and Syp.¹²  Activated PI3-K mediates Akt activation, which contributes to the survival signal of IGF-1.^14,15 

ADP is an important agonist for platelet activation and causes platelets to change their shape, aggregate, and release contents of granules. ADP-induced platelet aggregation requires coactivation of the G_q-coupled P2Y₁ and G_i-coupled P2Y₁₂ receptors.^16,17  The G_q-coupled P2Y₁ receptor is responsible for inositol phosphate formation through the activation of phospholipase C, leading to mobilization of calcium and platelet shape change.¹⁸  The G_i-coupled P2Y₁₂ receptor leads to the inhibition of adenylyl cyclase (AC)^19,20  and activates PI3-K,²¹  Akt,^22,23  and Rap1b.²⁴  Moreover, G_z signaling triggered by epinephrine mimicked the P2Y₁₂ receptor–mediated signaling events, thus restoring the ability of ADP to induce platelet aggregation in the presence of P2Y₁₂ receptor antagonists.²⁵ 

The activation of PI3-K has been shown to be implicated in platelet aggregation.²⁶  There are 3 families of PI3-K (classes I, II, and III). The class I PI3-K is responsible for agonist-induced PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ production and plays a role in the activation of integrin α_IIbβ₃. The class I PI3-K is subdivided into class IA (α, β, and δ) and class IB (γ) based on their distinct p110 catalytic subunits and modes of regulation.²⁷  The class IA isoforms have p55–85 regulatory subunits and are classically regulated by tyrosine kinases, whereas the class IB isoform has a p101 regulatory subunit and is activated by G protein–coupled receptors.²⁷  Recent studies have reported the selective inhibitors of these PI3-K isoforms.^28,,,,–33  Platelets express all class I PI3-K isoforms with lower levels of p110δ.³⁴  It is known that PI3-K γ activation is thought to be mediated by the βγ complexes dissociated from G_i proteins upon receptor activation,³⁵  and exerts a significant role in ADP-induced platelet aggregation.²¹  It is also shown that PI3-K β has an important role in ADP-induced platelet aggregation.³¹ 

Stimulation of platelets with various agonists results in Ser⁴⁷³ and Thr³⁰⁸ phosphorylation of Akt, which results in Akt activation.³⁶  The PI3-K products PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ trigger the simultaneous phosphorylation of Akt, and Akt functions as one of the important downstream effectors of PI3-K.^37,38  We and others have shown that the G_i-coupled P2Y₁₂ ADP receptor is responsible for a significant proportion of activation of Akt, and ADP-induced Akt phosphorylation requires G_i signaling.^22,23 

In this study, we have demonstrated a role for IGF-1 and its signaling mechanisms in platelet activation. Because IGF-1 is important for the proliferation and survival of numerous cell types, and PI3-K/Akt pathways are implicated in the survival of various cancer cells, we focused on the role of PI3-K/Akt in IGF-1–mediated signaling in platelets. We found that IGF-1 can potentiate platelet aggregation induced by a number of agonists, including ADP. The potentiating effect of IGF-1 on platelet aggregation occurs in the absence of protein kinase C (PKC) activation or intracellular calcium mobilization and abolished by the presence of the PI3-K inhibitor wortmannin or the PI3-Kα inhibitor PIK-75. IGF-1 induces Akt activation through PI3-Kα by supplementing G_i-dependent pathway in platelets. Therefore, we conclude that IGF-1 potentiates platelet aggregation by synergizing with G_i but not G_q signaling through the PI3-Kα pathway.

Approval for this study was obtained from the Institutional Review Board of Temple University (Philadelphia, PA). Informed consent was obtained in accordance with the Declaration of Helsinki.

IGF-1, epinephrine, 2-MeSADP, MRS-2179, apyrase (type V), CDP-Star chemiluminescent substrates, and bovine serum albumin (BSA; fraction V) were purchased from Sigma (St Louis, MO). SFLLRN and AYPGKF were custom synthesized by Invitrogen (Carlsbad, CA). Anti–phospho-Akt (Ser⁴⁷³) antibody was purchased from Cell Signaling Technology (Beverly, MA). Anti-PKCδ antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Alkaline phosphatase (AP)–labeled secondary antibody was from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Wortmannin and LY294002 were from Biomol Research Laboratories (Plymouth Meeting, PA). TGX-221 was purchased from Cayman Chemical (Ann Arbor, MI). PIK-75, AS-252424, and IC87114 were from the lab of Shaun Jackson (Monash University, Victoria, Australia). 5,5′-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (dimethyl BAPTA) was obtained from Molecular Probes (Eugene, OR). AR-C69931MX was gift from AstraZeneca (Loughborough, United Kingdom). Bisindolylmaleimide I (GF 109203X) and AG 1024 were from Calbiochem (San Diego, CA). YM-254890 was a generous gift from Yamanouchi Pharmaceutical (Ibaraki, Japan). All other reagents were reagent grade, and deionized water was used throughout.

