A Platelet Secretion Pathway Mediated by cGMP-dependent Protein Kinase*

Platelet secretion (exocytosis) is critical in amplifying platelet activation, in stabilizing thrombi, and in arteriosclerosis and vascular remodeling. The signaling mechanisms leading to secretion have not been well defined. We have shown previously that cGMP-dependent protein kinase (PKG) plays a stimulatory role in platelet activation via the glycoprotein Ib-IX pathway. Here we show that PKG also plays an important stimulatory role in mediating aggregation-dependent platelet secretion and secretion-dependent second wave platelet aggregation, particularly those induced via Gq-coupled agonist receptors, the thromboxane A2 (TXA2) receptor, and protease-activated receptors (PARs). PKG I knock-out mouse platelets and PKG inhibitor-treated human platelets showed diminished aggregation-dependent secretion and also showed a diminished secondary wave of platelet aggregation induced by a TXA2 analog and thrombin receptor-activating peptides that were rescued by the granule content ADP. Low dose collagen-induced platelet secretion and aggregation were also reduced by PKG inhibitors. Furthermore PKG I knockout and PKG inhibitors significantly attenuated activation of the Gi pathway that is mediated by secreted ADP. These data unveil a novel PKG-dependent platelet secretion pathway and a mechanism by which PKG promotes platelet activation.

Platelet secretion (exocytosis) is critical in amplifying platelet activation, in stabilizing thrombi, and in arteriosclerosis and vascular remodeling. The signaling mechanisms leading to secretion have not been well defined. We have shown previously that cGMP-dependent protein kinase (PKG) plays a stimulatory role in platelet activation via the glycoprotein Ib-IX pathway. Here we show that PKG also plays an important stimulatory role in mediating aggregation-dependent platelet secretion and secretion-dependent second wave platelet aggregation, particularly those induced via G q -coupled agonist receptors, the thromboxane A2 (TXA2) receptor, and protease-activated receptors (PARs). PKG I knock-out mouse platelets and PKG inhibitor-treated human platelets showed diminished aggregation-dependent secretion and also showed a diminished secondary wave of platelet aggregation induced by a TXA2 analog and thrombin receptor-activating peptides that were rescued by the granule content ADP. Low dose collageninduced platelet secretion and aggregation were also reduced by PKG inhibitors. Furthermore PKG I knockout and PKG inhibitors significantly attenuated activation of the G i pathway that is mediated by secreted ADP. These data unveil a novel PKG-dependent platelet secretion pathway and a mechanism by which PKG promotes platelet activation.
The secretion of granule contents (exocytosis) is an important cellular function shared by blood platelets, leukocytes, neurons, endocrine glands, and many other cell types. In platelets, the secretion of granules plays critical roles in amplifying platelet activation, recruitment of platelets into aggregates, and formation and stabilization of thrombi at the site of vascular injury (1). The mechanisms of granule secretion have not been totally clear but are known to require molecules that are shared by different secretory cells. These include soluble Nethylmaleimide-sensitive factor attachment protein receptors (SNAREs), 1

which mediate fusion between granules (vesicles)
and plasma membranes and cargo release (1)(2)(3). There are many other similarities between different cell types in the secretion of granule contents. For example, platelet-dense granules uptake, store, and secret the neurotransmitter serotonin in a way similar to certain neuron cell presynaptic vesicles (1). Secretion of granules is a regulated process in platelets and many other secretory cells and is known to involve common signaling mechanisms such as the elevation of intracellular calcium levels and phosphorylation of SNARE proteins (1). Platelet secretion can be induced by soluble agonists such as thromboxane A2 (TXA2) and thrombin that binds to G-proteincoupled heptahelical receptors (4 -8) or by adhesive proteins such as collagen and von Willebrand factor (9 -11). So called "weak agonists," or low concentrations of agonists, induce activation of integrins and platelet aggregation and subsequently require integrin outside-in signaling to induce aggregation-dependent platelet secretion. However, "strong agonists" such as collagen can induce aggregation-independent granule secretion via pathways that do not require integrin outside-in signaling. TXA2 induces a unique two-wave platelet secretion: a small peak of aggregation-independent platelet secretion, which precedes the first wave of platelet aggregation, and a large peak of aggregation-dependent second wave platelet secretion, which induces the second wave of platelet aggregation (12). Thus, TXA2 is a useful tool in differentiating signaling mechanisms associated with aggregation-dependent or -independent platelet secretion.
