Compartmentation and compartment-specific regulation of PDE5 by protein kinase G allows selective cGMP-mediated regulation of platelet functions

Abstract

It is generally accepted that nitric oxide (NO) donors, such as sodium nitroprusside (SNP), or phosphodiesterase 5 (PDE5) inhibitors, including sildenafil, each impact human platelet function. Although a strong correlation exists between the actions of NO donors in platelets and their impact on cGMP, agents such as sildenafil act without increasing global intra-platelet cGMP levels. This study was undertaken to identify how PDE5 inhibitors might act without increasing cGMP. Our data identify PDE5 as an integral component of a protein kinase G1β (PKG1β)-containing signaling complex, reported previously to coordinate cGMP-mediated inhibition of inositol-1, 4, 5-trisphosphate receptor type 1 (IP₃R1)-mediated Ca²⁺-release. PKG1β and PDE5 did not interact in subcellular fractions devoid of IP₃R1 and were not recruited to IP₃R1-enriched membranes in response to cGMP-elevating agents. Activation of platelet PKG promoted phosphorylation and activation of the PDE5 fraction tethered to the IP₃R1-PKG complex, an effect not observed for the nontethered PDE5. Based on these findings, we elaborate a model in which PKG selectively activates PDE5 within a defined microdomain in platelets and propose that this mechanism allows spatial and temporal regulation of cGMP signaling in these cells. Recent reports indicate that sildenafil might prove useful in limiting in-stent thrombosis and the thrombotic events associated with the acute coronary syndromes (ACS), situations poorly regulated with currently available therapeutics. We submit that our findings may define a molecular mechanism by which PDE5 inhibition can differentially impact selected cellular functions of platelets, and perhaps of other cell types.

Blood platelets prevent blood loss and promote wound healing by aggregating in response to injury and promoting thrombus formation. Although these processes are of significant adaptive advantage, chronic platelet activation results in altered blood flow patterns and promotes the formation of thrombus-based arterial occlusions. Arterial occlusions contribute to the pathogenesis of acute coronary syndromes (ACS) (1), a spectrum of conditions including unstable angina and myocardial infarctions. Although drug-eluting stents have largely mitigated the problem of in-stent restenosis after percutaneous coronary interventions, in-stent thrombosis, which can occur anytime after stenting, often presents catastrophically triggering death or acute myocardial infarctions (2–6). Although anti-platelet agents, including aspirin or thienopyridines, can reduce thrombosis in ACS or at stents, their weak potencies rarely eliminate platelet-mediated re-occlusions in response to strong platelet activation signals, including those associated with thrombolysis (7, 8). The anti-platelet actions of the selective cyclic nucleotide phosphodiesterase 5 (PDE5) inhibitors, including sildenafil citrate (Viagra), was suggested to represent a therapeutic option in controlling ACS and in-stent thrombosis (9–11). Although the anti-platelet actions of sildenafil support the use of PDE5 inhibitors as anti-thrombotic agents, other reports suggest that sildenafil might have proaggregatory effects (12). To date, no basis for these seemingly contradictory findings were offered.

A significant literature supports the concept that cellular cyclic nucleotide signaling is compartmented. Indeed, distinct macromolecular cAMP-signaling complexes have been shown to allow resolution of the distinct spatial and temporal effects of several cAMP-mediated actions in cells (13, 14). Although most cAMP-signaling complexes contain protein kinase A (PKA), and are defined based on the identity of the A-kinase anchoring protein (AKAP) tethering the PKA, others contain the exchange-protein activated by cAMP (EPAC) (14–16). More recently, a critically important role for integration of specific cyclic nucleotide phosphodiesterases (PDEs) into these complexes has emerged as a mechanism, allowing their coordinated actions in cells. Although much less extensively studied, growing evidence also supports an important role for compartmentation of cGMP-based cellular signaling. Indeed, putative PKG-binding proteins (GKAPs) have been identified in certain cells (17, 18) and selective subcellular distribution of cGMP-activated kinases (PKG) and cGMP-hydrolyzing PDEs has also been reported (19–21). For example, a model in which distinct PDEs selectively regulate either plasma membrane, or cytosolic, cGMP “pools,” has emerged from recent experiments using heterologously expressed cGMP biosensors (19, 21). Herein we report that PDE5 forms an intrinsic and critically important regulatory component of a previously identified IP₃R1-based, PKG-containing signaling complex in human platelets (22, 23). We propose that these data may further our understanding of the effects of PDE5 inhibitors reported in recent literature and support the concept that PDE5-inhibitors may be useful agents in inhibiting platelet activation.

