Two Waves of Platelet Secretion Induced by Thromboxane A2 Receptor and a Critical Role for Phosphoinositide 3-Kinases
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.m301838200
ISSN1083-351X
AutoresLI Zhen-yu, Guoying Zhang, Guy C. Le Breton, Xiaopei Gao, Asrar B. Malik, Xiaoping Du,
Tópico(s)Antiplatelet Therapy and Cardiovascular Diseases
ResumoThromboxane A2 (TXA2)-mediated platelet secretion and aggregation are important in thrombosis. Here, we present a novel finding that the stable TXA2 analogue, U46619, induces two waves of platelet secretion, each of which precedes a distinct wave of platelet aggregation. ADP released from platelets during the first wave of secretion played a major role in augmenting the first wave of platelet aggregation. The second wave of platelet secretion and aggregation required the first wave of both ADP secretion and aggregation and were blocked by either the integrin inhibitor RGDS or a P2Y12 receptor antagonist, indicating a requirement for both the integrin outside-in signal and ADP-activated Gi pathway. U46619 stimulated phosphoinositide 3-kinase (PI3K)-dependent phosphorylation of Akt, which was augmented by ADP but did not require integrin outside-in signaling. Platelets from PI3Kγ knock-out mice or PI3K inhibitor-treated platelets showed an impaired second wave of platelet secretion and aggregation. However, the second wave of platelet aggregation was restored by addition of exogenous ADP to PI3Kγ deficient or PI3K inhibitor-treated platelets. Thus, our data indicate that PI3K, together with the integrin outside-in signaling, play a central role in inducing the second wave of platelet secretion, which leads to the second wave of irreversible platelet aggregation. Thromboxane A2 (TXA2)-mediated platelet secretion and aggregation are important in thrombosis. Here, we present a novel finding that the stable TXA2 analogue, U46619, induces two waves of platelet secretion, each of which precedes a distinct wave of platelet aggregation. ADP released from platelets during the first wave of secretion played a major role in augmenting the first wave of platelet aggregation. The second wave of platelet secretion and aggregation required the first wave of both ADP secretion and aggregation and were blocked by either the integrin inhibitor RGDS or a P2Y12 receptor antagonist, indicating a requirement for both the integrin outside-in signal and ADP-activated Gi pathway. U46619 stimulated phosphoinositide 3-kinase (PI3K)-dependent phosphorylation of Akt, which was augmented by ADP but did not require integrin outside-in signaling. Platelets from PI3Kγ knock-out mice or PI3K inhibitor-treated platelets showed an impaired second wave of platelet secretion and aggregation. However, the second wave of platelet aggregation was restored by addition of exogenous ADP to PI3Kγ deficient or PI3K inhibitor-treated platelets. Thus, our data indicate that PI3K, together with the integrin outside-in signaling, play a central role in inducing the second wave of platelet secretion, which leads to the second wave of irreversible platelet aggregation. Platelets play a central role in hemostasis and thrombosis and help maintain the integrity of the vessel wall. At sites of vascular injury, platelets are activated by various soluble and immobilized agonists, leading to shape change, aggregation, and secretion of granule contents. Platelet activation includes a series of rapid positive feedback loops that greatly amplify the activation signals and enable robust platelet recruitment at the site of vascular injury. An important mechanism of this response amplification is the synthesis of thromboxane A2 (TXA2) 1The abbreviations used are: TXA2, thromboxane A2; TP, thromboxane-prostanoid; PI3K, phosphoinositide 3-kinase; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; A3P5P, adenosine-3′-phosphate-5′-phosphate; 2MeSAMP, 2-methylthioadenosine 5′-monophosphate triethylammonium salt. via the cyclooxygenase-mediated arachidonic metabolic pathway (1Samuelsson B. Goldyne M. Granstrom E. Hamberg M. Hammarstrom S. Malmsten C. Annu. 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In addition, dissociation of Gα subunits from Gβγ subunits induced by receptor occupancy allows Gβγ subunits to activate phosphoinositide 3 kinase (PI3K), resulting in increases in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, and activation of Akt (20Kucera G.L. Rittenhouse S.E. J. Biol. Chem. 1990; 265: 5345-5348Abstract Full Text PDF PubMed Google Scholar, 21Rittenhouse S.E. Blood. 