Regulation of Endogenous Reactive Oxygen Species in Platelets Can Reverse Aggregation
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《动脉硬化血栓血管生物学》
From the Department of Pharmacology (P.C., A.M.), Georgetown University Medical Center, Washington, DC, and the Whitaker Cardiovascular Institute and Evans Department of Medicine (J.E.F.), Boston University School of Medicine, Boston, Mass.
Correspondence to Jane E. Freedman, 700 Albany Street W507, Boston MA 02118. E-mail Freedmaj@bu.edu
Abstract
Objective— While much is known about the normal activation of platelets, there have been few observations demonstrating reversibility of the aggregation process. Inhibition of phosphoinositide 3-kinase (PI3-kinase) has been shown to cause platelet disaggregation. In addition, NO is a known potent inhibitor of platelet function. In this study, the role of PI3-kinase in the regulation of endogenous platelet NO and the relevance to platelet function was determined.
Methods and Results— Incubation of platelets with PI3-kinase inhibitors led to a dose-dependent increase in platelet NO and cGMP levels that were temporally related to the period of platelet disaggregation. Addition of ferroheme myoglobin eliminated both the augmented NO release and disaggregation. PI3-kinase inhibition decreased the functional activation of NADPH oxidase and this corresponded to decreased superoxide release. To confirm these findings, platelets from NOS III-deficient mice were studied. These platelets did not release NO, and PI3-kinase inhibition led to decreased superoxide but not platelet disaggregation.
Conclusion— Overall, these results indicate that platelet-derived NO contributes to the process of platelet disaggregation. PI3-kinase plays a role in regulating NADPH oxidase-generated superoxide in platelets and, by altering the bioactivity of platelet NO, may be a potential method for reversing platelet aggregation and thrombus formation.
Key Words: NADPH oxidase ? nitric oxide ? phosphoinositide 3-kinase ? platelets ? superoxide
Introduction
The activation of platelets involves a series of coordinated events that are tightly regulated to maintain hemostasis. This involves a conformational change in the extracellular domain of the integrin platelet glycoprotein (GP)IIb-IIIa facilitating the binding of soluble fibrinogen and enabling the formation of cross bridges between platelets essential for platelet aggregation. Engagement of GPIIb-IIIa, which serves as a bidirectional conduit, triggers outside-in signaling, causing platelet aggregation to become irreversible as a result of sustained GPIIb-IIIa activation. While much is known about the normal activation of platelets, there have been few observations demonstrating reversibility of the aggregation process. Although phosphoinositide 3-kinase (PI3-kinase) does not appear to be essential in the initial activation of GPIIb-IIIa, the sustained activation and thus maintenance of aggregation may be due to the generation of the PI3-kinase product PtdIns(3,4)P2 following GPIIb-IIIa engagement.1,2 Therefore, the activation of PI3-kinase is critical for strengthening platelet aggregation. PI3-kinase has been grouped into three different classes based on structural homology, substrate specificity, and regulation mode. Platelet stimulation results in the activation of two classes of PI3-kinase, class I and class II,3 that contribute to the production of the various D3 phosphoinositides.
The effects of NO, a well-established regulator of platelet function, are similar to the effects of PI3-kinase inhibition. Platelet-derived NO, generated by endothelial nitric oxide synthase (eNOS),4 regulates platelet aggregation and adhesion.5,6 Moreover, NO donors are able to reverse GPIIb-IIIa activation,7 suggesting NO plays a consequential role in mediating platelet aggregation. Inhibition of PI3-kinase and the addition of exogenous NO donors appear to have additive platelet inhibitory effects,8 however, the relevance of these findings to endogenous NO release is unclear.
The highly reactive oxygen species superoxide is produced by activated platelets and also acts to regulate platelet function.9 Superoxide generated by stimulated platelets can increase both platelet adhesion and aggregation,9 presumably as a consequence of its reaction with NO and the attenuation of NO bioactivity.
In phagocytotic cells, superoxide is generated via a one-electron reduction of molecular oxygen by the multicomponent NADPH oxidase. The NADPH oxidase complex consists of the membrane-bound cytochrome b558 (composed of the two subunits gp91-phox and p22-phox) and the cytosolic proteins, p47-phox, p67-phox, p40-phox, and a small GTP-binding protein Rac.10 Activation of this highly regulated enzyme involves the phosphorylation and translocation of the cytosolic components to the membrane-bound cytochrome b558, where the catalytically functional oxidase is assembled.10 Initially identified in phagocytotic cells, NADPH oxidase has also been identified in the intact vessel wall11–13 and several cultured vascular cells.14 Indirect evidence suggested the superoxide-generating NADPH oxidase is present in platelets,15 and recently the expression of p22-phox and p67-phox was shown.16 In addition, multiple lines of evidence implicate the lipid products of PI3-kinase in the activation of the phagocyte NADPH oxidase.17 Therefore, the purpose of the present study was to examine the role of PI3-kinase in the regulation of NADPH oxidase-generated superoxide and determine its implications on platelet-derived NO bioactivity and platelet function.
Methods
NOS III-Deficient Mice
All studies were approved by the Institutional Animal Care and Use Committee at Boston University. The generation of mice bearing NOS III gene deletion has been previously described in detail.18 Mice deficient in eNOS (-/-) were compared with c57 wild-type mice representing the strains from which the eNOS-deficient mice were derived (Charles River Laboratories, Cambridge Mass).
Measurement of Platelet Aggregation, NO, and Superoxide Release
Aggregation was induced in washed platelets prepared as previously described19 by the addition of thrombin receptor activating peptide (TRAP) (25 μmol/L). Washed platelets were preincubated with the indicated concentrations of LY294002, wortmannin, diphenyleneiodonium chloride, NG-nitro-L-arginine methyl ester, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol), or vehicle control.6 An NO-selective microelectrode was adapted for use in a standard platelet aggregometer (Payton Associates) to monitor platelet NO production and aggregation simultaneously.6 Platelet NO production was quantified as the integrated signal detected by the microelectrode after platelet activation. Platelet concentration for all experiments was 3x105 platelets/μL.
Ferroheme myoglobin was prepared by a previously described method.20 During the measurement of platelet aggregation and NO release, ferroheme myoglobin (10 μmol/L) was added approximately 100 seconds after TRAP-induced platelet activation at the peak of measured platelet NO release.
