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Originally published In Press as doi:10.1074/jbc.M506518200 on September 6, 2005

J. Biol. Chem., Vol. 280, Issue 45, 37430-37438, November 11, 2005
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Stimulatory Roles of Nitric-oxide Synthase 3 and Guanylyl Cyclase in Platelet Activation*

Jasna A. Marjanovic, Zhenyu Li, Aleksandra Stojanovic, and Xiaoping Du1

From the Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612

Received for publication, June 15, 2005 , and in revised form, August 29, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO) stimulates soluble guanylyl cyclase and, thus, enhances cyclic guanosine monophosphate (cGMP) levels. It is a currently prevailing concept that NO inhibits platelet activation. This concept, however, does not fully explain why platelet agonists stimulate NO production. Here we show that a major platelet NO synthase (NOS) isoform, NOS3, plays a stimulatory role in platelet secretion and aggregation induced by low doses of platelet agonists. Furthermore, we show that NOS3 promotes thrombosis in vivo. The stimulatory role of NOS is mediated by soluble guanylyl cyclase and results from a cGMP-dependent stimulation of platelet granule secretion. These findings delineate a novel signaling pathway in which agonists sequentially activate NOS3, elevate cGMP, and induce platelet secretion and aggregation. Our data also suggest that NO plays a biphasic role in platelet activation, a stimulatory role at low NO concentrations and an inhibitory role at high NO concentrations.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Development of thrombotic diseases involves the injury or dysfunction of the blood vessel wall and activation of blood platelets (1). Upon exposure to agonists such as thrombin, ADP, collagen, and von Willebrand factor (VWF),2 platelets become "activated" and aggregate to form primary thrombi (1, 2). Activated platelets secrete large quantities of ADP, serotonin, and other factors that amplify platelet activation and stabilize platelet aggregates (3). Activated platelets also secrete pro-coagulation, pro-inflammatory, and growth factors (3–5). Thus, platelet activation plays a major role not only in acute arterial thrombosis but also in the development of chronic vascular diseases, such as atherosclerosis, which in turn causes thrombosis (1, 6).

A major advance in the field of vascular biology in the last century was the discovery of the vessel dilator, nitric oxide (NO) (7–9). NO is a short-lived messenger molecule synthesized from L-arginine by a family of enzymes known as nitric-oxide synthases (NOS). Three isoforms of NOS enzymes are known (10–12): NOS1 (neuronal NOS), NOS2 (inducible NOS), and NOS3 (endothelial NOS). NOS3 is the major isoform known to be expressed in platelets (13). One of the major functions of NO is to stimulate soluble guanylyl cyclase (sGC) and increase the synthesis of cyclic guanosine monophosphate (cGMP) that serves as a secondary messenger regulating the function of cGMP-dependent protein kinase (PKG), cGMP-dependent ion channels, and cGMP-regulated phosphodiesterases (7). High concentrations of NO can also chemically modify (nitrosylation and nitration) proteins and, thus, affect cell functions in a cGMP-independent manner (7, 14–16). NO is involved in diverse processes, such as smooth muscle relaxation, neurotransmission, immune responses, and inflammation (7). It has been a prevailing concept that NO, by elevating intracellular cGMP, inhibits platelet activation (8). This concept is supported by data that high concentrations of NO donor compounds inhibit platelet activation (17–19). However, the concept that NO inhibits platelet activation does not fully explain why endogenous platelet NO production is stimulated by platelet agonists (20, 21). Thus, the physiological role of endogenous platelet NO synthesis by NOS during platelet activation has been unclear. In this study we show that the major NOS isoform expressed in platelets, NOS3, in fact plays a stimulatory role in low dose agonist-induced platelet activation and promotes in vivo thrombotic response in an injuryinduced arterial thrombosis model. The stimulatory roles of NOS3 are dependent on sGC activation and elevation of cGMP. Furthermore, we found that the NOS3-sGC pathway stimulates aggregation-dependent platelet secretion of granule contents, which is required for the stimulatory roles of the NOS3-sGC pathway in platelet activation. Thus, our data provide an important revision to the current concept of NO signaling in platelets. We propose a new concept that NO plays a biphasic role in platelet activation; low concentrations of NO (such as that produced by platelet NOS3) promote platelet secretion and aggregation, but high concentrations of NO, as previously reported, inhibit platelet activation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Soluble GC inhibitor 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), PKG activator 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP), nitric oxide donor sodium nitroprusside (SNP), NOS inhibitor NG-monomethyl-L-arginine (L-NMMA), and thromboxane A2 analog U46619 [GenBank] were from Calbiochem. FeCl3, o-phthalaldehyde, ADP, N{omega}-nitro-L-arginine methyl ester (L-NAME), the inactive stereoisomer of L-NAME, D-NAME, and NOS substrate L-arginine were from Sigma. Human {alpha}-thrombin was purchased from Enzyme Research Laboratories, South Bend, IN. Luciferin/luciferase reagent and collagen were purchased from Chronolog, Havertown, PA. NOS3-deficient mice back-crossed for more than 10 generations to C57BL background, and C57BL/6 control mice were obtained from The Jackson Laboratory, Bar Harbor, ME, and colonies were maintained at the University of Illinois Biological Resources Laboratory. Some experiments were performed using littermates obtained from heterozygotes breeding, with similar results.