1996; 88: 4401-4414Crossref PubMed Google Scholar). PI3K is a family of enzymes that phosphorylate the D3 hydroxyl group of phosphoinositides. Platelets express both the Class IA PI3K (PI3Kα, PI3Kβ) and Class IB (PI3Kγ) isoforms (21Rittenhouse S.E. Blood. 1996; 88: 4401-4414Crossref PubMed Google Scholar, 22Hirsch E. Bosco O. Tropel P. Laffargue M. Calvez R. Altruda F. Wymann M. Montrucchio G. FASEB J. 2001; 15: 2019-2021Crossref PubMed Scopus (207) Google Scholar). It has been established that Gβγ subunits activate PI3Kγ (23Stoyanov B. Volinia S. Hanck T. Rubio I. 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Blood. 2002; 99: 151-158Crossref PubMed Scopus (108) Google Scholar). However, the specific role of PI3K in signaling platelet activation and, in particular, in TXA2-induced platelet activation remains unclear. Among the unresolved issues in TXA2-induced platelet activation pathway is the relationship between the TXA2-induced platelet secretion and aggregation. Previous studies showed evidence that TXA2-induced secretion follows initial wave of platelet aggregation, and the initial wave of platelet aggregation is independent of platelet secretion (32Charo I.F. Feinman R.D. Detwilter T.C. Smith J.B. Ingerman C.M. Silver M.J. Nature. 1977; 269: 66-69Crossref PubMed Scopus (62) Google Scholar). However, it has also been shown that ADP and ADP-induced P2Y12-dependent Gαi pathway are important in TXA2-induced initial platelet aggregation (19Paul B.Z. Jin J. Kunapuli S.P. J. Biol. Chem. 1999; 274: 29108-29114Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 33Dangelmaier C. Jin J. Smith J.B. Kunapuli S.P. Thromb. Haemostasis. 2001; 85: 341-348Crossref PubMed Scopus (91) Google Scholar). To resolve the discrepancy, and to understand the specific role of PI3Ks in TXA2-induced platelet activation, we present a new finding that TXA2 receptor induces two waves of platelet secretion. ADP from the first wave of secretion is important in augmenting the first wave of platelet aggregation, which in turn is important in the triggering the second wave of platelet secretion, and thus the second wave of platelet aggregation. Furthermore, we present novel evidence that PI3K, by cooperating with the integrin outside-in signal, plays a critical role in signaling the induction of second wave of platelet secretion. These results suggest a novel response amplification mechanism in TXA2-induced platelet activation. Materials—The TXA2 analog U46619, and PI3K inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), were purchased from Calbiochem. TXA2 antagonists, SQ29,548 and BMS180,291, were obtained from Cayman Chemicals (Ann Arbor, MI) and Bristol-Myers Squibb Co., respectively. ADP, apyrase (grade III), adenosine-3′-phosphate-5′-phosphate (A3P5P), and 2-methylthioadenosine 5′-monophosphate triethylammonium salt (2MeSAMP) were purchased from Sigma. A rabbit polyclonal antibody against a recombinant human Akt 1 fragment (amino acid residues 345–480) was purchased from Santa Cruz Biotechnology Inc., and a polyclonal antibody against phosphorylated Ser473 of Akt was from Cell Signaling Technology. Mouse Platelet Preparation—PI3Kγ knock-out mice were generously provided by Dr. Josef M. Penninger, Amgen Institute, Toronto) (34Crackower M. Oudit G. Kozieradzki I. Sarao R. Sun H. Sasaki T. Hirsch E. Suzuki A. Shioi T. Irie-Sasaki J. Sah R. Cheng H. Rybin V. Lembo G. Fratta L. Oliveira-dos-Santos A. Benovic J. Kahn C. Izumo S. Steinberg S. Wymann M. Backx P. Penninger J. Cell. 2002; 110: 737-749Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). Wild type C57BL/6 mice (obtained from Jackson Laboratory, Bar Harbor, ME) were used as controls. Mice were bred and maintained in the University of Illinois Animal Care Facility following institutional and National Institutes of Health guidelines after approval by the Animal Care Committee. Washed platelets from PI3Kγ knock-out and wild type mice were prepared as described previously (35Li Z. Xi X. Gu M. Feil R. Ye R. Eigenthaler M. Hofmann F. Du X. Cell. 2003; 112: 77-86Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Briefly, male and female mice (6–8 weeks) were anesthetized by intraperitoneal injection of pentobarbital. Whole blood from homozygous PI3Kγ knockout and wild type mice was collected from the inferior vena cava using 17 volume of ACD (85 mm trisodium citrate, 83 mm dextrose, and 21 mm citric acid) as anticoagulant. For each experiment, platelets from blood were pooled from five