Results

Sodium Nitroprusside and Sildenafil Synergize to Increase Platelet cGMP and Inhibit Aggregation.

NO donors (i.e., sodium nitroprusside, SNP) inhibit platelet activation by increasing cGMP and inhibitors of the dominant platelet cGMP phosphodiesterase, PDE5, potentiate these effects (26–29). In our experiments, SNP (1–100 μM) inhibited platelet aggregation in a concentration-dependent manner; a selective PDE5 inhibitor, sildenafil (100 nM), potentiated this effect (Fig. 1A). Sildenafil alone did not inhibit platelet aggregation (Fig. 1A). SNP (10 μM) caused a rapid (3.3 ± 0.4-fold after 1 min) and transient (1.8 ± 0.4-fold after 3 min) increase in platelet cGMP but sildenafil (100 nM) did not increase platelet cGMP levels (0.96 ± 0.2-fold control values after 3 min). Together, SNP (10 μM) and sildenafil (100 nM) synergistically increased cGMP (24 ± 3-fold after 3 min).

Fig. 1.

Regulation of platelet functions. (A) PRP was incubated with SNP (10 μM), sildenafil (100 nM), or both agents (30 s, 37°C while stirring at 1,000 rpm). Subsequently, platelet aggregation was promoted [ADP (2 μM), 3 min, 1000 rpm]. *, significant difference (P < 0.05) against “Basal”; **, significant difference (P < 0.05) against SNP. Aggregation are expressed as mean ± SEM. (n = 5). (B) Fluo-4-loaded platelets were incubated with or without My-PKI (10 μM, 5 min) and then with 6BzcAMP (30 μM), SNP (10 μM), sildenafil (100 nM) either singly or in combination (3 min). Subsequently, thrombin (0.4 units/ml) was added and [Ca²⁺] measured at 520 nm (n = 4). *, significant differences (P < 0.05) between thrombin alone and thrombin with these agents; **, significant difference (P < 0.05) between SNP and SNP/sildenafil.

Sildenafil Inhibits Thrombin-Induced Ca²⁺ Release.

Thrombin generates intra-platelet Ca²⁺ transients by promoting opening of IP₃R1 channels and releasing endoplasmic reticulum (ER) Ca²⁺ stores (30). PKG activation inhibits this action of thrombin (23) and PKG-mediated phosphorylation of IP₃R1, and of an IP₃R1-associated PKG-substrate protein (IRAG), coordinates the inhibition (22, 23). In agreement with previous reports (23), SNP inhibited thrombin-induced Ca²⁺ release in human platelets (Fig. 1B). Thus, at a concentration that inhibited platelet aggregation by approximately 50% (10 μM), SNP inhibited thrombin-induced Ca²⁺ release by 39 ± 4% (Fig. 1B). In marked contrast to its effect on platelet aggregation, sildenafil alone significantly inhibited thrombin-induced platelet Ca²⁺ release. In fact, sildenafil (100 nM) had a more marked effect than SNP (10 μM) in this regard (Fig. 1B). Because thrombin did not alter platelet cGMP levels, or influence the ability of sildenafil to alter platelet cGMP (Table 1), a comparison of the effects of SNP and sildenafil are inconsistent with a direct correlation between changes in global intra-platelet cGMP and inhibition of Ca²⁺ release. Rather, these data are consistent with the concept that sildenafil acted locally to regulate this intra-platelet event.

Table 1.