Preparation of Washed Platelets—Fresh blood from healthy volunteers was anticoagulated with one-seventh volume of acid citrate dextrose (ACD) as described previously (22). For the preparation of mouse platelets, 6 – 8-week-old mice of either sex were anesthetized with an intraperitoneal injection of pentobarbital, and blood was drawn from the inferior vena cava (22). Blood from 5–6 mice of either genotype was pooled, and platelets were isolated by differential centrifugation as previously described (22). Platelets were washed 2 times with CGS buffer (0.12 M sodium chloride, 0.0129 M trisodium citrate, and 0.03 M D-glucose, pH 6.5), resuspended in modified Tyrode buffer at 3 x 108/ml, and allowed to rest for at least 1 h at room temperature before use (23). In some experiments 100 µM L-arginine, pH 7.4, was added to the platelet suspension.

Platelet Aggregation and Secretion—Platelet aggregation was measured in a turbidometric platelet aggregometer (Chronolog) at 37 °C with stirring (1000 rpm). Platelets were preincubated for 5 min at 37 °C with various concentrations of inhibitors or corresponding vehicle controls before the addition of platelet agonists. To investigate the effects of 8-pCPT-cGMP, SNP, or ADP on platelet aggregation, these agents were added to platelets immediately after the addition of the agonist. Platelet secretion was monitored in parallel with platelet aggregation as ATP release in a platelet lumiaggregometer (Chronolog) with the addition of luciferin-luciferase reagent to platelet suspension. Quantification was performed using the ATP standard. Experiments were repeated at least three times. Statistical significance was examined using a paired t test and data from three or more experiments. The amount of total ATP in mouse platelets was determined by measuring the luminescence of Triton X-100 platelet lysates in the presence of luciferin-luciferase reagent in a lumiaggregometer.

Measurement of Granule Contents—Mouse platelets were prepared as described for aggregation and secretion studies. The measurement of serotonin was done fluorometrically based on the reaction of serotonin with o-phthalaldehyde, as described previously (24). The fluorescence was measured on Wallac1420 Victor multitask reader (PerkinElmer Life Sciences) with the excitation wave length at 355 nm and emission wave length at 460 nm. The levels of an {alpha}-granule content, VWF, were estimated by immunoblotting with a polyclonal antibody (DakoCytomation, Carpinteria, CA).

Measurement of Platelet cGMP Levels—Washed platelets resuspended in modified Tyrode buffer were preincubated with NOS or sGC inhibitors for 5 min or an equal volume of vehicle control at 37 °C before the addition of different concentrations of thrombin and incubated in a platelet aggregometer with stirring (1000 rpm) for 5 min. The reaction was stopped by the addition of ice-cold 12% (w/v) trichloroacetic acid, the samples were centrifuged at 2000 x g for 15 min at 4 °C, and the supernatant was extracted 4 times with 5 volumes of water saturated diethyl ether. The samples were lyophilized, and cGMP concentrations were determined using a cGMP enzyme immunoassay kit from Amersham Biosciences. Results are expressed as the mean ± S.E. Statistical significance between groups was determined by t test.

Platelet NOS3 Levels—Washed platelets in Tyrode buffer were solubilized with 50 mM {beta}-octylglucoside, 10 mM Hepes, 10 mM EDTA, 0.15 M NaCl, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mM E64, pH 7.4. The lysates was incubated with 2', 5'-ADP-agarose beads, and bound proteins were analyzed by SDS-PAGE and immunoblotting with a NOS3-specific antibody (Transduction laboratories).