cGMP is an important secondary messenger synthesized by guanylyl cyclases (13,14). Elevation of cGMP activates cGMPdependent protein kinase (protein kinase G, PKG), which plays key roles in regulating physiological functions including vessel dilation, neuron function, and platelet activation (13,15). The roles of the cGMP-PKG pathway in platelet activation have been controversial (13, 16 -18). Although it has been believed for many years that the cGMP-PKG pathway inhibits platelet function (13), we have shown recently that the cGMP-PKG pathway in fact plays a biphasic role in platelet activation (19). An early stimulatory role of cGMP is important in the adhesion receptor, the glycoprotein Ib-IX (GPIb-IX)-, dependent platelet activation (19). A late inhibitory role of cGMP requires high concentrations of cGMP and is predominantly dependent upon cGMP-mediated activation of cAMP-dependent protein kinase (PKA) in humans (19 -21). However, the role of PKG in platelet activation remains apparently controversial because PKG knockout, although showing reduced GPIb-IX-dependent platelet activation, did not affect platelet aggregation induced by a relatively high concentration of collagen but is required for inhibition of collagen-induced platelet activation by a high concentration of a cGMP analog (22). Also, it is not clear whether PKG plays a role in platelet activation induced by GPIb-IXindependent, G-protein-coupled platelet agonists, which are critical in thrombus formation. More importantly, mechanisms of PKG involvement in platelet activation are not clear. In this study, we present a new finding that PKG is an important signaling mediator for aggregation-dependent secretion of platelet granules and that PKG-dependent secretion of dense granules is required in the second wave platelet aggregation induced by low doses of platelet agonists. These findings define a novel signaling mechanism that not only is important in understanding the roles of PKG in platelet activation but also has implications for understanding the common signaling mechanisms of degranulation and exocytosis.
Platelet Aggregation and Secretion-Fresh blood from healthy volunteers was anticoagulated with 1 ⁄7 volume of ACD (85 mM trisodium citrate, 83 mM dextrose, and 21 mM citric acid). Platelets were washed 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's solution at 3 ϫ 10 8 /ml, and allowed to incubate at 22°C for 1-2 h as described previously (23). In experiments using platelet-rich plasma (PRP), 1 ⁄10 volume of 3.8% trisodium citrate was used as anticoagulant. Platelet aggregation was measured by detecting changes in light transmission. Platelet secretion was determined by measuring the release of ATP using luciferin/luciferase reagent (Chrono-lume). Luciferin/luciferase reagent (12 l) was added to 238 l of washed platelet suspension within 1 min before stimulation. Platelet aggregation and secretion were recorded in real time in a Chrono-log lumiaggregometer at 37°C with stirring (1000 rpm). To examine the effects of PKG inhibitors, PRP or washed platelets were preincubated with KT5823 (5 M), Rp-pCPT-cGMPS (250 M), or Rp-Br-PET-cGMPS (250 M) for 5 min prior to the addition of the agonists. P-selectin Expression-Washed platelets from healthy human donors were resuspended in Tyrode's buffer and preincubated with or without Rp-pCPT-cGMPS or Rp-Br-PET-cGMPS for 5 min. The platelets were then incubated with different agonists at 37°C for 5 min and fixed by adding paraformaldehyde (final concentration of 1%). The platelets were incubated with a monoclonal anti-human P-selectin antibody, SZ51 (24). After washing, the platelets were further incubated with a fluorescein isothiocyanate-conjugated goat anti-mouse Ig antibody. Pselectin expression was analyzed using a FACScalibur flow cytometer.