Impact of SNP and sildenafil treatment on intracellular platelet cGMP and cAMP levels

Additions	cGMP measurement, pmol cGMP/mg protein		cAMP measurement, pmol cGMP/mg protein
	Without thrombin	0.4 unit/ml thrombin	Without thrombin	0.4 unit/ml thrombin
Basal	0.27 + 0.03	0.19 + 0.02	3.14 + 0.41	2.68 + 0.46
10 μM SNP 3 min	1.07 + 0.35*	1.90 + 0.21*	2.97 + 0.54	2.50 + 0.28
100 nM sildenafil 3 min	0.33 + 0.08	0.34 + 0.09	2.39 + 0.32	2.00 + 0.54
10 μM SNP+100 nM sildenafil 3 min	5.80 ± 1.19**	5.25 ± 1.3**	3.36 ± 0.80	2.41 ± 0.13

*P < 0.05 against basal;

**, P < 0.05 against SNP.

Previous reports showed that NO donors could alter cAMP levels in cells expressing the cGMP-sensitive cAMP-hydrolyzing PDEs, PDE2, or PDE3. Under the conditions of our study, neither SNP nor sildenafil altered human platelet cAMP and thrombin did not alter this fact (Table 1). To test directly whether PKA was involved in SNP-, or sildenafil-induced inhibition of thrombin-mediated Ca²⁺ release, we inhibited PKA in some experiments. Although the PKA-activator, 6BzcAMP (30 μM), inhibited thrombin-induced Ca²⁺ release, and the cell-permeable PKA inhibitory peptide, myristoylated PKI (My-PKI), reversed this effect, My-PKI did not attenuate the ability of SNP, or sildenafil, to inhibit thrombin-induced Ca²⁺ release (Fig. 1B). Together these data demonstrate that the cAMP-PKA system did not coordinate the effects of SNP, or sildenafil, on Ca²⁺ transients in human platelets.

PDE5 Is Resident in an ER cGMP-Signaling Complex.

Because sildenafil inhibited thrombin-induced Ca²⁺ transients without increasing global intra-platelet cGMP, we tested the hypothesis that PDE5 might be acting locally at the platelet ER. More precisely, we determined whether PDE5 formed a functional part of the IP₃R1-IRAG-PKG1β-signaling complex (23). Differential centrifugation of platelet lysates identified PKG1β (≈25%), PDE5 (≈45%), and IP₃R1 (≈100%) in particulate cellular fractions (Fig. 2A). Interestingly, selective immunoprecipitation of PKG1β, PDE5, or IP₃R1, allowed coimmunoprecipitation of all three proteins (Fig. 2C); a finding consistent with the idea that PDE5 was integral to the IP₃R1-IRAG-PKG1β intra-platelet complex. Consistent with our finding that PKA inhibition did not impact sildenafil-induced inhibition of Ca²⁺ transients, neither the platelet cAMP PDE (PDE3A), nor PKA were recovered in immune complexes containing the cGMP-signaling proteins. To ensure specificity of our immunoprecipitations, all experiments contained a control immunoprecipitation with rabbit IgG (control IPs) (Fig. 2C). Also, immune complexes were routinely probed for, and found deficient in, abundant ER proteins such as BiP (data not shown). We were unable to secure an aliquot of an anti-IRAG antibody and could not directly test for association between IRAG and PDE5 in our studies.

Fig. 2.

Identification of a cGMP signaling complex. (A) Washed platelets were fractionated, and PDE5, PKG, or IP₃R1 were detected by immunoblot analysis. (B) Incorporation of ³²P into anti-PKG immunoprecipitate proteins after incubation with cGMP (50 μM) and ATP (250 μM [γ-³²P]ATP) for 30 min. ³²P incorporation in the immune complex, or released by the treatment, were resolved by SDS/PAGE and detected by autoradiography (n = 3). (C) Precleared platelet lysates (1.5 mg) were incubated with either antisera against PDE5 (1 μg), PKG (1 μg), IP₃R1 (1 μg), or mouse IgG (1 μg) and Protein A/G beads (16 h, 4°C). Immune complexes and aliquots of cell lysates (30 μg) were each resolved by SDS/PAGE and immunoblotted for PDE5 (1:5,000), PKG (1:5,000), IP₃R1 (1:1,000), PDE3A (1:2,000), or PKA_c (1:1,000).