Bleeding Time— 4–5-Week-old littermates obtained from heterozygous breeding of NOS3+/– mice were anesthetized with intraperitoneal injection of avertin. The distal portion of the tail (5 mm) was amputated with a scalpel, and the tail was immersed in 0.15 M NaCl at 37 °C as previously described. Time to stable cessation of the bleeding was defined as the time where no re-bleeding for longer than 60 s was recorded. Statistical analysis was performed using the Mann-Whitney test.

In Vivo Thrombosis— 8–10-Week-old mice were anesthetized with intraperitoneal injection of pentobarbital. The left carotid artery was isolated from surrounding tissues (25). MA-0.5PSB nanoprobe (Transonic Systems, Ithaca, NY) was hooked to the artery, and blood flow was monitored with a TS420 flowmeter (Transonic Systems). After stabilization, 1.2 µl of 5% FeCl3 was applied to a filter paper disc (2-mm diameter) that was immediately placed on top of the artery for 3 min. After removing the filter paper, blood flow was monitored continuously until 5 min after occlusion. Time to occlusion was calculated as a difference in time between the removal of the filter paper and stable occlusion (no blood flow for 2 min). Statistical analysis was performed using the Mann-Whitney test for the evaluation of differences in median occlusion time (p = 0.04) or by t test for the mean occlusion time after exclusion of the outliers by Dixon outlier range test (3 in NOS3–/– and 2 in NOS3+/+ group) (p = 0.03). Similar results were obtained using both methods.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
NOS3 Knock-out Inhibits Low Dose Agonist-induced Platelet Aggregation in Mouse Platelets—Fig. 1A shows that NOS3 protein is present in wild type mouse platelets but is deficient in NOS3–/– mouse platelets. To investigate the role of NOS3 in platelet activation, washed wild type or NOS3-deficient mouse platelets (26) suspended in modified Tyrode solution were exposed to platelet agonists. NOS3-deficient platelets showed reduced and unstable platelet aggregation compared with wild type platelets in response to low concentrations of thrombin (at 0.017 units/ml thrombin, p < 0.01, n = 5) (Fig. 1, A and D). When exposed to a sub-threshold concentration of thrombin (0.0125 units/ml), wild type platelets only partially aggregated under the same buffer conditions as above but fully aggregated when a physiological concentration of L-arginine (a substrate for NOS (27)) was added, suggesting that L-arginine increased the sensitivity of wild type platelets to thrombin (Fig. 1, B and E). In contrast, the enhancing effect of L-arginine was absent in NOS3-deficient platelets (Fig. 1, B and E). Thus, NO synthesis by NOS3 plays an important role in promoting and stabilizing low dose thrombin-induced platelet aggregation. To examine the effect of NOS3 in platelet activation induced by other agonists, platelets were exposed to the thromboxane A2 analog U46619 [GenBank] or collagen. Similarly, NOS3-deficient platelets showed a reduced aggregation response to low doses of U46619 [GenBank] (Fig. 2A) and collagen (Fig. 2E). These data indicate that NOS3 is important in low dose agonistinduced platelet aggregation.

NOS3 Mediates Dense Granule Secretion—The reduced platelet aggregation in NOS3-deficient platelets was observed at low concentrations of agonists and was characteristic of defects in the secretion-dependent second wave of platelet aggregation (22). Thus, we also examined if NOS3 knock-out affected platelet secretion using a real time luciferase-luciferin assay of secreted dense granule ATP. Platelet secretion induced by low dose thrombin was significantly reduced in NOS3-deficient platelets (Fig. 1, B and F). Thromboxane A2 induced two waves of platelet secretion as we reported previously (22) (Fig. 2, A and B). The first wave secretion was aggregation-independent, but the second wave was aggregation-dependent. The aggregation-dependent second wave platelet secretion in response to low dose U46619 [GenBank] was absent in NOS3 knock-out mouse platelets (Fig. 2A). Also, low dose collagen-induced platelet secretion was inhibited in NOS3 knock-out platelets (Fig. 2E). At higher concentrations of thrombin (Fig. 1, C and G), U46619 [GenBank] (Fig. 2, B and D), or collagen (Fig. 2, F and H) platelet secretion was still significantly reduced by NOS3 deficiency, although the remaining secretion appeared to be sufficient to induce the second wave of platelet aggregation. To exclude the possibility that reduced response of NOS3–/– platelets was due to the "exhausted platelets" effect caused by prior platelet activation and secretion during preparation, we determined the total granule contents of wild type control as well as NOS3–/– platelets. The amounts of total ATP were not significantly different between the platelets from wild type and NOS3–/– mice (Fig. 3A). The amounts of dense granule-specific constituent serotonin were also similar between wild type and NOS3-deficient platelets (Fig. 3B). Additionally, platelet lysates contained comparable amounts of {alpha}-granule protein, VWF (Fig. 3C), and thrombospondin (data not shown). Therefore, NOS3 knockout platelets carry normal levels of granule cargo but are defective in agonist-stimulated secretion.