Whole blood was obtained from a pool of healthy volunteers in a one-sixth volume of acid-citrate-dextrose (2.5 g sodium citrate, 1.5 g citric acid, and 2.0 g glucose in 100 mL H₂O). Platelet-rich plasma (PRP) was prepared by centrifugation of citrated blood at 230g for 20 minutes at room temperature (RT). Acetylsalicylic acid was added to PRP to a final concentration of 1 mM, and the preparation was incubated for 45 minutes at 37°C followed by centrifugation at 980g for 10 minutes at RT. The platelet pellet was resuspended in Tyrode buffer (138 mM NaCl, 2.7 mM KCl, 2 mM MgCl₂, 0.42 mM NaH₂PO₄, 5 mM glucose, 10 mM HEPES; N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid [pH 7.4], and 0.2% BSA) containing 0.05 U/mL apyrase. This low concentration of apyrase is not enough to block responses to ADP, but will prolong the responsiveness of platelets to ADP by preventing desensitization of the P2Y receptors. These conditions have been standardized in the laboratory. The platelet count was adjusted to 2 × 10⁸ cells/mL.

Platelet aggregation was measured using a lumi-aggregometer (Chrono-Log, Havertown, PA) at 37°C under stirring conditions (900 rpm). A 0.5-mL sample of aspirin-treated washed platelets was stimulated with different agonists, and change in light transmission was measured. Platelets were preincubated with each inhibitor (where noted) as follows: 100 μM MRS-2179, 100 nM AR-C69931MX, 100 μM AG 1024, 10 μM dimethyl-BAPTA, 10 μM GF109203X, 100 nM wortmannin, 25 μM LY294002, 500 nM PIK-75, 500 nM TGX-221, 2 μM AS-252424, 1 μM IC87114, and 50 nM YM-254890 before agonist stimulation in some experiments. The chart recorder (Kipp and Zonen, Bohemia, NY) was set for 0.2 mm/second.

Platelets were stimulated with agonists under nonstirring condition for the appropriate time, and the reaction was stopped by the addition of 3 × sodium dodecyl sulfate (SDS) sample buffer. In some experiments, wortmannin (100 nM), LY294002 (25 μM), PIK-75 (500 nM), TGX-221 (500nM), AS-252424 (2 μM), IC87114 (1 μM), and YM-254890 (50 nM) were added and incubated for 5 minutes at 37°C without stirring before agonist stimulation. Samples were separated on 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membrane. Nonspecific binding sites were blocked by incubation in Tris-buffered saline/Tween (TBST; 20 mM Tris, 140 mM NaCL, 0.1% [vol/vol] Tween 20) containing 0.5% (wt/vol) milk protein and 3% (wt/vol) BSA for 30 minutes at RT, and membranes were incubated overnight at 4°C with primary antibody (1:1000 in TBST/2% BSA) with gentle agitation. After 3 washes for 5 minutes each with TBST, the membranes were probed with the AP-labeled goat anti-rabbit IgG (1:5000 in TBST/2% BSA) for 1 hour at RT. After additional washing steps, membranes were then incubated with CDP-Star chemiluminescent substrates for 10 minutes at RT, and chemiluminescence was measured using a Fujifilm LAS-3000 Luminescent Image Analyzer (Fuji, Tokyo, Japan).