Aggregation-dependent superoxide production was measured in a lumiaggregometer (aggregometer with lucigenin detection) as previously described using lucigenin.6 This permits simultaneous measurement of superoxide production and aggregation.
Measurement of Cytosolic Calcium Concentration
Lyophilized aequorin (1 mg) was reconstituted in Chelex-treated deionized H2O containing 7 mmol/L EGTA, 450 mmol/L KCl, and 15 mmol/L HEPES. After washing, the platelets were resuspended in HEPES buffer and 10 μL of 3 mg/mL aequorin solution. Platelet aggregation and aequorin luminescence were measured simultaneously using a lumiaggregometer. To measure intracellular calcium mobilization, EGTA (1 mmol/L) was added to the platelets to bind any free extracellular Ca2+ present prior to the addition of TRAP. Cytosolic calcium concentration was calculated as previously described.21
Phosphorylation of Platelet Proteins
Washed platelets incubated with [32P]orthophosphate (1 mCi/mL) were passed through PD-10 columns to remove free [32P]. Platelet aggregation was stopped by the addition of lysis buffer. In other experiments, the lysate was centrifuged (15 000 g, 5 minutes, 4°C) to separate the Triton X-100-soluble and -insoluble cytoskeletal (CSK) fractions. The CSK fraction was resuspended in LIPA buffer.22 The proteins were resolved on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and immunoblotted with the specified primary polyclonal antibody. Immunoblots were probed with species-specific secondary antibodies coupled to horseradish peroxidase and visualized with chemiluminescent substrate. Autoradiography was used to determine phosphorylation of [32P]-labeled proteins. Band intensities were determined by densitometry.
Measurement of cGMP
Briefly, washed platelets were mixed with 10-5 M volume of 3-isobutyl-methylxanthine (IBMX). Trichloracetic acid (final concentration, 5% vol/vol) was added. The supernatant was extracted, acetylated, and assayed for cGMP by an ELISA methodology using cGMP antiserum.
Statistics
All data are presented as mean±SEM. Significance of difference between groups was evaluated with 1-way ANOVA and an appropriate post hoc comparison. Statistical significance was accepted if the null hypothesis was rejected with a P<0.05.
Results
Effect of PI3-Kinase Inhibition on Platelet Aggregation and NO Release
Platelet aggregation induced by TRAP in the presence of the PI3-kinase inhibitor LY294002 was characterized by an initial primary phase of aggregation that was not significantly different than that observed in vehicle-treated platelets (Figure 1A). However, in LY294002-treated platelets, this primary aggregation phase was followed by progressive disaggregation (reversal of aggregation) of the platelets approximately 2 to 3 minutes after TRAP stimulation (Figure 1A). LY294002 induced a dose-dependent increase in platelet-NO release, primarily as the result of a significant secondary peak of NO released (Figure 1B). This secondary NO peak preceding platelet disaggregation increased platelet NO release from 64.5±17.5 pmol/108 platelets in vehicle-treated platelets to 143.1±22.9 pmol/108 platelets in platelets treated with 25 μmol/L LY294002 (n=5, P<0.04). The dose-dependent increase in NO release and platelet disaggregation was confirmed in experiments using the noncompetitive, structurally dissimilar PI3-kinase inhibitor wortmannin (31±3.9 for control versus 60±1pmol/108 platelets; n=3, *P<0.002 compared with the control). Maximal platelet aggregation was not significantly changed with PI3-kinase inhibition.
Figure 1. PI3-kinase inhibition causes platelet disaggregation. Platelets were preincubated with (A) 25 μmol/L LY294002 or vehicle control for 20 minutes prior to activation with 25 μmol/L TRAP. B, Reversal of aggregation and enhanced NO release induced by PI3-kinase inhibition can be reversed by myoglobin. Platelets were preincubated with 25 μmol/L LY294002 for 20 minutes prior to activation with 25 μmol/L TRAP. Platelet aggregation and NO release were measured. Myoglobin was added at the point of secondary NO release while measuring aggregation and NO release. Platelet aggregation is measured as change in light transmission (%) over time (seconds). The tracings are representative of 8 experiments.
Effect of PI3-Kinase Inhibition on Platelet cGMP Levels
Increased NO is reflected by an accumulation of cGMP as a direct result of the activation of soluble guanylate cyclase. Inhibition of PI3-kinase did not significantly increase platelet cGMP levels in nonstimulated platelets (Figure 2). Thirty seconds after TRAP stimulation, which corresponds in time to the initial release of NO, cGMP levels rapidly increased in vehicle- and LY294002-treated platelets. Seven minutes after platelet stimulation, the platelet cGMP levels remained significantly elevated in the LY294002-treated platelets as compared with the vehicle-treated platelets (220.7±11.7 versus 137.4±21.8 pmol/mL, respectively, n=3, P<0.03), and with the LY294002-treated unstimulated platelets (220.7±11.7 versus 129.6±17.4 pmol/mL, respectively, n=3, P<0.02). The sustained increase in cGMP levels mirrors the increase in platelet-derived NO as a result of the secondary NO release in LY294002-treated platelets.
Figure 2. Effect of PI3-kinase inhibition on platelet cGMP levels. IBMX-treated platelets were stimulated with 25 μmol/L TRAP after a 20-minute incubation with 25 μmol/L LY294002 (black bars) or vehicle control (gray bars). cGMP was measured in nonstimulated platelets, or in platelets in which the reaction was stopped 30 seconds or 7 minutes after TRAP stimulation. The cGMP concentration is expressed as the mean±SEM for three experiments. *P<0.03 compared with control at 7 minutes; P<0.02 compared with nonstimulated LY294002-treated.
Elimination of Platelet-Derived NO and Disaggregation with Ferroheme Myoglobin
The ferrous heme iron of myoglobin binds NO acting as an NO scavenger. To determine if the disaggregation was the result of the LY294002-induced secondary release of NO, ferroheme myoglobin (10 μmol/L) was added to LY294002-treated platelet approximately 100 seconds after TRAP stimulation. The added myoglobin effectively captured the platelet-derived NO, eliminating both the LY294002-induced secondary NO peak and platelet disaggregation (Table and Figure 1B).