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  		FIGURE 1.
Stimulatory roles of NOS3 in low dose thrombin induced platelet aggregation and secretion. A, a representative aggregation trace showing different responses of NOS3–/– (–/–) or NOS3+/+ (+/+) mouse platelets to a low concentration of thrombin. The inset shows the immunoblot (IB) result that NOS3 is expressed in NOS3+/+ but not in NOS3–/– platelets. B, concomitantly recorded aggregation and ATP secretion of NOS3+/+ or NOS3–/– platelets suspended in buffer without or with 100 µM L-arginine (+Arg) in response to a sub-threshold concentration of thrombin. C, platelet aggregation and ATP secretion in response to a higher concentration of thrombin. Note the reduced secretion in NOS3–/– platelets. D, quantitative presentation (mean ± S.E.) of platelet aggregation data as shown in A (p = 0.0056, n = 5). E and F, the experiment described in B was repeated three times, and data are presented as mean ± S.E. of % light transmission (E) and concentration of secreted ATP (F). Note that the addition of L-arginine enhanced the aggregation and secretion response only in the wild type but not in NOS3 knock-out platelets. G, quantitative data of ATP secretion experiment as shown in C (mean ± S.E., p = 0.0002, n = 7).

 
Thus, it appears that NOS3 deficiency primarily affects platelet secretion. Insufficient secretion consequently abolishes or reduces the second wave of platelet aggregation and stability of aggregates. These data explain why NOS3 knock-out only affects platelet aggregation induced by low dose agonists. These data also suggest that NOS3 mediates an important (but not the only) platelet secretion signaling pathway.

NOS3 Promotes in Vivo Thrombosis—NOS3–/– mice are hypertensive, suggesting a strong vasoconstrictive effect of NOS3 deficiency. However, despite these strong vascular effects of NOS3 knock-out that favor in vivo hemostasis, we found no statistically significant difference in tail bleeding time between wild type (n = 29) and NOS3 knock-out (n = 33) littermate mice generated by heterozygous breeding (p = 0.5919). Rather, NOS3 knock-out mice showed a tendency of increased re-bleeding after initial unstable cessation of bleeding (data not shown).

To determine the in vivo physiological relevance of the role of NOS3 in promoting platelet secretion and aggregation, we compared in vivo thrombosis of wild type and NOS3–/– mice using the FeCl3-injured carotid artery thrombosis model. FeCl3-induced arterial thrombi are known to be platelet-rich thrombi. The time to the formation of stable thrombus in NOS3–/– mice is significantly prolonged compared with wild type mice (p = 0.03, n = 20 for NOS3–/– mice, n = 21 for wild type controls) (Fig. 4). These data suggest that NOS3 participates in thrombus formation and stability in vivo, which is in agreement with the observed stimulatory role of NOS3 in platelet activation.



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  		FIGURE 2.
Stimulatory roles of NOS3 in platelet activation induced by low dose U46619 [GenBank] and collagen. A and B, concomitantly recorded aggregation and secretion of NOS3+/+ and NOS3–/– platelets stimulated with 250 nM (A) or 500 nM U46619 [GenBank] (B). C and D, quantitative data of aggregation (C) and secretion (D) data from three experiments as shown in A and B, respectively. E and F, platelet aggregation and secretion induced by 0.5 µg/ml (E) or 1 µg/ml (F) collagen. G and H, quantitative data from three different experiments as shown in E and F, respectively. Note that at higher agonist concentrations secretion is still significantly reduced in NOS3–/– platelets.