to six mice of each genotype. Platelets were then washed twice with CGS (0.12 m sodium chloride, 0.0129 m trisodium citrate, and 0.03 m d-glucose, pH 6.5), resuspended in modified Tyrode's buffer (36Du X.P. Plow E.F. Frelinger A.L. O'Toole T.E. Loftus J.C. Ginsberg M.H. Cell. 1991; 65: 409-416Abstract Full Text PDF PubMed Scopus (420) Google Scholar) at 3 × 108/ml, and incubated at room temperature for 2 h before use. Platelet Aggregation and Secretion—Platelet aggregation was measured by detecting changes in light transmission. Platelet secretion was determined by measuring the release of ATP using luciferin/luciferase reagent (Chrono-lume) (Chrono-Log, Havertown, PA). Luciferin/luciferase (12 μl) was added to 238 μl of washed platelet suspension within 2 min before stimulation. Platelet aggregation and secretion were recorded in real time in a Chrono-Log lumi-aggregometer at 37 °C with stirring (1000 rpm). To examine the effects of PI3K inhibitors, platelets were preincubated with wortmannin (100 nm) or LY294002 (20 μm) for 5 min prior to the addition of U46619. Assessment of Fibrinogen Binding—Washed mouse platelets (200 μl, 2 × 108/ml in Tyrode's buffer) were preincubated with LY294002 (20 μm), Me2SO, or RGDS (2 mm) at room temperature for 5 min. Platelets were then incubated with Oregon Green 488-labeled fibrinogen (30 μg/ml) in the presence or absence of U46619 (1 μm) at 37 °C in the aggregometer for various lengths of time and then analyzed by flow cytometry using FACScalibur. Western Blot Analysis of Akt Phosphorylation in Platelets—Washed platelets (3 × 108/ml) were resuspended in modified Tyrode's buffer and incubated at room temperature for 2 h before use. Platelets were stimulated with U46619 (1 μm) in a platelet aggregometer at 37° for 5 min and then solubilized in SDS-PAGE sample buffer. Platelet lysates were analyzed by SDS-PAGE on 4–15% gradient gel and electrotransfered to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk in TBS (20 mm Tris-HCl, 150 mm NaCl, pH 7.5) and incubated with a polyclonal anti-Akt antibody (Santa Cruz) or a polyclonal antibody specific for the phosphorylated Ser473 of Akt (New England Biolabs) at 22 °C for 2 h. After three washes in TBS containing 0.05% Tween 20, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (0.5 μg/ml) for 45 min. After further washing, reactions were visualized using an Amersham-Pharmacia enhanced chemiluminescence kit. Human Platelet Preparation and Aggregation—Fresh blood from healthy volunteers was anticoagulated with 17 volume of ACD (36Du X.P. Plow E.F. Frelinger A.L. O'Toole T.E. Loftus J.C. Ginsberg M.H. Cell. 1991; 65: 409-416Abstract Full Text PDF PubMed Scopus (420) Google Scholar). Platelets were washed with CGS buffer, resuspended in Tyrode's buffer, and allowed to incubate at room temperature for 1 h as described previously (37Li Z. Xi X. Du X. J. Biol. Chem. 2001; 276: 42226-42232Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Platelet aggregation and secretion was measured in a Chrono-Log lumi-aggregometer as described above for mouse platelets. To examine the effects of PI3K, platelets were preincubated with wortmannin (100 nm) or LY294002 (20 μm) for 5 min at 37 °C. U46619 Induces Two Waves of Secretion and Two Waves of Aggregation in Mouse and Human Platelets—To determine the roles of platelet secretion in TXA2-induced platelet aggregation, we examined platelet secretion and aggregation induced by a stable TXA2 analog, U46619, using washed platelets from wild type C57BL/6 mice and healthy human donors. Platelet aggregation and ATP release as a marker for secretion from dense granules were monitored in a lumi-aggregometer in real time. Using this method, we found that U46619 induced two waves of secretion and two waves of aggregation in mouse (Fig. 1) and human platelets (Fig. 2). The first wave of platelet secretion occurred very quickly following addition of U46619, which is followed sequentially by the first wave of platelet aggregation, the second wave of platelet secretion, and the second wave of platelet aggregation. Both waves of secretion and platelet aggregation were abolished by TXA2 receptor antagonists, SQ29548 and BMS180291, indicating that they were induced via the platelet TP receptor (Figs. 1, A and B, and 2A).Fig. 2U46619-induced two waves of secretion and aggregation in human platelets. Washed platelets from healthy donors (3 × 108/ml) in modified Tyrode's buffer were preincubated with buffer (Control) or 1 μm SQ29548 (SQ) (A) or buffer (Control), 10 μm 2MeSAMP, 1 unit/ml apyrase, or 0.5 mm A3P5P at 37 °C for 5 min (B). Luciferin/luciferase reagent and then 1 μm U46619 were added to platelets to induce aggregation and secretion, which were recorded in real time using the lumi-aggregometer.