PKG Phosphorylates and Activates PKG-Associated PDE5 in Vitro.

Because PDE5 was a known PKG substrate (31, 32), and phosphorylation activated this enzyme, we determined whether PKG-associated PDE5 was a PKG substrate. Thus, in vitro kinase assays with PKG1β-immunoprecipitates allowed phosphorylation of several proteins with electrophoretic mobilities consistent with IP₃R1 (≈250 kDa), IRAG (≈120 kDa), and PDE5 (≈95 kDa) (Fig. 2B). The approximate 250-kDa and 95-kDa phosphoproteins were recognized by IP₃R1- or PDE5-selective antisera, respectively (Fig. 2C). Also, in vitro kinase assays with PKG1β-immunoprecipitates showed that cGMP (50 μM) and ATP (250 μM) resulted in robust phosphorylation of PDE5 at S¹⁰² (Fig. 3A), and a significant level of activation of the tethered PDE5 (≈3-fold, Fig. 3B). Addition of cGMP alone to PKG-immune complexes did not cause PDE5-S¹⁰² phosphorylation or PDE5 activation (Fig. 3 A and B). In marked contrast, in vitro kinase assays of anti-PDE5 immunoprecipitates did not result in PDE5 phosphorylation at S¹⁰², nor PDE5 activation (Fig. 3 A and B).

Fig. 3.

In vitro phosphorylation of PKG-bound PDE5. Anti-PKG or anti-PDE5 immunoprecipitates were incubated with either cGMP (50 μM) or both cGMP (50 μM) and ATP (250 μM) (30 min, 30°C). (A) Immune complexes were resolved by SDS-PAGE and immunoblotted with a S¹⁰²-specific phospho-PDE5 antibody (1:1,000). (B) Effect of treatments in (A) on PDE5 activities in the immunoprecipitates. *, significant difference (P < 0.05) between PKG-immunoprecipitate treated with or without ATP. Immunoblots and PDE5 activity values are from the same experiment and are representative of three experiments.

PKG-Associated PDE5 Is Selectively Activated by PKG in Platelets.

Although previous work indicated that PDE5 was activated upon cGMP-binding to a PDE5 GAF-A domain, or PKG phosphorylation of PDE5 at S¹⁰² (31–33), these studies were silent on the relative importance of these mechanisms in cells. To address this issue, we compared the phosphorylation and activation of the PKG-associated and non-PKG-associated forms of PDE5 in 8BrcGMP (1 mM, 15 min)-treated platelets. Strikingly, 8BrcGMP treatment of platelets markedly increased the S¹⁰² phosphorylation status and activity of the PKG-associated form of PDE5, but not that which was not associated (Fig. 4 and Table 2). Consistent with the idea that the phosphorylated PDE5 was resident within the IP₃R1/IRAG/PKG1β complex, IP₃R1 was recovered in the anti-PKG immune complexes but not in those representing bulk PDE5 (Fig. 4). Similarly, when anti-IP₃R1 immune complexes were obtained from control or 8BrcGMP-treated platelets, only the IP₃R1-associated PDE5 was activated by 8BrcGMP (Fig. 5). An identical pattern of PDE5 activation was obtained when PDE5 was isolated using the method used originally to isolate and characterize the IP₃R1/IRAG/PKG1β complex in platelets (Fig. 5). Taken together, these data were consistent with the novel idea that only PKG-associated PDE5 was subject to PKG-catalyzed phosphorylation and activation in cells treated with 8BrcGMP. Consistent with this, addition of alkaline phosphatase to anti-PKG immune complexes dephosphorylated and further reduced their PDE5 activity. In contrast, phosphatase treatment was without effect on the PDE5 activity present in anti-PDE5 immunoprecipitates (Table 2).