Elimination of LY294002-Induced Secondary Release of NO and Disaggregation by Ferroheme Myoglobin
Effect of PI3-Kinase Inhibition on Platelet Cytosolic Calcium
An increase in cytosolic Ca2+ concentration, [Ca2+]i, within the platelets as a result of calcium influx and mobilization of intracellular stores is essential for platelet and eNOS activation. Stimulation induced a transient rise in platelet [Ca2+]i that coincided with the onset of aggregation. There was no significant difference in the transient increase in platelet [Ca2+]i in LY294002-treated platelets and in vehicle-treated platelets (4.6±0.5 and 5.6±0.5 μmol/L, respectively, n=6, P=NS).
Effect of PI3-Kinase Inhibition on Platelet eNOS Phosphorylation
Phosphorylation of eNOS alters the catalytic activity of this enzyme. In platelets, protein kinase C phosphorylation of eNOS is believed to decrease platelet-derived NO.23 Therefore, we evaluated the effect of PI3-kinase inhibition on eNOS phosphorylation in TRAP-stimulated platelets. No significant change in the phosphorylation of eNOS could be detected in the presence of LY294002 (data not shown).
Recent studies have demonstrated that the phosphorylation of eNOS at Ser-1177 in endothelial cells has a significant effect on the activity of the enzyme.24,25 Therefore, the effect of LY294002 on the phosphorylation of Ser-1177 in platelets stimulated with TRAP for increasing durations was examined. The phosphorylation of eNOS Ser-1177 in the CSK fraction increased gradually, peaking 3 minutes after platelet activation and gradually decreasing thereafter (data not shown). However, the PI3-kinase inhibitor LY294002 did not alter the Ser-1177 phosphorylation.
Effect of LY294002 on Platelet-Derived Superoxide
Superoxide produced by aggregating platelets readily reacts with NO,26 reducing its bioactivity. To determine if change in superoxide release played a role in the PI3-kinase dependent increase in NO, platelet-derived superoxide was measured from TRAP-activated platelets. Superoxide release was significantly decreased in stimulated LY294002-treated platelets as compared with stimulated vehicle-treated platelets (0.3±0.03 versus 1.1±0.3 arb. units, respectively, n=3, P<0.05).
To confirm that the effect of PI3-kinase inhibition was the consequence of reduced superoxide, the effects of the membrane-permeable superoxide dismutase (SOD) mimetic tempol (1 mmol/L) on TRAP-stimulated platelets were assessed. Tempol significantly increased TRAP-stimulated NO release from 35.7±4.9 to 60.9±7.7 pmol/108 platelets (n=4, P<0.04) in addition to causing platelet disaggregation (n=4, 11.7±3.2 arb. units, P<0.03 compared with control). Treatment with tempol also decreased platelet superoxide release from 1.1±0.2 to 0.3±0.1 arb. units (n=4, P<0.01 as compared with control).
Role of PI3-Kinase in NADPH Oxidase Activation
Initially identified in phagocytic cells, NADPH oxidase has since been identified as the primary producer of superoxide in vascular tissues.13 To verify NADPH oxidase was present in platelets, the Triton-soluble and CSK fractions isolated from TRAP-stimulated platelets were immunoblotted with antibodies to p67-phox and p47-phox. The presence of both p67-phox and p47-phox in platelets was confirmed in both fractions (data not shown).
Data suggest that PI3-kinase plays a role in the activation of NADPH oxidase27 that requires the translocation of the cytosolic subunits to the membrane-bound cytochrome b558.28 To investigate whether inhibition of PI3-kinase inhibits NADPH oxidase activation in platelets, the CSK fraction of TRAP-stimulated platelets treated with either LY294002 or vehicle were immunoblotted with the anti-p67-phox antibody. Western blot analysis revealed that 3 minutes after TRAP stimulation there was a significant increase in the amount of p67-phox present in the CSK of platelets (Figure 3). However, there was significantly less p67-phox present in the CSK fraction isolated from LY29402-treated platelets 3 minutes after TRAP stimulation.
Figure 3. Effect of PI3-kinase inhibition on translocation of p67-phox to the platelet cytoskeleton. Isolated platelets were preincubated with 25 μmol/L LY294002 (black bars) or vehicle control (gray bars) for 20 minutes and stimulated with 25 μmol/L TRAP for the time periods indicated. The Triton X-100-insoluble fraction was analyzed by Western blot using an antibody specific for p67-phox. Densitometry was performed to quantify the amount of p67-phox protein present and expressed as the mean±SEM (n=3, *P<0.002 compared with nonstimulated control, P<0.03 compared with control at 3 minutes).
Effect of PI3-Kinase Inhibition on NOS III-Deficient Platelets
To confirm the previous findings and determine the relative contribution of NO and superoxide to platelet disaggregation, platelets isolated from mice deficient in eNOS (NOS III) were treated with LY294002 and tested for aggregation, NO, and superoxide release after 25 μmol/L TRAP stimulation. As compared with platelets from control mice, platelets from NOS III-deficient mice did not release NO (Figure 4). Incubation with LY294002 caused disaggregation in the control platelets but not the NOS III-deficient platelets. Interestingly, there was still increased superoxide release in stimulated platelets from NOS III-deficient animals although it was less as compared with control (Figure 4). As compared with LY294002-treated platelets, platelets incubated with vehicle control from either wild-type or NOS III-deficient mice did not demonstrate any disaggregation (data not shown). These data suggest that PI3-kinase regulates superoxide release but NO is required for disaggregation to occur.
Figure 4. Effect of PI3-kinase inhibition on platelets from NOS III-deficient mice. Platelets from control or NOS III-deficient mice were isolated, incubated with LY294002, and stimulated with 25 μmol/L TRAP. Peak platelet aggregation and extent of disaggregation were determined (% light transmittance; n=3; *P<0.05 compared with control). Platelet NO release represents total NO release (arb units; n=3; *P<0.001 compared with control). Platelet superoxide release represents total release (arb. units; n=3; P=ns).