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To study the causal links between the two waves of secretion and aggregation, we examined the role of secreted ADP and integrin outside-in signaling in U46619-induced platelet secretion and aggregation using an ADP scavenger, apyrase, antagonists of two different ADP receptors (P2Y1 and P2Y12) and an integrin inhibitor, RGDS. We found that the first wave of secretion was not affected by 2MeSAMP, a selective antagonist of ADP P2Y12 receptor (Gi-coupled) (Figs. 1C and 2B) or by A3P5P, a selective ADP P2Y1 receptor antagonist (Fig. 2B). The first wave of platelet secretion also occurred when platelet aggregation was inhibited by RGDS (Fig. 1E) or in the absence of stirring (not shown), indicating that the first wave of platelet secretion does not require integrin outside-in signaling. On the other hand, the first wave of platelet aggregation induced by U46619 was significantly inhibited by 2MeSAMP or apyrase (Figs. 1, C and D, and 2B), but not by A3P5P (Fig. 2B). However, inhibition of the first wave of platelet aggregation by 2MeSAMP or apyrase appeared to be incomplete as a small change in the light transmission was still visible (Figs. 1, C and D, and 2B) and was more obvious at a higher U46619 concentration (data not shown). Thus, ADP from the first wave secretion, by activating Gi-coupled P2Y12 receptor pathway, plays important roles in augmenting TP receptor-mediated first wave platelet aggregation. In addition to its effect on the first wave of platelet aggregation, the first wave of platelet ADP secretion was also required for the U46619-induced second wave of platelet secretion, as the second wave of platelet secretion was completely inhibited by 2MeSAMP (Figs. 1C and 2B). The second wave of secretion was not only inhibited by ADP receptor antagonists, but also abolished by an inhibitor of integrin, RGDS peptide (Fig. 1E). Thus, both the ADP released during the first wave of secretion and the integrin outside-in signaling during the first wave of platelet aggregation are required for the U46619-induced second wave of secretion. It is important to note that the second wave of platelet secretion induced by U46619, unlike that induced by other agonists such as ADP, does not require endogenous TXA2 synthesis as aspirin-treated platelets also showed two waves of secretion and aggregation in response to U46619 (cf. Fig. 6). As expected, the second wave of platelet aggregation was dependent on platelet secretion, because it was inhibited by 2MeSAMP and apyrase (Figs. 1, C and D, and 2B). U46619-induced, PI3K-dependent Akt Phosphorylation—The data presented above indicate that the secreted ADP plays important roles in TXA2-induced platelet secretion and aggregation. As previous studies showed that the ADP-induced platelet activation involved PI3-Kγ, we examined whether and how PI3Kγ is involved in TXA2-induced platelet activation. To do this, we first examined the U46619-induced Akt phosphorylation, which is a known marker of PI3K activity. Phosphorylation of Akt was detected by immunoblotting using an anti-Akt antibody recognizing the phosphorylated Ser473 site of Akt. As shown in Fig. 3, Akt was phosphorylated in U46619- or ADP-stimulated wild type mouse platelets. Phosphorylation of Akt induced by ADP was completely inhibited by the ADP scavenger, apyrase (Fig. 3A). U46619-induced Akt phosphorylation, however, was only partially inhibited by apyrase (Fig. 3A), indicating that U46619-induced PI3K activation was augmented by ADP secretion, but was not totally ADP-dependent. The P2Y12 inhibitor, 2MeSAMP, but not P2Y1 inhibitor, A3P5P, also partially inhibited U46619-induced Akt phosphorylation (Fig. 3B). Thus, ADP augments PI3K activation and subsequent Akt phosphorylation via P2Y12-coupled pathway. Akt phosphorylation induced by either ADP or U46619 was not inhibited by RGDS (Fig. 3A), indicating that activation of PI3K by ADP or U46619 was independent of integrin outside-in signaling. U46619-induced phosphorylation of Akt was markedly inhibited in PI3Kγ-deficient platelets (Fig. 3C) and completely inhibited by PI3K inhibitors. Thus, U46619-induced Akt phosphorylation indicates PI3K activation, and PI3K is activated during U46619-induced platelet