Determination of cAMP-Washed platelets from healthy human donors and wild type or PKG I knock-out mice (1 ϫ 10 8 /ml) were resuspended in Tyrode's buffer and preincubated with or without apyrase, Rp-pCPT-cGMPS, RGDS, or PTX at 37°C for 5 min. Forskolin (10 M) alone or forskolin and U46619 (1 M) were then added to the platelets. After incubation for an additional 5 min in a platelet lumiaggregometer, the reactions were stopped by adding equal volumes of ice-cold 12% (w/v) trichloroacetic acid. Samples were mixed and centrifuged at 2000 ϫ g for 15 min at 4°C. The supernatant was removed and washed with 5 volumes of water-saturated diethyl ether four times and then lyophilized. cAMP levels were measured using a cAMP enzyme immunoassay kit (Amersham Biosciences).
PKG I Knock-out Mice and Mouse Platelet Preparation-The generation of a PKG I-null (Ϫ) allele by homologous recombination has been described previously (25). Male and female mice (6 -8 weeks) were anesthetized by intraperitoneal injection of pentobarbital. Whole blood from homozygous PKG I knock-out mice or wild type mice was collected from the inferior vena cava using 1 ⁄7 volume of ACD as anticoagulant as described previously (12). For each experiment, blood was pooled from 5-6 mice of each genotype. The platelets were then washed twice with CGS, resuspended in modified Tyrode's buffer at 3 ϫ 10 8 /ml, and incubated at room temperature for 1 h before use.
Calcium Mobilization-Calcium mobilization was measured in Indo-1/AM-labeled cells. Briefly, washed platelets were resuspended in CGS at 1 ϫ 10 9 /ml and incubated with 5 M Indo-1/AM at 37°C for 45 min. After washing with CGS once more, platelets were resuspended to 2 ϫ 10 8 /ml in modified Tyrode's solution. Continuous fluorescent measurements of calcium-bound and free Indo-1/AM were made using a PTI (Photon Technology International, Monmouth Junction, NJ) spectrofluorometer, detecting at 405 and 485 nm, respectively, with an excitation wavelength of 340 nm. The intracellular Ca 2ϩ level was expressed as relative fluorescence, calculated based on the ratio of Indo-1 fluorescence at 405 and 485 nm and standardized for Indo-1 loading and cell responsiveness.

PKG Plays a Stimulatory Role in Platelet Aggregation Induced by Several G-protein-coupled Receptor
Agonists-Platelets can be activated via several different types of signaling pathways. G-protein-coupled signaling pathways play a major role in platelet activation induced by soluble platelet agonists such as thrombin, TXA2, and ADP (6 -8, 26, 27). The glycoprotein VI/Fc receptor ␥ chain pathway mediates collagen-induced platelet activation (28 -30). The GPIb-IX-dependent activation pathway mediates von Willebrand factor-induced platelet activation (11,(31)(32)(33) and is also important in low dose thrombininduced platelet activation (34 -37). We reported recently that PKG plays a stimulatory role in GPIb-IX-dependent integrin activation (19,20). To investigate whether and how PKG plays roles in platelet activation via various pathways, we examined the platelet aggregation response of PKG I knock-out mouse or PKG inhibitor-treated human platelets to GPIb-IX-independent platelet agonists. These include U46619 (a stable thromboxane A2 analog) and ADP (Fig. 1). PKG I knockout (Fig. 1A) or PKG inhibitors Rp-pCPT-cGMPS, KT5823 (Fig. 1C), or Rp-Br-PET-cGMPS (see below) significantly reduced platelet aggregation induced by U46619 but had no significant effect on platelet aggregation induced by ADP (Fig. 1D). Thus, PKG plays an important role in promoting platelet aggregation in- duced via the G-protein-coupled TXA2 receptor that is independent of the GPIb-IX pathway. It is important to note that the aggregation responses of PKG I knock-out mouse platelets and PKG inhibitor-treated platelets are very similar, further confirming the specificity and effectiveness of the PKG inhibitors used in this study (19, 21, 38 -40). However, PKG inhibitors are slightly more effective in reducing mouse