Discussion
In this study PI3-kinase was found to play a role in regulating NADPH oxidase-generated superoxide in platelets, altering the bioactivity of platelet NO that contributes to platelet disaggregation. The progressive reversal of aggregation following initial aggregation as the result of PI3-kinase inhibition in stimulated platelets is consistent with the findings of previous studies.2 Given that disaggregation resulting from PI3-kinase inhibition in TRAP-stimulated platelets correlated with a dose-dependent inhibition of the integrin GPIIb-IIIa activation,2 the activation of PI3-kinase is considered essential for maintaining GPIIb-IIIa in its activated state to sustain aggregation. Likewise, the ability of NO donors to induce platelet disaggregation has been linked to the diminished activation of GPIIb-IIIa and fibrinogen binding.29 The stimulation of platelets induces a conformational change in GPIIb-IIIa (inside-out signaling) leading to the binding of soluble fibrinogen and the onset of platelet aggregation. The prolonged activation of GPIIb-IIIa activation is essential for irreversible aggregation. The real time measurement of platelet-derived NO release concurrent with aggregation shows that platelet disaggregation is invariably preceded by an increase in NO release from platelets, suggesting the increased release of platelet-NO due to the inhibition of PI3-kinase is the cause of the platelet disaggregation. The ability of the NO-scavenger ferroheme myoglobin, which effectively traps NO, to eliminate the platelet disaggregation evoked by PI3-kinase inhibition demonstrates that this secondary release of NO is intimately involved in regulating platelet aggregation. Platelets are anuclear structures, making transfection experiments impossible; therefore, these observations were confirmed using NOS III-deficient platelets.
The secondary release of NO, paralleled by elevated intraplatelet cGMP levels, is unique to inhibition of PI3-kinase. One explanation for this increase in platelet-derived NO is alteration in the activation of eNOS. Phosphorylation is important in the regulation of eNOS activity. Depending on the kinase and residue involved, phosphorylation can result in either an increase or a decrease in enzyme activity. The serine/threonine kinase Akt phosphorylates eNOS, activating the enzyme,24 whereas protein kinase C-mediated phosphorylation has the opposite effect, inhibiting eNOS activation.30 The lack of a significant change in the phosphorylation of eNOS caused by PI3-kinase inhibition indicates the increase in platelet-derived NO as a result of PI3-kinase inhibition is not mediated by eNOS phosphorylation. The importance of eNOS phosphorylation by several serine/threonine kinases downstream of PI3-kinase in the regulation of eNOS function in endothelial cells24 warranted further examination of eNOS phosphorylation at Ser-1177 in platelets using antibodies that detect eNOS only when phosphorylated at this site. Although the phosphorylation of Ser-1177 did change to some extent over time as platelet aggregation progressed, we found no evidence indicating that inhibition of PI3-kinase had any consequence on eNOS phosphorylation at Ser-1177. There are, however, significant differences between endothelial cells and platelets. In addition to lacking nuclei, platelets lack caveolin-1 found within the caveolae and Golgi apparatus of endothelial cells, which binds the majority of inactive eNOS.31 The activation of eNOS requires the disruption of the eNOS-caveolin-1intereaction by the Ca2+/calmodulin complex, eNOS phosphorylation, or possibly both.32 Thus, it is not unreasonable to surmise that the regulation of eNOS by PI3-kinase in the endothelial cell will not be precisely the same as in the platelet. The possibility that PI3-kinase is not involved in the phosphorylation of specific sites of eNOS24 in the platelet cannot be completely ruled out. The resolution of this must await a detailed analysis in the future, as more knowledge regarding these specific sites becomes known.
The significant decrease in platelet superoxide suggests that the increase in platelet-derived NO might be the result of a decrease in the production of the radical oxygen species superoxide. Superoxide and NO react to form peroxynitrite (ONOO-), diminishing NO bioactivity.33 Therefore, one possible explanation for the NO-induced disaggregation caused by PI3-kinase inhibition is a decrease in platelet superoxide. This concept is consistent with a recent study that demonstrated a 50% inhibition of thrombin-induced superoxide release in platelets treated with the PI3-kinase inhibitor wortmannin.34 Moreover, the ability of the SOD mimetic tempol to increase platelet NO and inhibit platelet aggregation, in addition to decreasing platelet superoxide release, indicates that superoxide can modulate the inhibitory action of NO on platelet aggregation. This observation is strengthened by the finding that PI3-kinase inhibition alters superoxide release but does not reverse platelet aggregation in eNOS deficient platelets.
Although the production of platelet-derived superoxide and its proaggregatory effects have been established, the molecular identities of the cellular enzymes responsible for this platelet superoxide production are not well defined. Superoxide production has been ascribed to a variety of different sources including the lipoxygenase pathway, xanthine oxidase, eNOS, and NADPH oxidase.15,16 The change in translocation of NADPH oxidase and the marked decrease in superoxide suggests that NADPH oxidase is partially responsible for the generation of platelet superoxide that regulates the bioactivity of platelet NO. This is consistent with the evidence showing the inhibition of both thrombin- and ADP-induced platelet aggregation, and the potentiation of the antiplatelet effect of the NO donor sodium nitroprusside in diphenyleneiodonium chloride-treated platelets.15
Immunoblot analysis confirmed the presence of two of the cytosolic components, p67-phox and p47-phox, in platelets, although a more detailed identification of the different components of NADPH oxidase will be necessary to fully characterize the molecular structure of this oxidase in platelets. Furthermore, the marked translocation of p67-phox in TRAP-stimulated platelets, blocked by PI3-kinase inhibition, demonstrates the functional activation of NADPH oxidase is regulated in part by PI3-kinase (Figure 3). In neurophils, PI3-kinase has been implicated in the activation of NADPH oxidase.35 Both wortmannin and LY294002 effectively inhibit fMLP-stimulated neutrophil-generated superoxide.35 The diminished release of superoxide caused by PI3-kinase inhibition could be explained by the blunted translocation of p67-phox that precludes the catalysis of the reactive oxygen species by NADPH oxidase.36 Moreover, the temporal correlation of the inhibition of p67-phox translocation, the secondary release of platelet NO, and disaggregation as the result of PI3-kinase inhibition indicate that the effect of PI3-kinase inhibition in TRAP-stimulated platelets can be explained by the abated production of NADPH oxidase-derived superoxide, which serves to limit the bioactivity of platelet-derived NO.
In summary, these results support the conclusion that the disaggregation of platelets produced by PI3-kinase inhibition is due to the decrease in NADPH oxidase-generated superoxide, which increases the bioavailability of platelet-derived NO. The demonstration of a PI3-kinase-regulated NADPH oxidase in platelets that is partially responsible for platelets-derived superoxide suggests an intriguing model for explaining the changes in the bioavailability of platelet NO. The full identification of all the components of the NADPH oxidase in platelets and a better understanding of how PI3-kinase regulates this oxidase may be useful in the development of novel interventions for the control of thrombosis.