activation. U46619-stimulated Second Wave of Secretion and Aggregation Is Inhibited in PI3Kγ-deficient Platelets—To determine whether PI3Kγ is important in TXA2-induced platelet secretion and aggregation, we examined U46619-induced aggregation and secretion of platelets from PI3Kγ knock-out mice and control wild type mice. As shown in Fig. 4, U46619, at lower concentrations (250–500 nm), induced a typical two-wave platelet secretion in wild type mice, which preceded the onset of two-waves of platelet aggregation (Fig. 4, A and B). The first wave of secretion of PI3Kγ knock-out mouse platelets induced by low concentrations of U46619 was not significantly different from the wild type platelets, but the second wave of platelet secretion was abolished in PI3Kγ knock-out mouse platelets (Fig. 4, A and B). The low dose U46619-induced second wave of platelet aggregation in PI3Kγ-deficient platelets was also reduced (Fig. 4, A and B). These results indicate that PI3Kγ plays an important role in the second wave of platelet secretion and aggregation induced by low dose U46619. At higher U46619 concentrations, however, the second wave of platelet secretion and aggregation in PI3Kγ knock-out platelets became increasing similar to wild type platelets (Fig. 4C), suggesting that there might be alternative pathways or other PI3K isoforms that were also involved in platelet activation under these conditions. Effects of PI3K Inhibitors on U46619-induced Platelet Secretion and Aggregation in Mice—To determine whether other isoforms of PI3K such as class IA PI3K may also play roles in TXA2-induced platelet aggregation, we examined the effects of PI3K inhibitors, Wortmannin (Fig. 5) and LY294002 (not shown), on U46619-induced aggregation and secretion of mouse platelets. These inhibitors have been shown to inhibit not only PI3Kγ, but also class IA PI3Ks. Wortmannin (and LY294002, data not shown) completely abolished U46619-induced second wave of secretion, but had no significant effect on the first wave of secretion (Fig. 5). At a low U46619 concentration, wild type platelets treated with wortmannin (Fig. 5A) were similar to PI3Kγ knock-out platelets in secretion and aggregation (see Fig. 4), suggesting that, at low U46619 concentrations, PI3Kγ is the major enzyme responsible for the role of PI3K in inducing the second wave of platelet secretion and aggregation. In contrast to the PI3Kγ knock-out, PI3K inhibitors, wortmannin or LY294002, also blocked the second wave of platelet secretion and aggregation induced by high concentrations of U46619 (>1 μm) (Fig. 5B). Furthermore, these inhibitors inhibited high dose U46619-induced second wave of secretion and aggregation in PI3Kγ knock-out platelets (Fig. 5C). These results indicate that at high concentrations of U46619, the isoforms of PI3K other than PI3Kγ can substitute the role of PI3Kγ in inducing the second wave of platelet secretion and aggregation. Effects of PI3K Inhibitors on U46619-induced Integrin Activation—It is known that platelet aggregation is the consequence of activation of fibrinogen binding function of integrin αIIbβ3. Thus we also examined the effects of PI3K inhibitors on integrin-dependent fibrinogen binding to platelets at 1, 2, and 5 min time points following addition of U46619. As shown in Fig. 5A, at the 1-min time point, the first wave, but not the second wave, of platelet secretion and aggregation occurred. At this time, there is an increase in fibrinogen binding to platelets, which is minimally affected by PI3K inhibitor LY294002 (Fig. 5D). At the 2-min time point, just after onset of the second wave of platelet secretion and aggregation, fibrinogen binding is only partially inhibited by LY294002. At 5 min after the peak of the second wave of platelet secretion, however, fibrinogen binding to platelets was dramatically increased, which was inhibited by LY294002 to the level equivalent to before the onset of the second wave of platelet secretion and aggregation. These results indicate that the PI3K inhibitor only affects the second wave secretion-dependent integrin activation. These data indicate that integrin activation that coincides with the second wave of platelet aggregation is subsequent to the second wave of platelet secretion. These data also indicate that PI3K plays a minimal role in the first wave of integrin activation and aggregation, but plays an important role in the second wave secretion-dependent amplification of integrin activation. The Effects of PI3K Inhibitors in U46619-induced Platelet