As previously reported,⁹  IGF-1 alone was unable to induce platelet shape change and aggregation. In contrast, IGF-1 potentiated platelet aggregation induced by a low dose of 2-MeSADP, whereas IGF-1 had no effect on platelet aggregation induced by a higher dose of 2-MeSADP. Figure 1A shows that partial aggregation induced by 30 nM 2-MeSADP was potentiated by increasing concentrations of IGF-1, where the potentiaing effect of IGF-1 was already detectable at 100 nM IGF-1. However, 100 nM 2-MeSADP–induced full aggregation was not affected, even in the presence of a higher concentration of IGF-1 (1 μM). The tyrphostins are a family of synthetic protein tyrosine kinase inhibitors, and AG 1024 has been shown to be highly selective against IGF-1, among other tyrphostins.³⁹  Platelet aggregation induced by 30 nM 2-MeSADP was inhibited by the IGF-1 receptor selective tyrosine kinase inhibitor AG 1024 (Figure 1MB), and the potentiating effect of IGF-1 on 2-MeSADP–induced platelet aggregation was also suppressed by AG 1024. In addition, 100 nM 2-MeSADP–induced full aggregation that was not enhanced by exogenous IGF-1 was not affected by AG 1024 (data not shown). These results demonstrate that IGF-1 potentiates 2-MeSADP–induced platelet aggregation upon its release from α granules.

2-MeSADP requires signaling through both G_q-coupled P2Y₁ and G_i-coupled P2Y₁₂ receptors to cause platelet aggregation.¹⁷  As shown in Figure 2, selective activation of G_q pathways by 2-MeSADP in the presence of the G_i-coupled P2Y₁₂ receptor antagonist AR-C69931MX resulted in platelet shape change. IGF-1 in the presence of G_q-selective stimulation caused platelet aggregation, suggesting the contribution of IGF-1 in platelet aggregation by substituting for G_i-signaling pathways.

2-MeSADP (30 nM) was unable to cause platelet aggregation in the presence of G_q-coupled P2Y₁ receptor antagonist MRS-2179. However, the combination of G_i stimulation with IGF-1 caused partial platelet aggregation without shape change (Figure 2). It has been shown that a higher dose of ADP causes partial platelet aggregation without shape change in G_q- or P2Y₁-deficient mouse platelets, indicating that P2Y₁₂-mediated activation of G_i induces platelet aggregation.^40,–42  Thus, these results suggest that IGF-1 causes partial platelet aggregation by enhancing G_i-signaling pathways.

Because it is also possible that potentiation of platelet aggregation induced by IGF-1 can be mediated by complementing G_z-signaling pathways, we tested the effect of IGF-1 with the combination of G_z signaling. We have previously shown that costimulation of G_i and G_z signaling is sufficient to cause aggregation in both human and mouse platelets.⁴³  Selective stimulation of G_z signaling through α_2A adrenergic receptor with 10 μM epinephrine did not cause platelet aggregation, but the combination of IGF-1 (100 nM) with epinephrine caused partial platelet aggregation without shape change (Figure 2) in a dose-dependent manner from 100 nM to 1 μM (data not shown). These results are similar to the costimulation of G_i signaling with ADP and G_z signaling with epinephrine,⁴³  confirming that IGF-1 substitutes for G_i-dependent pathways to trigger platelet aggregation in combination with G_z signaling.

In order to identify the intracellular signaling mediators that contribute to the potentiating effect of IGF-1 on platelet aggregation, we tested effects of inhibiting calcium, PKC, and PI3-K on the aggregation induced by IGF-1. As shown in Figure 3A, IGF-1 was able to potentiate 2-MeSADP–induced platelet aggregation in the presence of the pan-PKC inhibitor GF109203X (10 μM) or the intracellular calcium chelator dimethyl BAPTA (10 μM). Despite its potentiating effect on 2-MeSADP–induced platelet aggregation, IGF-1 failed to potentiate platelet aggregation induced by 2-MeSADP in the presence of the PI3-K inhibitor wortmannin (100 nM), suggesting that IGF-1 requires PI3-K activation to induce potentiation of platelet aggregation.

To better understand the role of individual PI3-K isoforms, we have evaluated the effect of specific PI3-K isoforms on IGF-1–mediated potentiation of 2-MeSADP–induced platelet aggregation by using PI3-Kα inhibitor PIK-75, PI3-Kβ inhibitor TGX-221, PI3-Kγ inhibitor AS-252424, and PI3-Kδ inhi-bitor IC87114.^30,,–33  IGF-1 failed to potentiate 2-MeSADP–induced platelet aggregation in the presence of PIK-75, whereas IGF-1 was able to potentiate 2-MeSADP–induced platelet aggregation in the presence of TGX-221, AS-252424, or IC87114(Figure 3B,C), indicating the important role of the PI3-Kα isoform in this potentiating event by IGF-1. When platelets were treated with another PI3-Kα inhibitor, compound 15e,²⁸  IGF-1 was also unable to potentiate 2-MeSADP–induced platelet aggregation (data not shown), confirming a critical role for PI3-Kα.