platelet aggregation than PKG I knockout (19), suggesting a possible compensatory mechanism in PKG I knock-out mice. Also, PKG inhibitors appear to be more effective in inhibiting platelet aggregation in humans than mice (19), suggesting possible differences between species. We have shown previously that PKG I knockout or PKG inhibitors inhibited low dose thrombin-induced platelet aggregation (19). To investigate whether PKG inhibitors affect GPIb-IX-independent thrombin receptor pathways, we also examined the effects of PKG inhibitors on thrombin receptor-activating peptides (TRAPs) for PAR1 and PAR4 (41,42). Rp-pCPT-cGMPS inhibited both PAR1 and PAR4 TRAP-induced platelet aggregation (Fig. 2), indicating that PKG also promotes PAR-dependent platelet activation pathways. Interestingly, although PKG inhibitors almost totally inhibited GPIb-IX-dependent low dose thrombin-induced human platelet aggregation as we have shown previously (19), they only partially inhibited human platelet aggregation induced by PAR1 or PAR4 TRAPs (Fig. 2), suggesting that PKG plays distinct roles in GPIb-IX-dependent platelet response and PAR-induced platelet responses.
PKG Mediates Secretion of Dense Granules-The partial inhibitory effects of PKG inhibitors on U46619 and TRAP-induced platelet aggregation resemble the pattern of platelet aggregation in the absence of aggregation-dependent platelet secretion. Thus, we further investigated the roles of PKG in platelet dense granule secretion using real time luciferin-luciferase assay. As we have shown recently (12), U46619 sequentially induced the first wave of dense granule secretion, the first wave of platelet aggregation, and then the second wave of aggregation-dependent secretion followed by the second wave of platelet aggregation (Fig. 2). The second wave of dense granule secretion and the subsequent second wave of platelet aggregation were abolished in PKG I knock-out mouse platelets ( Fig. 2A) and in PKG inhibitor-treated human platelets (Fig.  2B). Thrombin or TRAPs induce both aggregation-dependent and aggregation-independent secretion (not shown). At low concentrations, the aggregation-dependent secretion is predominant. Low dose TRAP1-or TRAP4-induced platelet secretion was also significantly inhibited by PKG inhibitors (Fig. 2,  C and D). Thus, PKG plays an important role in mediating a common aggregation-dependent secretion pathway induced by several G-protein-coupled receptors.
The Roles of PKG in Collagen-induced Platelet Secretion of Dense Granules-Collagen can induce significant platelet secretion in an aggregation-independent manner, and platelet aggregation induced by a relatively high dose of collagen is not significantly affected by knockout of PKG I (22). However, these findings do not exclude the possibility that aggregationdependent secretion can still occur. Indeed, platelet secretion induced by a low dose collagen (0.5 g/ml) is mostly aggregationdependent because the integrin inhibitor RGDS almost completely inhibited ATP secretion induced by collagen at this low concentration (Fig. 3A). Thus, to investigate whether the role of PKG is common in aggregation-dependent platelet secretion induced by agonists other than the G-protein-coupled receptors, we examined the effect of PKG inhibitors on collageninduced platelet secretion of dense granules. Both Rp-pCPT-cGMPS and Rp-Br-PET-cGMPS inhibited platelet secretion and aggregation induced by 0.5 g/ml collagen in a manner similar to RGDS, suggesting that PKG is important for platelet secretion and aggregation induced by this low concentration of collagen. Consist with previous reports, platelet aggregation induced by a higher concentration of collagen (5 g/ml) was not significantly affected by PKG inhibitors. However, platelet secretion in PKG inhibitor-treated platelets was partially but significantly reduced even at the higher collagen concentration (Fig. 3B). These results indicate that PKG is important in platelet secretion induced by collagen.