 



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  		FIGURE 3.
Granule contents of wild type and NOS3 knock-out platelets. The total amounts of dense granule contents, ATP (A) and serotonin (B), in NOS3+/+ and NOS3–/– platelets were determined as described under "Experimental Procedures" (mean ± S.E., n = 3). C, a representative immunoblot (IB) showing that the total VWF ({alpha} granule constituent) levels are similar between NOS3+/+ (+/+) and NOS3–/– (–/–) platelets.

 
Effects of NOS Inhibitors on Human Platelet Secretion and Aggregation—To determine whether NOS is important in human platelet aggregation and secretion, washed human platelets were preincubated with NOS inhibitors L-NAME or NG-monomethyl-L-arginine (L-NMMA), then stimulated with low doses of thrombin. Both inhibitors reduced platelet aggregation (Fig. 5, A and E). The inhibitory effect of L-NAME was specific for the biologically relevant L form of the compound, since aggregation was not affected by D-NAME (Fig. 5D). Furthermore, NOS substrate L-arginine significantly enhanced low dose thrombin-induced platelet aggregation (Fig. 5F). Similar to the results in platelets from NOS3-deficient mice, L-NAME significantly inhibited dense granule release in human platelets (Fig. 5, A and C), whereas L-arginine significantly enhanced platelet secretion (Fig. 5F).



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  		FIGURE 4.
NOS3 deficiency delays formation of stable occlusive thrombi in vivo. FeCl3-induced carotid artery injury was performed, and time to occlusive thrombosis was recorded as described under "Experimental Procedures." The occlusion time of each mouse is shown as circles (NOS3+/+, n = 21) and triangles (NOS3–/–, n = 20). The bars represent the mean occlusion time ± S.D. (249.8 ± 101.7 s for NOS3+/+ and 327.4 ± 118.4 s for NOS3–/–; p = 0.0299).

 
One of the major substances secreted from dense granules is ADP. ADP is known to stimulate integrin activation and amplify platelet aggregation. Thus, it is possible that inhibition of ADP secretion is primarily responsible for the decreased platelet aggregation in NOS inhibitor-treated platelets. Indeed, supplementation with a low concentration of ADP, insufficient to induce aggregation on its own, reversed the inhibitory effects of L-NAME (Fig. 6). Thus, NOS plays an important role in mediating platelet secretion of dense granule contents (mainly ADP), which induces the second wave of platelet aggregation.

NOS3 Plays Stimulatory Roles in a cGMP-dependent Manner—Nitric oxide activates sGC, an enzyme that produces cGMP (7, 8). To investigate whether the role of NOS in platelet activation is upstream of sGC, we examined the effects of NOS inhibitors and NOS3 knock-out on thrombin-stimulated cGMP elevation. Thrombin induced an elevation of cGMP, which was partially but significantly inhibited in NOS3-deficient mouse platelets (Fig. 7A) as well as in L-NAME-treated human platelets (Fig. 7B), suggesting that thrombin induces NOS-dependent elevation of cGMP during platelet activation. These results also suggest that NOS3 is the major but not the only mechanism responsible for agonist-stimulated cGMP elevation. To further determine whether NOS-induced cGMP production was important in the NOS-dependent platelet aggregation, we investigated whether a cell-permeable cGMP analog could reverse the inhibitory effects of L-NAME. The addition of 8-pCPT-cGMP immediately after the addition of thrombin reversed the inhibition of platelet aggregation by L-NAME (Fig. 7C). It is important to note that the effects of cGMP in restoring platelet aggregation in L-NAME-treated platelets could be achieved only if the concentrations of 8-pCPT-cGMP were in a 0.5–10 µm range. Higher concentrations of 8-pCPT-cGMP failed to restore platelet aggregation, which is in agreement with our previous observations that the effects of cGMP on platelet aggregation are biphasic (28).

If NOS stimulates platelet activation via the cGMP pathway, inhibition of NO-stimulated sGC should result in inhibition of platelet activation. Indeed, preincubation of human platelets with a selective inhibitor of sGC, 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (29), dose-dependently inhibited low dose thrombin-induced platelet aggregation (Fig. 8A). Additionally, this compound completely inhibited cGMP elevation in thrombin-stimulated platelets (Fig. 8B). Together, these data indicate a novel signaling pathway in which platelet agonists activate platelet NOS3, releasing NO, which in turn activates sGC, leading to elevation of intracellular cGMP. cGMP stimulates platelet secretion via a PKG-dependent pathway that induces the second wave of platelet aggregation (30).