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Nisimoto Y, Motalebi S, Han CH, Lambeth JD. The p67(phox) activation domain regulates electron flow from NADPH to flavin in flavocytochrome b(558). J Biol Chem. 1999; 274: 22999–23005.(Patricia Clutton; Anne Mi)
Correspondence to Jane E. Freedman, 700 Albany Street W507, Boston MA 02118. E-mail Freedmaj@bu.edu
Abstract
Objective— While much is known about the normal activation of platelets, there have been few observations demonstrating reversibility of the aggregation process. Inhibition of phosphoinositide 3-kinase (PI3-kinase) has been shown to cause platelet disaggregation. In addition, NO is a known potent inhibitor of platelet function. In this study, the role of PI3-kinase in the regulation of endogenous platelet NO and the relevance to platelet function was determined.
Methods and Results— Incubation of platelets with PI3-kinase inhibitors led to a dose-dependent increase in platelet NO and cGMP levels that were temporally related to the period of platelet disaggregation. Addition of ferroheme myoglobin eliminated both the augmented NO release and disaggregation. PI3-kinase inhibition decreased the functional activation of NADPH oxidase and this corresponded to decreased superoxide release. To confirm these findings, platelets from NOS III-deficient mice were studied. These platelets did not release NO, and PI3-kinase inhibition led to decreased superoxide but not platelet disaggregation.
Conclusion— Overall, these results indicate that platelet-derived NO contributes to the process of platelet disaggregation. PI3-kinase plays a role in regulating NADPH oxidase-generated superoxide in platelets and, by altering the bioactivity of platelet NO, may be a potential method for reversing platelet aggregation and thrombus formation.
Key Words: NADPH oxidase ? nitric oxide ? phosphoinositide 3-kinase ? platelets ? superoxide
Introduction
The activation of platelets involves a series of coordinated events that are tightly regulated to maintain hemostasis. This involves a conformational change in the extracellular domain of the integrin platelet glycoprotein (GP)IIb-IIIa facilitating the binding of soluble fibrinogen and enabling the formation of cross bridges between platelets essential for platelet aggregation. Engagement of GPIIb-IIIa, which serves as a bidirectional conduit, triggers outside-in signaling, causing platelet aggregation to become irreversible as a result of sustained GPIIb-IIIa activation. While much is known about the normal activation of platelets, there have been few observations demonstrating reversibility of the aggregation process. Although phosphoinositide 3-kinase (PI3-kinase) does not appear to be essential in the initial activation of GPIIb-IIIa, the sustained activation and thus maintenance of aggregation may be due to the generation of the PI3-kinase product PtdIns(3,4)P2 following GPIIb-IIIa engagement.1,2 Therefore, the activation of PI3-kinase is critical for strengthening platelet aggregation. PI3-kinase has been grouped into three different classes based on structural homology, substrate specificity, and regulation mode. Platelet stimulation results in the activation of two classes of PI3-kinase, class I and class II,3 that contribute to the production of the various D3 phosphoinositides.
The effects of NO, a well-established regulator of platelet function, are similar to the effects of PI3-kinase inhibition. Platelet-derived NO, generated by endothelial nitric oxide synthase (eNOS),4 regulates platelet aggregation and adhesion.5,6 Moreover, NO donors are able to reverse GPIIb-IIIa activation,7 suggesting NO plays a consequential role in mediating platelet aggregation. Inhibition of PI3-kinase and the addition of exogenous NO donors appear to have additive platelet inhibitory effects,8 however, the relevance of these findings to endogenous NO release is unclear.
The highly reactive oxygen species superoxide is produced by activated platelets and also acts to regulate platelet function.9 Superoxide generated by stimulated platelets can increase both platelet adhesion and aggregation,9 presumably as a consequence of its reaction with NO and the attenuation of NO bioactivity.
In phagocytotic cells, superoxide is generated via a one-electron reduction of molecular oxygen by the multicomponent NADPH oxidase. The NADPH oxidase complex consists of the membrane-bound cytochrome b558 (composed of the two subunits gp91-phox and p22-phox) and the cytosolic proteins, p47-phox, p67-phox, p40-phox, and a small GTP-binding protein Rac.10 Activation of this highly regulated enzyme involves the phosphorylation and translocation of the cytosolic components to the membrane-bound cytochrome b558, where the catalytically functional oxidase is assembled.10 Initially identified in phagocytotic cells, NADPH oxidase has also been identified in the intact vessel wall11–13 and several cultured vascular cells.14 Indirect evidence suggested the superoxide-generating NADPH oxidase is present in platelets,15 and recently the expression of p22-phox and p67-phox was shown.16 In addition, multiple lines of evidence implicate the lipid products of PI3-kinase in the activation of the phagocyte NADPH oxidase.17 Therefore, the purpose of the present study was to examine the role of PI3-kinase in the regulation of NADPH oxidase-generated superoxide and determine its implications on platelet-derived NO bioactivity and platelet function.
Methods
NOS III-Deficient Mice
All studies were approved by the Institutional Animal Care and Use Committee at Boston University. The generation of mice bearing NOS III gene deletion has been previously described in detail.18 Mice deficient in eNOS (-/-) were compared with c57 wild-type mice representing the strains from which the eNOS-deficient mice were derived (Charles River Laboratories, Cambridge Mass).
Measurement of Platelet Aggregation, NO, and Superoxide Release
Aggregation was induced in washed platelets prepared as previously described19 by the addition of thrombin receptor activating peptide (TRAP) (25 μmol/L). Washed platelets were preincubated with the indicated concentrations of LY294002, wortmannin, diphenyleneiodonium chloride, NG-nitro-L-arginine methyl ester, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol), or vehicle control.6 An NO-selective microelectrode was adapted for use in a standard platelet aggregometer (Payton Associates) to monitor platelet NO production and aggregation simultaneously.6 Platelet NO production was quantified as the integrated signal detected by the microelectrode after platelet activation. Platelet concentration for all experiments was 3x105 platelets/μL.
Ferroheme myoglobin was prepared by a previously described method.20 During the measurement of platelet aggregation and NO release, ferroheme myoglobin (10 μmol/L) was added approximately 100 seconds after TRAP-induced platelet activation at the peak of measured platelet NO release.