Inhibition of ADP Secretion Is Responsible for Decreased Platelet Aggregation in PKG Inhibitor-treated Platelets-A ma-
jor substance secreted from dense granules is ADP, which induces integrin activation and thus promotes platelet aggregation. As shown in Fig. 1, ADP-induced platelet aggregation was not significantly affected by PKG I knockout or PKG inhibitors, suggesting that ADP-induced integrin activation and platelet aggregation do not require PKG signaling. Therefore, we hypothesized that the inhibitory effects of PKG I knockout or PKG inhibitors on TXA2-or TRAP-induced platelet aggregation result from inhibition of ADP secretion. To investigate this hypothesis, we examined whether restoration of low concentrations of ADP can reverse the inhibitory effects of PKG inhibitors during U46619-induced platelet aggregation. Fig. 4 shows that PKG inhibitors diminished secretion and second wave platelet aggregation. Addition of a low concentration of ADP (0.5 M), although insufficient by itself to induce platelet aggregation (not shown), restored the second wave of aggregation induced by U46619 in PKG inhibitor-treated platelets. These results suggest that the inhibitory effects of PKG inhibitors on the second wave of platelet aggregation mainly result from inhibition of secretion of ADP from dense granules.
PKG-dependent Secretion of ADP Promotes Platelet Aggregation via the G i Signaling Pathway-The above data show that a major role for PKG in platelet aggregation is to mediate signal transduction leading to secretion of dense granule ADP. ADP binds to two different receptors on platelets, the G i -coupled P2Y12 receptor and the G q -coupled P2Y1 receptor. G q is also the major G-protein mediating signaling of receptors for TXA2 and TRAPs. The role of ADP in potentiating platelet aggregation induced by TXA2 or low dose TRAP only requires the G i -coupled P2Y12 pathway because it is inhibited by P2Y12 antagonists but not P2Y1 antagonists (12,43). Therefore, we reason that low concentrations of G-proteincoupled agonists other than ADP may require secreted ADP to enhance the G i signaling pathway and thus allow full scale platelet aggregation and that PKG inhibition reduces secretion of ADP and thus inhibits the ADP-dependent G i -coupled pathway. To test this hypothesis, we examined the effects of PKG inhibitors or PKG I knockout on TXA2-induced inhibition of adenylyl cyclase function. To increase the sensitivity of the assay, we used forskolin (a standard reagent for elevating cAMP levels) to elevate cAMP and examined the effects of agonists and PKG inhibitors. Fig. 5 shows that U46619 caused inhibition of forskolin-stimulated adenylyl cyclase (Fig. 5, A and B), which was completely reversed by PTX (Fig. 5A), an inhibitor of the G i pathway, indicating a  (n ϭ 3). C, wild type (PKG ϩ/ϩ ) or PKG I Ϫ/Ϫ mouse platelets were stimulated with or without U46619 or with ADP in the presence of forskolin. Levels of cAMP were then determined as described in A. requirement for G i signaling. Addition of an ADP scavenger, apyrase, also reversed U46619-induced inhibition of adenylyl cyclase (Fig. 5B), suggesting that U46619-induced activation of the G i pathway is dependent on secreted ADP. U46619induced inhibition of adenylyl cyclase was completely reversed by the PKG inhibitor Rp-pCPT-cGMPS (Fig. 5B) and in PKG I knock-out platelets (Fig. 5C). In contrast, ADPinduced inhibition of adenylyl cyclase was not affected by PKG knockout. These data indicate that PKG mediates signals leading to ADP secretion that plays important roles in activating the G i pathway and in inducing second wave platelet aggregation.