NO Plays Biphasic Roles in Platelet Activation—It seems that our conclusion of a stimulatory role for NOS3 contradicts previous data that NO donors inhibit platelet aggregation. However, endogenous NO production in platelets is at nanomolar range (21), and NOS-dependent cGMP elevation is also at nanomolar levels (Fig. 7). In contrast, relatively high concentrations of NO donors (micromolar levels) or high concentrations of cGMP analogs (0.1–5 mM) have to be used to achieve inhibitory effects (17, 18, 31–33). To determine whether NO plays concentration-dependent biphasic roles in platelet activation, we examined the effects of low concentrations of NO donors on platelet aggregation. The addition of a low concentration of NO donor (10 nM), SNP, enhanced platelet aggregation-induced by a sub-threshold concentration of thrombin (Fig. 9, A and B). In contrast, higher concentrations of SNP reduced platelet activation (Fig. 9C) (17). Therefore, our results suggest that, similar to cGMP, NO plays biphasic roles in platelet activation. Low concentrations of NO produced endogenously by NOS3 during platelet activation promote platelet secretion and aggregation, but further rising of NO or high concentrations of exogenous NO may be inhibitory.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The data described in this study provide evidence for a major revision of the currently accepted concept of NO signaling in platelets. Our data support a new concept that NOS3-mediated low level NO production in platelets stimulates platelet secretion and, thus, amplifies and stabilizes platelet aggregation.

The conclusion that endogenous NO production by NOS3 promotes platelet activation is supported by data that 1) NOS3 knock-out mouse platelets showed reduced platelet secretion and aggregation in response to low concentrations of thrombin, U46619 [GenBank] , and collagen, 2) NOS3 knock-out mice showed reduced ability to form occlusive arterial thrombus in an in vivo thrombosis model, 3) NOS inhibitors reduced platelet secretion and aggregation, and 4) a NOS substrate, L-arginine, that enhances NO production promotes platelet secretion and aggregation. These data appear to contradict previous results that NO plays an inhibitory role in platelet activation (17, 18, 34). However, previous studies were performed under conditions of high NO concentrations. For example, the inhibitory effects of NO donors on platelet activation require relatively high concentrations of NO donors (micromolar levels), which cause a 100 – 200-fold elevation of intra-platelet cGMP levels (18). In contrast, we show that physiologically low levels of NO produced endogenously by NOS3 or exogenous NO donor SNP at concentrations comparable with endogenous NO production have a stimulatory effect on platelet activation. Furthermore, we show that a major mechanism for the stimulatory role of NOS3 is the NO- and cGMP-dependent activation of aggregation-dependent platelet secretion that amplifies and stabilizes platelet aggregation and thrombus formation. Thus, the in vitro stimulatory role of NOS3 is manifested mainly when platelets were stimulated by low concentrations of platelet agonists, which require secretion for the full scale platelet aggregation. In contrast, some previous studies were performed under the condition where platelets responded poorly to the low concentrations of platelet agonists and were characteristic of a lack of the secretion-dependent second wave platelet aggregation (35). Therefore, combined with previous findings that high concentrations of NO inhibit platelet activation, our data indicate a biphasic role for NO in platelet activation. At low concentrations, NO stimulates platelet secretion and, thus, enhances platelet response to low concentrations of platelet agonists and stabilizes platelet aggregates. At high concentrations NO becomes inhibitory.



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  		FIGURE 5.
Effects of NOS inhibitors and the NOS substrate L-arginine on human platelet aggregation and secretion. A, thrombin-induced aggregation and secretion of platelets pretreated with or without L-NAME. B and C, quantitative data of the effects of L-NAME on platelet aggregation (p < 0.0001, n = 7) (B) and secretion (p = 0.0135, n = 5) (C). D and E, representative traces of aggregation of platelets pretreated with L-NAME or D-NAME (p < 0.01, n = 3) (D) or platelets pretreated with or without a different NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA)(p < 0.05, n = 3) (E). F, aggregation and ATP secretion of human platelets in response to thrombin in the presence (Arg) or absence of 100 µM L-arginine (p < 0.0001, n = 3).

 



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  		FIGURE 6.
The reversal effect of ADP on L-NAME-induced inhibition of platelet aggregation. Washed human platelets preincubated with L-NAME or buffer (Control) were stimulated with a low dose of thrombin. L-NAME-treated platelets were also stimulated with thrombin followed by the addition of a sub-threshold concentration of ADP (500 nM) (in washed platelets ADP is not sufficient to induce platelet secretion or induce aggregation without exogenous fibrinogen).