Aggregation-dependent superoxide production was measured in a lumiaggregometer (aggregometer with lucigenin detection) as previously described using lucigenin.6 This permits simultaneous measurement of superoxide production and aggregation.
Measurement of Cytosolic Calcium Concentration
Lyophilized aequorin (1 mg) was reconstituted in Chelex-treated deionized H2O containing 7 mmol/L EGTA, 450 mmol/L KCl, and 15 mmol/L HEPES. After washing, the platelets were resuspended in HEPES buffer and 10 μL of 3 mg/mL aequorin solution. Platelet aggregation and aequorin luminescence were measured simultaneously using a lumiaggregometer. To measure intracellular calcium mobilization, EGTA (1 mmol/L) was added to the platelets to bind any free extracellular Ca2+ present prior to the addition of TRAP. Cytosolic calcium concentration was calculated as previously described.21
Phosphorylation of Platelet Proteins
Washed platelets incubated with [32P]orthophosphate (1 mCi/mL) were passed through PD-10 columns to remove free [32P]. Platelet aggregation was stopped by the addition of lysis buffer. In other experiments, the lysate was centrifuged (15 000 g, 5 minutes, 4°C) to separate the Triton X-100-soluble and -insoluble cytoskeletal (CSK) fractions. The CSK fraction was resuspended in LIPA buffer.22 The proteins were resolved on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and immunoblotted with the specified primary polyclonal antibody. Immunoblots were probed with species-specific secondary antibodies coupled to horseradish peroxidase and visualized with chemiluminescent substrate. Autoradiography was used to determine phosphorylation of [32P]-labeled proteins. Band intensities were determined by densitometry.
Measurement of cGMP
Briefly, washed platelets were mixed with 10-5 M volume of 3-isobutyl-methylxanthine (IBMX). Trichloracetic acid (final concentration, 5% vol/vol) was added. The supernatant was extracted, acetylated, and assayed for cGMP by an ELISA methodology using cGMP antiserum.
Statistics
All data are presented as mean±SEM. Significance of difference between groups was evaluated with 1-way ANOVA and an appropriate post hoc comparison. Statistical significance was accepted if the null hypothesis was rejected with a P<0.05.
Results
Effect of PI3-Kinase Inhibition on Platelet Aggregation and NO Release
Platelet aggregation induced by TRAP in the presence of the PI3-kinase inhibitor LY294002 was characterized by an initial primary phase of aggregation that was not significantly different than that observed in vehicle-treated platelets (Figure 1A). However, in LY294002-treated platelets, this primary aggregation phase was followed by progressive disaggregation (reversal of aggregation) of the platelets approximately 2 to 3 minutes after TRAP stimulation (Figure 1A). LY294002 induced a dose-dependent increase in platelet-NO release, primarily as the result of a significant secondary peak of NO released (Figure 1B). This secondary NO peak preceding platelet disaggregation increased platelet NO release from 64.5±17.5 pmol/108 platelets in vehicle-treated platelets to 143.1±22.9 pmol/108 platelets in platelets treated with 25 μmol/L LY294002 (n=5, P<0.04). The dose-dependent increase in NO release and platelet disaggregation was confirmed in experiments using the noncompetitive, structurally dissimilar PI3-kinase inhibitor wortmannin (31±3.9 for control versus 60±1pmol/108 platelets; n=3, *P<0.002 compared with the control). Maximal platelet aggregation was not significantly changed with PI3-kinase inhibition.
Figure 1. PI3-kinase inhibition causes platelet disaggregation. Platelets were preincubated with (A) 25 μmol/L LY294002 or vehicle control for 20 minutes prior to activation with 25 μmol/L TRAP. B, Reversal of aggregation and enhanced NO release induced by PI3-kinase inhibition can be reversed by myoglobin. Platelets were preincubated with 25 μmol/L LY294002 for 20 minutes prior to activation with 25 μmol/L TRAP. Platelet aggregation and NO release were measured. Myoglobin was added at the point of secondary NO release while measuring aggregation and NO release. Platelet aggregation is measured as change in light transmission (%) over time (seconds). The tracings are representative of 8 experiments.
Effect of PI3-Kinase Inhibition on Platelet cGMP Levels
Increased NO is reflected by an accumulation of cGMP as a direct result of the activation of soluble guanylate cyclase. Inhibition of PI3-kinase did not significantly increase platelet cGMP levels in nonstimulated platelets (Figure 2). Thirty seconds after TRAP stimulation, which corresponds in time to the initial release of NO, cGMP levels rapidly increased in vehicle- and LY294002-treated platelets. Seven minutes after platelet stimulation, the platelet cGMP levels remained significantly elevated in the LY294002-treated platelets as compared with the vehicle-treated platelets (220.7±11.7 versus 137.4±21.8 pmol/mL, respectively, n=3, P<0.03), and with the LY294002-treated unstimulated platelets (220.7±11.7 versus 129.6±17.4 pmol/mL, respectively, n=3, P<0.02). The sustained increase in cGMP levels mirrors the increase in platelet-derived NO as a result of the secondary NO release in LY294002-treated platelets.
Figure 2. Effect of PI3-kinase inhibition on platelet cGMP levels. IBMX-treated platelets were stimulated with 25 μmol/L TRAP after a 20-minute incubation with 25 μmol/L LY294002 (black bars) or vehicle control (gray bars). cGMP was measured in nonstimulated platelets, or in platelets in which the reaction was stopped 30 seconds or 7 minutes after TRAP stimulation. The cGMP concentration is expressed as the mean±SEM for three experiments. *P<0.03 compared with control at 7 minutes; P<0.02 compared with nonstimulated LY294002-treated.
Elimination of Platelet-Derived NO and Disaggregation with Ferroheme Myoglobin
The ferrous heme iron of myoglobin binds NO acting as an NO scavenger. To determine if the disaggregation was the result of the LY294002-induced secondary release of NO, ferroheme myoglobin (10 μmol/L) was added to LY294002-treated platelet approximately 100 seconds after TRAP stimulation. The added myoglobin effectively captured the platelet-derived NO, eliminating both the LY294002-induced secondary NO peak and platelet disaggregation (Table and Figure 1B).