The Role of PKG in Platelet Secretion of ␣-Granules-The above results indicate that PKG plays an important role in aggregation-dependent secretion of platelet dense granules. To determine whether PKG also plays a role in platelet secretion of ␣-granules, we examined the effect of PKG inhibitors on surface expression of P-selectin induced by platelet agonists. Expression of P-selectin was examined by flow cytometry using the monoclonal anti-human P-selectin antibody SZ51. Fig. 6 shows that both Rp-pCPT-cGMPS and Rp-Br-PET-cGMPS significantly inhibited P-selectin expression induced by U46619. P-selectin expression induced by TRAPs was also partially but significantly inhibited by PKG inhibitors, although to a lesser degree. Thus, PKG also plays an important role in platelet secretion from ␣-granules.
Effects of PKG Inhibitors and PKG Knockout on Calcium Mobilization-A common signaling event upstream from secretion is the elevation of intracellular calcium levels. To investigate the relationship between PKG and calcium elevation, we examined the effects of PKG inhibitors and PKG deficiency on U46619-induced intracellular calcium mobilization. As shown in Fig. 7A, U46619-induced calcium mobilization in PKG I knock-out mouse platelets was identical to that of wild type platelets. Similarly, U46619-induced calcium mobilization was not affected by the PKG inhibitor Rp-pCPT-cGMPS in human platelets (Fig. 7B). These results suggest that PKG is not required for agonist-induced calcium mobi-lization. Thus, PKG is likely to be involved in signaling events either downstream from or parallel to calcium mobilization in platelet secretion signaling. DISCUSSION In this study, we show that PKG plays an important role in mediating aggregation-dependent platelet secretion of dense granules and ␣-granules. We also show that by inducing secretion of ADP, PKG plays a general stimulatory role in promoting platelet activation and stabilizing platelet aggregation. These findings suggest a novel signaling mechanism mediating platelet granule secretion and provide a new mechanism for the stimulatory roles of the cGMP-PKG pathway in platelet activation. Because regulated secretion is important not only in platelet function but also in leukocytes, neurons, and other cell types, identification of a novel PKG-dependent signaling pathway that stimulates exocytosis is of general significance to the understanding of the regulatory mechanisms of degranulation, exocytosis, and vesicle trafficking.
Although it has been believed for the past 30 years that the cGMP-PKG pathway inhibits platelet activation, we have shown recently that cGMP promotes GPIb-IX-dependent integrin activation and platelet aggregation. GPIb-IX is a unique receptor that, unlike thromboxane A2 or collagen receptors, does not appear to require secretion of granules to induce activation of integrin. GPIb-IX-mediated integrin activation can be reconstituted in the cultured Chinese hamster ovary cells that do not have platelet-specific granules (19,20,33,44), although it is significantly amplified by ADP secre- tion like other weak platelet agonists (11). In contrast, integrin activation induced by TXA2, thrombin, and collagen has not been reconstituted in Chinese hamster ovary cells. Thus, our finding that PKG is important in mediating platelet secretion induced by GPIb-IX-independent agonists indicates a novel role for PKG in platelet activation and suggests that PKG is important not only in GPIb-IX-dependent platelet activation but also in promoting platelet activation induced by various platelet activation pathways. It is interesting to note that the stimulatory effects of PKG occur in the absence of exogenous cGMP, suggesting that the levels of cGMP produced by agonist-stimulated endogenous guanylyl cyclases (at nanomolar range) are sufficient to mediate granule secretion. In contrast, high concentrations of cGMP analogs (100 -3000 M) are required to show inhibitory effects on platelet aggregation and secretion (22,45). Thus, our data provide further evidence for biphasic roles of cGMP in platelet activation; that is, cGMP promotes platelet activation at low concentrations but becomes inhibitory at high concentrations. More importantly, we have identified a novel PKG-dependent signaling pathway leading to platelet secretion and a mechanism by which PKG promotes platelet activation.