Activation in Human Platelets—The above results showed that PI3K plays important roles in inducing the second wave of platelet secretion and aggregation in mouse platelets. To determine the role of PI3K in TXA2-induced human platelet activation, washed human platelets were preincubated with PI3K inhibitors, wortmannin and LY294002, and then exposed to U46619 to induce platelet aggregation and secretion. Both wortmannin and LY294002 inhibited the U46619-induced second wave of secretion and aggregation (Fig. 6, A and B). Furthermore, human platelets treated with aspirin also showed two waves of platelet secretion and aggregation (Fig. 6C), and the second wave of platelet secretion and aggregation was inhibited by PI3K inhibitors (Fig. 6D), indicating that PI3K-dependent second wave platelet secretion and aggregation were independent of endogenous TXA2 synthesis pathway. These results indicate that PI3K plays an important role in U46619-induced second wave of platelet secretion and aggregation in human platelets. PI3Kγ Knock-out and PI3K Inhibitors Inhibit Platelet Aggregation Indirectly by Abolishing the Second Wave of ADP Secretion—As shown above, PI3K plays important roles in the second wave of platelet secretion and aggregation. To determine whether the inhibitory effect of PI3K deficiency on the second wave of platelet aggregation resulted from its inhibitory effect on secretion, we examined whether exogenous ADP could induce the second wave of aggregation in PI3Kγ knock-out mouse platelets and wortmannin-treated human platelets. Fig. 7A shows that wild type platelets showed a typical two-wave platelet aggregation, and PI3Kγ knock-out platelets showed a significantly reduced second wave of platelet aggregation. Adding exogenous ADP to PI3Kγ knock-out platelets completely reversed the inhibitory effect of PI3Kγ deficiency and restored full scale platelet aggregation. This effect of exogenous ADP was only slightly affected by the P2Y1 ADP receptor inhibitor, A3P5P, but was totally blocked by a P2Y12 ADP receptor inhibitor, 2MeSAMP, which is similar to U46619-induced wild type platelet aggregation in the absence of exogenous ADP. Similarly, the inhibitory effects of PI3K inhibitor, wortmannin, was also reversed by addition of exogenous ADP, and this effect of exogenous ADP was completely inhibited by P2Y12 antagonist, 2MeSAMP (Fig. 7B). These data suggest that diminished second wave of platelet aggregation in PI3Kγ knock-out or wortmannin-treated platelets results from inhibition of the second wave secretion of ADP and ADP-induced P2Y12 signaling pathway. Thus, the second wave of platelet secretion is required for the second wave of platelet aggregation. PI3Ks play important roles in inducing the second wave of platelet secretion and thus are indirectly involved in the second wave of platelet aggregation. In this study, we present the new finding that TXA2-mediated platelet activation involves two waves of platelet secretion which, respectively, precede two waves of platelet aggregation. We have identified the sequential relationship between the two waves of secretion and aggregation. More importantly, we present novel evidence that PI3K plays a critical role in signaling the induction of second wave of platelet secretion. These data allow us to propose a new concept in the TXA2-induced platelet activation mechanism as depicted in Fig. 7C. Previous studies on the TXA2-induced platelet secretion using platelet-rich plasma or radiolabeled serotonin showed evidence of platelet secretion following the first wave of platelet aggregation and that the first wave of platelet aggregation occurred in the absence of detectable secretion (32Charo I.F. Feinman R.D. Detwilter T.C. Smith J.B. Ingerman C.M. Silver M.J. Nature. 1977; 269: 66-69Crossref PubMed Scopus (62) Google Scholar). However, recent studies using newly developed ADP receptor antagonists suggested that the ADP receptor- and P2Y12-mediated and Gαi-dependent signaling pathway may also play a role in the first wave of platelet aggregation induced by low dose U46619 (19Paul B.Z. Jin J. Kunapuli S.P. J. Biol. Chem. 1999; 274: 29108-29114Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar), although it is unclear whether P2Y12 receptor is activated by ADP secreted prior to the first wave of platelet aggregation, since platelet secretion was believed to follow the first wave of platelet aggregation. By detecting real time platelet secretion using the more sensitive luciferase assay in washed platelets, we