Elimination of LY294002-Induced Secondary Release of NO and Disaggregation by Ferroheme Myoglobin
Effect of PI3-Kinase Inhibition on Platelet Cytosolic Calcium
An increase in cytosolic Ca2+ concentration, [Ca2+]i, within the platelets as a result of calcium influx and mobilization of intracellular stores is essential for platelet and eNOS activation. Stimulation induced a transient rise in platelet [Ca2+]i that coincided with the onset of aggregation. There was no significant difference in the transient increase in platelet [Ca2+]i in LY294002-treated platelets and in vehicle-treated platelets (4.6±0.5 and 5.6±0.5 μmol/L, respectively, n=6, P=NS).
Effect of PI3-Kinase Inhibition on Platelet eNOS Phosphorylation
Phosphorylation of eNOS alters the catalytic activity of this enzyme. In platelets, protein kinase C phosphorylation of eNOS is believed to decrease platelet-derived NO.23 Therefore, we evaluated the effect of PI3-kinase inhibition on eNOS phosphorylation in TRAP-stimulated platelets. No significant change in the phosphorylation of eNOS could be detected in the presence of LY294002 (data not shown).
Recent studies have demonstrated that the phosphorylation of eNOS at Ser-1177 in endothelial cells has a significant effect on the activity of the enzyme.24,25 Therefore, the effect of LY294002 on the phosphorylation of Ser-1177 in platelets stimulated with TRAP for increasing durations was examined. The phosphorylation of eNOS Ser-1177 in the CSK fraction increased gradually, peaking 3 minutes after platelet activation and gradually decreasing thereafter (data not shown). However, the PI3-kinase inhibitor LY294002 did not alter the Ser-1177 phosphorylation.
Effect of LY294002 on Platelet-Derived Superoxide
Superoxide produced by aggregating platelets readily reacts with NO,26 reducing its bioactivity. To determine if change in superoxide release played a role in the PI3-kinase dependent increase in NO, platelet-derived superoxide was measured from TRAP-activated platelets. Superoxide release was significantly decreased in stimulated LY294002-treated platelets as compared with stimulated vehicle-treated platelets (0.3±0.03 versus 1.1±0.3 arb. units, respectively, n=3, P<0.05).
To confirm that the effect of PI3-kinase inhibition was the consequence of reduced superoxide, the effects of the membrane-permeable superoxide dismutase (SOD) mimetic tempol (1 mmol/L) on TRAP-stimulated platelets were assessed. Tempol significantly increased TRAP-stimulated NO release from 35.7±4.9 to 60.9±7.7 pmol/108 platelets (n=4, P<0.04) in addition to causing platelet disaggregation (n=4, 11.7±3.2 arb. units, P<0.03 compared with control). Treatment with tempol also decreased platelet superoxide release from 1.1±0.2 to 0.3±0.1 arb. units (n=4, P<0.01 as compared with control).
Role of PI3-Kinase in NADPH Oxidase Activation
Initially identified in phagocytic cells, NADPH oxidase has since been identified as the primary producer of superoxide in vascular tissues.13 To verify NADPH oxidase was present in platelets, the Triton-soluble and CSK fractions isolated from TRAP-stimulated platelets were immunoblotted with antibodies to p67-phox and p47-phox. The presence of both p67-phox and p47-phox in platelets was confirmed in both fractions (data not shown).
Data suggest that PI3-kinase plays a role in the activation of NADPH oxidase27 that requires the translocation of the cytosolic subunits to the membrane-bound cytochrome b558.28 To investigate whether inhibition of PI3-kinase inhibits NADPH oxidase activation in platelets, the CSK fraction of TRAP-stimulated platelets treated with either LY294002 or vehicle were immunoblotted with the anti-p67-phox antibody. Western blot analysis revealed that 3 minutes after TRAP stimulation there was a significant increase in the amount of p67-phox present in the CSK of platelets (Figure 3). However, there was significantly less p67-phox present in the CSK fraction isolated from LY29402-treated platelets 3 minutes after TRAP stimulation.
Figure 3. Effect of PI3-kinase inhibition on translocation of p67-phox to the platelet cytoskeleton. Isolated platelets were preincubated with 25 μmol/L LY294002 (black bars) or vehicle control (gray bars) for 20 minutes and stimulated with 25 μmol/L TRAP for the time periods indicated. The Triton X-100-insoluble fraction was analyzed by Western blot using an antibody specific for p67-phox. Densitometry was performed to quantify the amount of p67-phox protein present and expressed as the mean±SEM (n=3, *P<0.002 compared with nonstimulated control, P<0.03 compared with control at 3 minutes).
Effect of PI3-Kinase Inhibition on NOS III-Deficient Platelets
To confirm the previous findings and determine the relative contribution of NO and superoxide to platelet disaggregation, platelets isolated from mice deficient in eNOS (NOS III) were treated with LY294002 and tested for aggregation, NO, and superoxide release after 25 μmol/L TRAP stimulation. As compared with platelets from control mice, platelets from NOS III-deficient mice did not release NO (Figure 4). Incubation with LY294002 caused disaggregation in the control platelets but not the NOS III-deficient platelets. Interestingly, there was still increased superoxide release in stimulated platelets from NOS III-deficient animals although it was less as compared with control (Figure 4). As compared with LY294002-treated platelets, platelets incubated with vehicle control from either wild-type or NOS III-deficient mice did not demonstrate any disaggregation (data not shown). These data suggest that PI3-kinase regulates superoxide release but NO is required for disaggregation to occur.
Figure 4. Effect of PI3-kinase inhibition on platelets from NOS III-deficient mice. Platelets from control or NOS III-deficient mice were isolated, incubated with LY294002, and stimulated with 25 μmol/L TRAP. Peak platelet aggregation and extent of disaggregation were determined (% light transmittance; n=3; *P<0.05 compared with control). Platelet NO release represents total NO release (arb units; n=3; *P<0.001 compared with control). Platelet superoxide release represents total release (arb. units; n=3; P=ns).