To verify the effect of IGF-1 on platelet function, we have evaluated the effect of IGF-1 on other agonist-induced platelet aggregation. Platelet aggregation induced by low concentrations of the PAR1-activating peptide SFLLRN was potentiated by IGF-1, which was abolished by the presence of AG 1024 (Figure 4A). IGF-1 had no effect on platelet aggregation induced by higher concentrations of SFLLRN (data not shown). Inhibition of PI3-K by wortmannin completely abolished the potentiating effect of IGF-1 on SFLLRN-mediated platelet aggregation (Figure 4B), confirming an important role for PI3-K in IGF-1 signaling. G_i stimulation by secreted ADP has been shown to contribute to low concentrations of PAR agonist-mediated platelet aggregation. IGF-1 was able to potentiate SFLLRN-induced platelet aggregation in the presence of the P2Y₁₂ receptor antagonist AR-C69931MX, indicating that the potentiating effect of IGF-1 on SFLLRN-induced platelet aggregation is independent of secreted ADP. Similar results were observed with the PAR4-activating peptide AYPGKF (data not shown).

It is known that Akt functions as 1 of the major downstream effectors of PI3-K,^37,38  and PI3-K/Akt plays an important role in the G_i-dependent potentiation of platelet aggregation.^22,26  It is also known that ADP-induced Akt phosphorylation requires G_i signaling.^22,23  To determine the effect of IGF-1 on Akt phosphorylation, we exposed platelets to different concentrations of IGF-1 ranging from 100 nM to 1 μM, and Akt phosphorylation (Ser⁴⁷³) was measured at 3 minutes after the addition of agonist. IGF-1 induced a concentration-dependent increase in Akt phosphorylation (Figure 5A). A weak Ser⁴⁷³ phosphorylation was detectable at a concentration of 100 nM IGF-1, and higher concentrations revealed increased Akt phosphorylation. In order to determine the potentiating effect of IGF-1 on Akt phosphorylation, we exposed platelets to different concentrations of IGF-1 in the presence of 2-MeSADP. Figure 5B shows that IGF-1 potentiated, in a concentration-dependent manner, Akt phosphorylation mediated by 2-MeSADP. Simultaneous addition of IGF-1 even at the concentration of 100 nM to 2-MeSADP–stimulated platelets resulted in a significant increase in Akt phosphorylation compared with that induced by 2-MeSADP alone, suggesting that IGF-1 is capable of potentiating Akt phosphorylation mediated by G_i pathways.

As PI3-K is the main upstream regulator of Akt, we studied the role of PI3-K in IGF-1–induced Akt phosphorylation. We evaluated the contribution of PI3-K isoforms to Akt phosphorylation by using different PI3-K selective inhibitors.^30,,–33  It is known that ADP-induced Akt phosphorylation depends on G_i stimulation through PI3-K.^22,23  Immunoblot analysis revealed that Akt phosphorylation induced by IGF-1 was completely abolished by the PI3-K inhibitors LY204002 and wortmannin (Figure 6A), confirming the essential role of PI3-K in this event. IGF-1-induced Akt phosphorylation was completely inhibited by the PI3-K p110α inhibitor PIK-75, whereas the PI3-K p110β inhibitor TGX-221, the PI3-K p110γ inhibitor AS-252424, and the PI3-K p110δ inhibitor IC87114 showed little or no effect on Akt phosphorylation induced by IGF-1. It appears that IGF-1 requires PI3-K p110α for Akt phosphorylation. In addition, IGF-1 failed to potentiate Akt phosphorylation mediated by 2-MeSADP in the presence of wortmannin and PIK-75 (Figure 6B), confirming the essential role of PI3-K, especially PI3-K p110α, in the potentiation of 2-MeSADP–mediated Akt phosphorylation by IGF-1.

We have shown that G_12/13 signaling has a potentiating effect on Akt phosphorylation when combined with selective G_i stimulation through PI3-K in platelets.⁴⁴  To confirm the contribution of IGF-1 to Akt phosphorylation, we selectively activated G_12/13 signaling with SFLLRN or AYPGKF in the presence of the G_q-selective inhibitor YM-254890⁴⁵  and measured the effect of combined stimulation of G_12/13 and IGF-1 on Akt phosphorylation. As shown in Figure 7, selective stimulation of G_12/13 signaling with SFLLRN and AYPGKF in the presence of YM-254890 failed to induce Akt phosphorylation. However, IGF-1 in the presence of YM-254890 restored Akt phosphorylation to the extent achieved by these agonists, confirming that G_12/13 signaling potentiates Akt phosphorylation induced by IGF-1 through supplemental G_i signaling.