Despite the important roles of secretion in platelet function, signaling pathways regulating platelet granule secretion are poorly defined (1). Available evidence suggests several possible mechanisms that may be important in the regulation of secretion. 1) Elevation of intracellular calcium and activation of the cytoskeletal contractile apparatus may be important in inducing and facilitating granule secretion. 2) Phosphorylation of SNARE proteins and the roles of protein kinase C (PKC) and small G-protein Rab have been hypothesized in regulating SNARE complex formation and membrane fusion required for secretion. However, signaling pathways regulating these events or molecules remain obscure. Also, there have been various platelet functional disorders with characteristics of unidentified secretion defects, suggesting the existence of unidentified secretion signaling molecules and mechanisms (46). Thus, identification of the PKG-dependent secretion pathway in this study provides a novel signaling mechanism regulating the platelet secretion process. How PKG is activated is currently under our investigation. It is known that platelets express nitric oxide synthases (NOSs) and soluble guanylyl cyclase (13,15,47). These enzymes play an important role in cGMP elevation. It has also been reported that PKG can be activated by protein kinase C-mediated PKG phosphorylation (48). It is interesting to note that both protein kinase C and NOS3 are activated by the elevation of intracellular calcium, which is also required for platelet secretion. In addition, aggregation-dependent platelet secretion requires integrin outside-in signals. Integrin outside-in signaling causes dramatic elevation of intracellular calcium levels (49). Thus it is tempting to hypothesize that integrin-dependent elevation of calcium may activate endogenous platelet nitric oxide synthesis, subsequently elevating the cGMP level and activating PKG. However, cGMP elevation in platelets stimulated by TXA2 is not dependent upon integrin outside-in signals because RGDS peptides that inhibit integrin-ligand interaction do not significantly inhibit the cGMP elevation induced by this agonist (not shown). Also, most platelet agonists can directly cause calcium elevation (although at lower levels) without requiring integrin outside-in signaling. Therefore, it appears that agonists induce activation of the PKG pathway by an integrin-independent mechanism. The PKG signaling pathway then converges with the integrin outside-in signaling to induce aggregation-dependent secretion. In this respect, it is interesting to note that endothelial nitric oxide synthase can be activated in endothe-lial cells by Akt (50), a serine/threonine protein kinase that is a downstream effector of phosphatidylinositol 3-kinase. We and others have shown that phosphatidylinositol 3-kinase and Akt play important roles in platelet secretion and second wave platelet aggregation (12,51,52). Thus, it will be interesting to investigate further whether activation of cGMP/PKG is downstream from the phosphatidylinositol 3-kinase-Akt pathway during platelet activation. Because granule secretion in platelets shares similarity to secretion (exocytosis) and degranulation in other cell types, it is possible that the PKG-dependent secretion pathway is not only important in platelets but also shared in other secretory cells.
Our finding that PKG is important in aggregation-dependent secretion explains why there have been apparent agonistspecific differences in the effects of PKG inhibition on platelets. PKG knockout or inhibitors have no significant effect on platelet aggregation induced by high concentrations of collagen (Fig. 3) or thrombin (data not shown). These are so called strong agonists that can induce platelet secretion via the aggregation-independent secretion pathway. Although PKGmediated aggregation-dependent secretion may also occur in platelet responses to these agonists (Fig. 3), levels of aggregation-independent secretion are already sufficient to mediate full scale platelet aggregation. Thus, PKG deficiency would have no significant effect on platelet aggregation induced by these agonists. On the other hand, PKG knockout or inhibitors significantly inhibited the second wave of platelet aggregation induced by low concentrations of TXA2, thrombin, TRAPs, and collagen, which requires aggregation-dependent platelet secretion of ADP (Fig. 4). Abolishing the aggregation-dependent platelet secretion of granules by PKG inhibitors or PKG deficiency thus inhibits the second wave of platelet aggregation induced by low concentrations of these agonists. Also, because ADP can induce platelet aggregation without requiring secretion, our data explain why PKG inhibitors and PKG I knockout have no significant effect on ADP-induced platelet aggregation. Therefore, we have not only identified a novel secretion signaling pathway but also provided significant new insights into the complex interaction between secretion and aggregation and between different platelet activation pathways.