 
NO mediates signaling via cGMP-dependent and cGMP-independent pathways. In the cGMP-dependent pathway previous studies showed that NO stimulates sGC and elevates cGMP levels. cGMP stimulates several intracellular molecules, including PKG, cGMP-gated ion channel, and cGMP-regulated phosphodiesterases. Although it was believed that cGMP inhibits platelet activation by activating PKG, we have shown that PKG in fact plays an important stimulatory role in platelet activation (28, 30, 36), as PKG knock-out mouse platelets and PKG inhibitor-treated human platelets showed significantly reduced platelet aggregation in response to low dose agonists including soluble agonists thrombin, thromboxane A2, and adhesive proteins VWF and collagen. On the other hand, the inhibitory effects of NO donors and cGMP analogs on human platelets were reversed by inhibitors of cAMP-dependent protein kinase (37) and adenylyl cyclase (38), indicating that these effects require elevation of cAMP, possibly induced by inhibition of cGMP-regulated phosphodiesterase 3 (31). Thus, NO exerts biphasic effects on platelets via distinct downstream signaling pathways. This new concept helps explain previous controversies on the roles of the NO-cGMP pathway in platelets and is important in further understanding the physiological roles of the NO-cGMP pathway in the vascular system.



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  		FIGURE 7.
The roles of cGMP in NOS-mediated platelet aggregation. A and B, NOS3–/– or NOS3+/+ mouse platelets (A) or human platelets pretreated with or without 1 mML-NAME (B) were stimulated with thrombin for 5 min. cGMP levels (mean ± S.E., n = 6) were then determined. C and D, washed human platelets pretreated with 1 mML-NAME or buffer (Control) were stimulated with 0.02 units/ml thrombin and followed by adding various concentrations of 8-pCPT-cGMP or vehicle. A representative graph is shown in C, and quantitative data are shown in D (p = 0.0167, n = 4). Note that the inhibitory effect of L-NAME on aggregation was reversed by 10 µM 8-pCPT-cGMP.

 



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  		FIGURE 8.
The stimulatory role of sGC in platelet activation. Human platelets were preincubated for 5 min with various concentrations of the sGC inhibitor 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or Me2SO (DMSO, control). Platelet aggregation (p < 0.01, n = 3) (A) and cGMP levels (mean ± S.E.) (B) were then examined after stimulation with thrombin. U, units.

 
There are two types of platelet secretion. So called "strong agonists" such as collagen and thrombin at high concentrations induce "aggregation-independent secretion." The "weak" agonists such as ADP or low concentrations of strong agonists induce integrin-dependent platelet aggregation and require integrin "outside-in" signaling to induce "aggregation-dependent platelet secretion." The thromboxane A2 analog U46619 [GenBank] induces a unique two-wave secretion, the first wave aggregation-independent secretion that precedes the first wave platelet aggregation and the second wave aggregation-dependent secretion that induces the second wave platelet aggregation (22). NOS3 knock-out and NOS inhibitors inhibited the second wave aggregation-dependent secretion and the subsequent second wave platelet aggregation (Fig. 2), indicating that the primary role for NOS3 is to stimulate aggregation-dependent platelet secretion. This is consistent with our data on the role of PKG in platelet secretion, indicating NOS3 is upstream of cGMP elevation and PKG activation in stimulating platelet secretion (30). Interestingly, a previous study indicated a role for NOS3 in insulin-induced secretion of ATP and other vasodilator compounds. However, insulin is not known to stimulate platelet activation, and thus, it was postulated that insulin-induced secretion rather than being a proaggregatory signal, results in the release of potent vasodilator compounds (39). Thus, our study establishes an important role for NOS3 in platelet secretionduringplateletactivation. Because it is known that aggregation-dependent platelet secretion serves to amplify platelet activation induced by low concentrations of agonists and to stabilize platelet aggregates, our data explain why NOS3 and PKG are required only in platelet aggregation induced by low concentrations of platelet agonists. It is known that circulating platelets are often exposed to low level stimulation such as high shear stress, adrenaline, low levels of coagulation, and inflammatory factors such as thrombin and platelet-activating factors that are produced in response to minor vascular injury and inflammation. Thus, it is possible that NOS3-generated NO serves to increase the sensitivity of platelets in response to vascular injuries and to stabilize thrombi at sites of vascular injury. Indeed, NOS3-deficient mice had prolonged time to occlusion in carotid artery injury model compared with wild type controls. Therefore, NOS3 plays a physiologically relevant role in promoting in vivo thrombosis.