IGF-1 in platelets is important because IGF-1 is released from the platelet immediately after tissue injury directly into the tissue, where additive and synergistic actions with other growth factors in α granules, including platelet-derived growth factor, epidermal growth factor, and transforming growth factor-β, can promote tissue repair.⁴⁶  The IGF-1 in platelets is important not only as early determinants of wound healing, but could be involved in other platelet functions, such as the maintenance of vascular integrity and atherogenesis.⁴⁷  Therefore, the primary aim of present study was to determine the functional role of IGF-1 and its intracellular signaling events in platelet activation.

Even though IGF-1 alone fails to induce platelet aggregation, platelet aggregation induced by submaximal concentrations of 2-MeSADP, SFLLRN, and AYPGKF is potentiated by the addition of IGF-1 (100 nM), suggesting that IGF-1 plays an important role in potentiating platelet aggregation. This potentiating effect is observed with a much lower concentration of exogenously added IGF-1, as low as 10 nM (76 ng/mL) IGF-1 (data not shown). The normal physiologic range of IGF-1 in plasma is 150 to 400 ng/mL, suggesting the physiologic relevance of the potentiating effect of IGF-1. ADP has been shown to mediate α granule release in washed human platelets.⁷  2-MeSADP–induced platelet aggregation and the potentiaing effect of IGF-1 on 2-MeSADP–induced platelet aggregation was inhibited in the presence of AG 1024, suggesting that IGF-1 may have a direct role in platelet function, and IGF-1 secreted from the α granule can potentiate platelet aggregation under physiologic conditions.

ADP-induced platelet aggregation requires coactivation of both the G_q-coupled P2Y₁ receptor and the G_i-coupled P2Y₁₂ receptor.¹⁷  The P2Y₁ receptor is essential for intracellular calcium mobilization and platelet shape change, whereas the P2Y₁₂ receptor leads to inhibition of AC.^18,48  Even though selective P2Y₁ receptor or P2Y₁₂ receptor activation alone has been shown to be unable to cause platelet aggregation, we found that IGF-1 can trigger platelet aggregation in combination with selective P2Y₁ receptor activation, suggesting that IGF-1 supplements G_i-signaling pathways. Our results also show that the combination of IGF-1 and P2Y₁₂ receptor activation triggered platelet aggregation without shape change. It is known that high concentrations of ADP can induce partial platelet aggregation without calcium mobilization or platelet shape change in P2Y₁ receptor– or G_q-deficient mouse platelets,^41,42  indicating that P2Y₁₂ receptor/G_i signaling can induce this partial platelet aggregation that is dependent on PI3-K.⁴⁰  In addition, IGF-1 was able to potentiate platelet aggregation in the presence of calcium chelator or PKC inhibitor, indicating that the potentiating effect of IGF-1 on platelet aggregation is independent of intracellular calcium and PKC activation. These results suggest that IGF-1 may exert its potentiating effect on platelet aggregation by synergizing G_i, but not G_q, signaling. While G_12/13 signaling does not have a direct effect on Akt phosphorylation, it plays an important role in potentiating Akt phosphorylation mediated by G_i pathways in platelets.⁴⁴  Selective stimulation of G_12/13 signaling significantly increases the Akt phosphorylation in combination with IGF-1 stimulation, further indicating that IGF-1 can supplement G_i-signaling pathways.

Since G_z signaling triggered by epinephrine through the α_2A adrenergic receptor can supplement G_i signaling,²⁵  it was necessary to differentiate the effect of IGF-1 on G_z-signaling pathways. G_z signaling alone is not able to cause platelet aggregation.⁴⁹  It has been shown that combined stimulation of G_i signaling through the P2Y₁₂ receptor and G_z signaling through the α_2A adrenergic receptor causes platelet aggregation, where G_i or G_z signaling alone did not cause platelet aggregation.^40,43  Epinephrine-mediated signaling through G_z pathways or IGF-1 stimulation per se did not induce platelet aggregation, but the combination of epinephrine and IGF-1 was able to induce platelet aggregation without shape change. These results confirm that IGF-1 triggers platelet aggregation in combination with G_z signaling by selectively supplementing G_i signaling. However, the effect of IGF-1 may not be due to the inhibition of AC through G_i signaling because the platelet aggregation induced by combination of G_i and G_z signaling is independent of inhibition of AC.⁴⁰ 