show that TXA2 receptor induces two waves of platelet secretion and that the first wave of platelet secretion occurs almost immediately after addition of the agonist and precedes the first wave of platelet aggregation. Furthermore, the first wave platelet aggregation induced by low dose U46619 is significantly inhibited by P2Y12 antagonists and by apyrase. Thus, our data explain a previous discrepancy on the roles of ADP secretion in TXA2-induced platelet activation and suggest that ADP released from the TXA2-induced first wave secretion plays a critical role in augmenting the first wave platelet aggregation induced by TXA2. Furthermore, these data demonstrate a previously unknown kinetics of platelet secretion induced by TXA2 pathway, which allows further dissection of the different mechanisms associated with different waves of platelet secretion. ADP is important not only in augmenting first wave of platelet aggregation, but also in inducing the second wave of platelet secretion. This new role of ADP in TXA2-induced platelet activation is shown by the data that the second wave of U46619-induced platelet secretion is abolished by the P2Y12 antagonist. One may argue that this role of ADP is secondary to the effects of ADP on the first wave of platelet aggregation, because the integrin antagonist, RGDS, also prevented the second wave of platelet secretion. However, we found that ADP plays an important role in augmenting the TXA2-induced activation of PI3Ks, which is independent of first wave of platelet aggregation but is necessary for the induction of the second wave of platelet secretion (Figs. 3 and 4). It should be noted that it has been well established that PI3K can be activated by the Gβγ-coupled signaling pathway (23Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. et al.Science. 1995; 269: 690-693Crossref PubMed Scopus (642) Google Scholar, 24Stephens L.R. Eguinoa A. Erdjument-Bromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwallader K. Tempst P. Hawkins P.T. Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 25Zhang J. Benovic J.L. Sugai M. Wetzker R. Gout I. Rittenhouse S.E. J. Biol. Chem. 1995; 270: 6589-6594Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Thus, the presence of G protein-coupled agonist, TXA2, in combination of secreted ADP (also G protein-coupled) is sufficient to activate PI3Ks, although this does not exclude that integrin outside-in signaling may also be involved in PI3K activation as indicated previously (28Kovacsovics T.J. Bachelot C. Toker A. Vlahos C.J. Duckworth B. Cantley L.C. Hartwig J.H. J. Biol. Chem. 1995; 270: 11358-11366Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 38Banfic H. Tang X. Batty I.H. Downes C.P. Chen C. Rittenhouse S.E. J. Biol. Chem. 1998; 273: 13-16Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Since we show that the integrin inhibitor RGDS had no effect on PI3K activation induced by U46619, it appears that in U46619-stimulated platelets, PI3K activation is independent of integrin outside-in signaling. Thus our finding suggests a novel mechanism of induction of the second wave of platelet secretion, in which both the G protein-coupled PI3K activation (augmented by ADP) and integrin outside-in signaling are required in parallel to induce the second wave of platelet secretion. As the aggregation-dependent secretion of granule contents occurs in response to not only TXA2 but also many other agonists, our finding may be a general mechanism of aggregation-dependent platelet secretion. In this regard, previous data have shown that aggregation of PI3Kγ knock-out platelets in response to ADP was decreased in a manner consistent with the loss of the secondary wave of platelet aggregation (22Hirsch E. Bosco O. Tropel P. Laffargue M. Calvez R. Altruda F. Wymann M. Montrucchio G. FASEB J. 2001; 15: 2019-2021Crossref PubMed Scopus (207) Google Scholar). PI3K has been previously known to be important in platelet activation (21Rittenhouse S.E. Blood. 1996; 88: 4401-4414Crossref PubMed Google Scholar, 22Hirsch E. Bosco O. Tropel P. Laffargue M. Calvez R. Altruda F. Wymann M. Montrucchio G. FASEB J. 2001; 15: 2019-2021Crossref PubMed Scopus (207) Google Scholar, 28Kovacsovics T.J. Bachelot C. Toker A. Vlahos C.J. Duckworth B. Cantley L.C. Hartwig J.H. J. Biol. Chem. 1995; 270: 11358-11366Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 29Trumel C. Payrastre B. Plantavid M. Hechler B. Viala C. Presek P. Martinson E.A. Cazenave J.P. Chap H. Gachet C. Blood. 