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  		FIGURE 9.
The biphasic effects of NO donor on platelet activation and a novel NOS3-mediated platelet activation pathway. A and B, stimulatory effects of low concentrations of NO donor. Human platelets were stimulated with a sub-threshold concentration of thrombin followed by adding buffer (Control) or 10 nM SNP (A). Quantitative data (mean ± S.D.) from three independent experiments (p < 0.05) are shown in B. U, units. C, a representative trace showing inhibitory effect of micromolar concentrations of an NO donor, SNP (1 and 10 µM), on human platelet aggregation. D, a novel NOS3-mediated platelet activation pathway.

 
Platelets not only secrete agonists, receptors, and pro-coagulant factors to amplify and stabilize primary thrombus but also secrete inflammatory and growth factors (such as platelet-derived growth factor (5) and cytokines (4)). These factors play important physiological roles in vascular remodeling and wound repair and important pathological roles in vascular inflammation and in the development of atherosclerosis. In this respect it is interesting to note that despite the belief that NO is anti-atherosclerotic, recent studies showed that NOS3 promoted the development of atherosclerosis (40, 41), as NOS3 knock-out mice are less prone to atherosclerosis, whereas overexpression of NOS3 in mice promotes atherosclerosis. Thus, the finding that NOS3 plays an important role in stimulating platelet secretion provides a potential mechanism for the role of NOS3 in promoting the development of atherosclerosis.

The currently accepted concept that NO inhibits platelet activation contains intrinsic contradictions. Although high concentrations of NO donors inhibit platelet activation, endogenous NO production in platelets occurs only when platelets are stimulated with various platelet agonists. Furthermore, potential NOS3 activation signals suggested in other cell types, namely, calcium elevation and the phosphoinositide 3-kinase-Akt pathway (42), all play roles in stimulating platelet activation but not in inhibiting platelet activation (22, 43–46). Thus, our data that NO in fact plays biphasic roles in platelet activation provide an explanation for this apparent contradiction. It is important to note that we have recently found that phosphoinositide 3-kinase, like NOS3, plays a critical role in aggregation-dependent platelet secretion and secretion-dependent second wave platelet aggregation (22). Others have reported a stimulatory role for Akt in platelet secretion and aggregation (45, 46). Also, calcium elevation is induced by platelet agonists and aggregation-dependent integrin outside-in signaling (47). Thus, it would be logical to hypothesize a novel platelet secretion and activation pathway in which agonist-induced calcium elevation and/or a possible agonist-induced activation of the phosphoinositide 3-kinase signaling pathway induce NOS3 activation, which subsequently activates the cGMP-PKG pathway. The NOS3-cGMP-PKG pathway coordinates with integrin outside-in signaling, inducing platelet secretion and the second wave of platelet activation. Because the NOS3-cGMP-PKG pathway is important in promoting platelet secretion and aggregation under low agonist conditions, this pathway is also likely to play a major role during minor or chronic vascular injury and, thus, in the development of atherosclerosis.


   FOOTNOTES
 
* This work was supported by NHLBI, National Institutes of Health Grants HL62350 and HL68819 (to X. D), by an American Heart Association Midwest Affiliate fellowship (to J. A. M.), and by an American Heart Association Scientist Development Award (to Z. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pharmacology, University of Illinois at Chicago, 835 South Wolcott Ave., Rm. E403, Chicago, IL 60612. Tel.: 312-355-0237; Fax: 312-996-1225; E-mail: xdu{at}uic.edu.

2 The abbreviations used are: VWF, von Willebrand factor; NOS, nitric-oxide synthase; sGC, soluble guanylyl cyclase; PKG, cGMP-dependent protein kinase; 8-pCPT-cGMP, 8-(4-chlorophenylthio)-guanosine 3', 5'-cyclic monophosphate; SNP, sodium nitroprusside; L-NAME, N{omega}-nitro-L-arginine methyl ester. Back


   ACKNOWLEDGMENTS
 
We thank Dr. William Fay for help with the thrombosis model and Drs. Randal Skidgel and Guy Le Breton for helpful discussions.



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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