The activation of PI3-K downstream of G_i pathways is implicated in stabilization of platelet aggregation.²⁶  It has been shown that PI3-K activity is required for P2Y₁₂ receptor–mediated platelet aggregation.⁴⁰  Our results using the PI3-K inhibitor wortmannin show that PI3-K activity is necessary for the potentiating effect of IGF-1 on platelet aggregation. However, it was not clear which subtype of PI3-K (α, β, δ, or γ) is involved in this potentiating effect because of the lack of selectivity of wortmannin on PI3-K subtypes. It is reported that PI3-Kγ exerts a significant role in ADP-induced platelet aggregation.²¹  It is also shown that PI3-Kβ has an important role in sustaining platelet aggregation in response to weak agonist stimulation.³¹  To identify the subtype of PI3-K involved in platelet aggregation induced by IGF-1, we have used several PI3-K inhibitors.^29,,,–33  Our results show that IGF-1 is unable to potentiate platelet aggregation in the presence of the PI3-K p110α inhibitors PIK-75 and compound 15e, whereas IGF-1 can exert its potentiating effect in the presence of the PI3-K p110β inhibitor TGX-221, the PI3-K p110γ inhibitor AS-252424, or the PI3-K p110δ inhibitor IC87114, indicating the essential role of PI3-K p110α in this potentiating effect by IGF-1. Thus, IGF-1 and 2-MeSADP may use different signaling pathways to activate different isoforms of PI3-K and potentiate each other to induce platelet aggregation.

As PI3-K is known to be an upstream regulator of Akt, one would expect Akt phosphorylation to occur downstream of IGF-1. It has been shown that activation of the P2Y₁₂ receptor leads to Akt activation through IGF-1 receptor cross-talk in C6 glioma cells.¹⁰  In fact, stimulation of platelets with IGF-1 resulted in Akt phosphorylation. We and others have reported that ADP depends on the G_i-coupled P2Y₁₂ receptor for Akt activation in platelets, and PI3-K activity is necessary for Akt activation in platelets.^22,23  Our results using LY294002 and wortmannin show that PI3-K is required for IGF-1–mediated Akt phosphorylation. However, it is not clear which PI3-K isoforms are involved in Akt activation in platelets. Previous study has shown that Akt phosphorylation induced by ADP is partially inhibited in PI3-Kγ knockout mouse platelets.²¹  It is also shown that PI3-K p110β is the major regulator of the P2Y₁₂/G_i-signaling process linked to sustained platelet aggregation and G_i activation of Rap1b.³¹  We have found that Akt phosphorylation induced by IGF-1 was completely inhibited in the presence of PIK-75, whereas TGX-221, AS-252424, or IC87114 had little or no effect on Akt phosphorylation. We have also found that PIK-75 prevented Akt phosphorylation induced by costimulation of IGF-1 and 2-MeSADP. These results suggest that IGF-1 and 2-MeSADP may use different signaling events that synergize to Akt activation, and IGF-1 depends on PI3-K p110α to induce Akt phosphorylation. Thus, IGF-1 may require PI3-K p110α to cause PtdIns(3,4,5)P₃ production, and PtdIns(3,4,5)P₃ production downstream of IGF-1 is abolished in the absence of PI3-K p110α, which results in the complete inhibition of Akt phosphorylation.

In conclusion, we demonstrate that IGF-1 potentiates platelet aggregation induced by low concentrations of agonists, including 2-MeSADP. These effects occur in the absence of intracellular Ca²⁺ increase or PKC activation. IGF-1 induces Akt phosphorylation through PI3-K p110α and mediates its potentiating effect by supplementing G_i signaling through PI3-K p110α pathways.

This work was supported by research grants HL60683 and HL80444 from the National Institutes of Health (NIH; to S.P.K.) and by NIH training grant HL00777 in thrombosis (to A. G.).

National Institutes of Health

Contribution: S.K. designed and performed experiments, analyzed data, and wrote the paper. A.G. performed experiments. S.P.J. contributed vital new reagents and analyzed data. S.P.K. was responsible for the overall direction, designed experiments, and analyzed data.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Satya P. Kunapuli, Department of Physiology, Temple University School of Medicine, 3420 N Broad St, Philadelphia, PA 19140; e-mail:spk@temple.edu.