Discussion
In this study PI3-kinase was found to play a role in regulating NADPH oxidase-generated superoxide in platelets, altering the bioactivity of platelet NO that contributes to platelet disaggregation. The progressive reversal of aggregation following initial aggregation as the result of PI3-kinase inhibition in stimulated platelets is consistent with the findings of previous studies.2 Given that disaggregation resulting from PI3-kinase inhibition in TRAP-stimulated platelets correlated with a dose-dependent inhibition of the integrin GPIIb-IIIa activation,2 the activation of PI3-kinase is considered essential for maintaining GPIIb-IIIa in its activated state to sustain aggregation. Likewise, the ability of NO donors to induce platelet disaggregation has been linked to the diminished activation of GPIIb-IIIa and fibrinogen binding.29 The stimulation of platelets induces a conformational change in GPIIb-IIIa (inside-out signaling) leading to the binding of soluble fibrinogen and the onset of platelet aggregation. The prolonged activation of GPIIb-IIIa activation is essential for irreversible aggregation. The real time measurement of platelet-derived NO release concurrent with aggregation shows that platelet disaggregation is invariably preceded by an increase in NO release from platelets, suggesting the increased release of platelet-NO due to the inhibition of PI3-kinase is the cause of the platelet disaggregation. The ability of the NO-scavenger ferroheme myoglobin, which effectively traps NO, to eliminate the platelet disaggregation evoked by PI3-kinase inhibition demonstrates that this secondary release of NO is intimately involved in regulating platelet aggregation. Platelets are anuclear structures, making transfection experiments impossible; therefore, these observations were confirmed using NOS III-deficient platelets.
The secondary release of NO, paralleled by elevated intraplatelet cGMP levels, is unique to inhibition of PI3-kinase. One explanation for this increase in platelet-derived NO is alteration in the activation of eNOS. Phosphorylation is important in the regulation of eNOS activity. Depending on the kinase and residue involved, phosphorylation can result in either an increase or a decrease in enzyme activity. The serine/threonine kinase Akt phosphorylates eNOS, activating the enzyme,24 whereas protein kinase C-mediated phosphorylation has the opposite effect, inhibiting eNOS activation.30 The lack of a significant change in the phosphorylation of eNOS caused by PI3-kinase inhibition indicates the increase in platelet-derived NO as a result of PI3-kinase inhibition is not mediated by eNOS phosphorylation. The importance of eNOS phosphorylation by several serine/threonine kinases downstream of PI3-kinase in the regulation of eNOS function in endothelial cells24 warranted further examination of eNOS phosphorylation at Ser-1177 in platelets using antibodies that detect eNOS only when phosphorylated at this site. Although the phosphorylation of Ser-1177 did change to some extent over time as platelet aggregation progressed, we found no evidence indicating that inhibition of PI3-kinase had any consequence on eNOS phosphorylation at Ser-1177. There are, however, significant differences between endothelial cells and platelets. In addition to lacking nuclei, platelets lack caveolin-1 found within the caveolae and Golgi apparatus of endothelial cells, which binds the majority of inactive eNOS.31 The activation of eNOS requires the disruption of the eNOS-caveolin-1intereaction by the Ca2+/calmodulin complex, eNOS phosphorylation, or possibly both.32 Thus, it is not unreasonable to surmise that the regulation of eNOS by PI3-kinase in the endothelial cell will not be precisely the same as in the platelet. The possibility that PI3-kinase is not involved in the phosphorylation of specific sites of eNOS24 in the platelet cannot be completely ruled out. The resolution of this must await a detailed analysis in the future, as more knowledge regarding these specific sites becomes known.
The significant decrease in platelet superoxide suggests that the increase in platelet-derived NO might be the result of a decrease in the production of the radical oxygen species superoxide. Superoxide and NO react to form peroxynitrite (ONOO-), diminishing NO bioactivity.33 Therefore, one possible explanation for the NO-induced disaggregation caused by PI3-kinase inhibition is a decrease in platelet superoxide. This concept is consistent with a recent study that demonstrated a 50% inhibition of thrombin-induced superoxide release in platelets treated with the PI3-kinase inhibitor wortmannin.34 Moreover, the ability of the SOD mimetic tempol to increase platelet NO and inhibit platelet aggregation, in addition to decreasing platelet superoxide release, indicates that superoxide can modulate the inhibitory action of NO on platelet aggregation. This observation is strengthened by the finding that PI3-kinase inhibition alters superoxide release but does not reverse platelet aggregation in eNOS deficient platelets.
Although the production of platelet-derived superoxide and its proaggregatory effects have been established, the molecular identities of the cellular enzymes responsible for this platelet superoxide production are not well defined. Superoxide production has been ascribed to a variety of different sources including the lipoxygenase pathway, xanthine oxidase, eNOS, and NADPH oxidase.15,16 The change in translocation of NADPH oxidase and the marked decrease in superoxide suggests that NADPH oxidase is partially responsible for the generation of platelet superoxide that regulates the bioactivity of platelet NO. This is consistent with the evidence showing the inhibition of both thrombin- and ADP-induced platelet aggregation, and the potentiation of the antiplatelet effect of the NO donor sodium nitroprusside in diphenyleneiodonium chloride-treated platelets.15
Immunoblot analysis confirmed the presence of two of the cytosolic components, p67-phox and p47-phox, in platelets, although a more detailed identification of the different components of NADPH oxidase will be necessary to fully characterize the molecular structure of this oxidase in platelets. Furthermore, the marked translocation of p67-phox in TRAP-stimulated platelets, blocked by PI3-kinase inhibition, demonstrates the functional activation of NADPH oxidase is regulated in part by PI3-kinase (Figure 3). In neurophils, PI3-kinase has been implicated in the activation of NADPH oxidase.35 Both wortmannin and LY294002 effectively inhibit fMLP-stimulated neutrophil-generated superoxide.35 The diminished release of superoxide caused by PI3-kinase inhibition could be explained by the blunted translocation of p67-phox that precludes the catalysis of the reactive oxygen species by NADPH oxidase.36 Moreover, the temporal correlation of the inhibition of p67-phox translocation, the secondary release of platelet NO, and disaggregation as the result of PI3-kinase inhibition indicate that the effect of PI3-kinase inhibition in TRAP-stimulated platelets can be explained by the abated production of NADPH oxidase-derived superoxide, which serves to limit the bioactivity of platelet-derived NO.
In summary, these results support the conclusion that the disaggregation of platelets produced by PI3-kinase inhibition is due to the decrease in NADPH oxidase-generated superoxide, which increases the bioavailability of platelet-derived NO. The demonstration of a PI3-kinase-regulated NADPH oxidase in platelets that is partially responsible for platelets-derived superoxide suggests an intriguing model for explaining the changes in the bioavailability of platelet NO. The full identification of all the components of the NADPH oxidase in platelets and a better understanding of how PI3-kinase regulates this oxidase may be useful in the development of novel interventions for the control of thrombosis.
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