1999; 94: 4156-4165Crossref PubMed Google Scholar). However, how PI3K is involved in platelet activation is not clear. We conclude that PI3K is important in mediating the second wave of platelet secretion, which augments integrin activation and thus induces the second wave of platelet aggregation. In support of this conclusion, we have shown that 1) PI3Kγ knock-out or PI3K inhibitors diminished the second wave of platelet secretion and aggregation, but had minimal effects on the first wave of platelet secretion, integrin activation, and aggregation; 2) PI3K inhibitors inhibited the dramatic increase in the second wave secretion-dependent fibrinogen binding to platelets to the level before the onset of the second wave of platelet secretion; and 3) the effects of PI3Kγ knock-out or PI3K inhibitors on the second wave of aggregation was reversed by exogenous ADP. Thus PI3K provides the necessary signal cooperating with the integrin outside-in signals to induce platelet secretion. The effect of PI3K in augmenting integrin activation, and thus platelet aggregation, is the consequence of the role of PI3K in inducing the second wave of platelet secretion of ADP. Platelets express both type IA (PI3Kα and PI3Kβ) and IB (PI3Kγ) classes of PI3K (21Rittenhouse S.E. Blood. 1996; 88: 4401-4414Crossref PubMed Google Scholar, 22Hirsch E. Bosco O. Tropel P. Laffargue M. Calvez R. Altruda F. Wymann M. Montrucchio G. FASEB J. 2001; 15: 2019-2021Crossref PubMed Scopus (207) Google Scholar). It is known that PI3Kγ can be activated by G protein-coupled receptors by binding to the Gβγ subunits (23Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. et al.Science. 1995; 269: 690-693Crossref PubMed Scopus (642) Google Scholar, 24Stephens L.R. Eguinoa A. Erdjument-Bromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwallader K. Tempst P. Hawkins P.T. Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 25Zhang J. Benovic J.L. Sugai M. Wetzker R. Gout I. Rittenhouse S.E. J. Biol. Chem. 1995; 270: 6589-6594Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). We conclude here that PI3Kγ is the major isoform responsible for the PI3K signaling function particularly at low U46619 concentrations, because PI3Kγ knock-out mice showed a prominent defective second wave of platelet secretion and the consequent second wave of platelet aggregation. This conclusion is consistent with a recent report that PI3K IA knock-out did not affect platelet activation induced by G protein-coupled agonists, but only affected the GPIV/Fc receptor γ pathway (39Watanabe N. Nakajima H. Suzuki H. Oda A. Matsubara Y. Moroi M. Terauchi Y. Kadowaki T. Koyasu S. Ikeda Y. Handa M. Blood. 2003; 102: 541-548Crossref PubMed Scopus (82) Google Scholar). Interestingly, PI3K activation is significantly augmented by ADP secretion via the P2Y12-coupled Gi pathway, but is not totally ADP-dependent, suggesting that TXA2 induces PI3K activation via ADP-dependent and -independent mechanisms. Thus, it is possible that TXA2, by activating the TXA2 receptor-coupled G proteins, and more importantly by activating P2Y12-coupled G protein pathway, induces release of Gβγ subunits, which in turn activate PI3Kγ. PI3Kγ by cooperating with the integrin outside-in signal pathway induces the second wave of platelet secretion. At higher U46619 concentrations, however, the second wave of platelet secretion is not inhibited in PI3Kγ knock-out platelets, but is prevented by PI3K inhibitors that also block PI3K IA. Thus, TXA2-induced activation of PI3K IA isoforms is likely to be important under these conditions or to be able to compensate for PI3Kγ. This is consistent with previous reports that PI3K IA is important in platelet activation induced by other agonists such as thrombin (40Zhang J. Shattil S.J. Cunningham M.C. Rittenhouse S.E. J. Biol. Chem. 1996; 271: 6265-6272Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). It is not clear how PI3K IA can be activated by the TXA2 receptor. Although a classic pathway of activation of those PI3K isoforms is the receptor tyrosine kinase pathway, it has been shown that PI3K IA can also be activated by via G protein pathways (26Kurosu H. Maehama T. Okada T. Yamamoto T. Hoshino S. Fukui Y. Ui M. Hazeki O. Katada T. J. Biol. Chem. 1997; 272: 24252-24256Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 27Graness A. Adomeit A. Heinze R. Wetzker R. Liebmann C. J. Biol. Chem. 1998; 273: 32016-33222Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar).
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