Fig. 4.

PKG-associated PDE5 is selectively phosphorylated by PKG in intact platelets. Washed human platelets were incubated with, or without, 8BrcGMP (1 mM) and processed for immunoblot analysis as described in the legend to Fig. 2. Data are representative of observations made in three experiments.

Table 2.

Impact of 8BrcGMP treatment of human platelets on PKG- and PDE5-immunoprecipitated PDE5 activities

Sample	Addition	cGMP PDE activity (−sildenafil), pmol·mg−1·min−1	cGMP PDE activity (+sildenafil), pmol·mg−1·min−1	cGMP PDE activity (+1 unit CIAP), pmol·mg−1·min−1
Total lysate	None	31.0 + 1.1	3.3 + 0.8
Total lysate	8BrcGMP	40.0 + 6.2	1.2 + 0.6
PKG IP	None	4.6 + 0.7	0.9 + 0.8	0.9 + 0.7#
PKG IP	8BrcGMP	7.6 + 0.9*	−0.5 + 0.8	0.0 + 0.7 #
PDE5 IP	None	626.3 + 13.4	25.9 + 1.1	572.7 + 22.4
PDE5 IP	8BrcGMP	660.9 + 21.6	80.8 + 5.9	567.4 + 27.1

*, P < 0.05 against basal PKG-IP;

#, P < 0.05 against basal cGMP PDE activities.

Fig. 5.

Particulate, IP₃R1-associated PDE5, is selectively activated by PKG. Control or 8BrcGMP-treated human platelet lysates were fractionated and processed as described in the legend to Fig. 2. Data are cGMP PDE activities obtained in representative IP₃R1-immunoprecipitates or cGMP-agarose precipitates from three experiments. *, significant difference (P < 0.05) between cGMP PDE activity in IP₃R1-immunoprecipitates, or cGMP-agarose precipitates, derived from the 100,000 × g pellet of 8BrcGMP-treated platelets compared with this fraction in control platelets.

In addition to being selectively activated by PKG in cells, our data indicate that the IP₃R1-IRAG-PKG1β-associated PDE5 had a lower specific activity than that isolated from the bulk cytosol (Fig. 6). Indeed, the IP₃R1-IRAG-PKG1β-associated PDE5 was only approximately 14% as active as that isolated in anti-PDE5 immunoprecipitates (i.e., ≈21 vs. ≈140 pmol/mg/min, respectively). Because we routinely assessed PDE5 activity in our studies under nonsaturating substrate conditions, further studies will be required to assess the kinetic basis of the lower specific activity of the tethered PDE5 in platelets.

Fig. 6.

Low specific activity of compartmented PDE5. Precleared platelet lysates were processed as described in the legend to Fig. 2. Immunoblotting data (A) and cGMP PDE activities with cGMP (1 μM) (B) are representative of seven experiments. *, significant difference (P < 0.05) between the cGMP PDE activity in anti-PKG and anti-PDE5-derived immune complexes.

Impact of PKG-Activation on IP₃R1-IRAG-PKG-PDE5 Complex Dynamics in Platelets.

To determine whether PDE5 and PKG formed a stable complex in platelets, or only interacted within the ER-based IP₃R1-signaling complex, we assessed whether these proteins could be coimmunoprecipitated in the IP₃R1-devoid cytosolic fractions. Our data are inconsistent with the idea that PDE5 and PKG form a stable complex in platelet cellular fractions devoid of IP₃R1. Indeed, although PKG, or PDE5, were each individually immunoprecipitated from platelet supernatants, in the four separate experiments in which it was tested, these proteins never coimmunoprecipitated from this fraction [supporting information (SI) Fig. S1]. Because PDE5, PKG, or IP₃R1 was not enriched in IP₃R1-containing subcellular fraction in cells incubated with a cGMP analogue, or with an NO donor, our data are inconsistent with the idea that cGMP elevation triggers movement of these proteins between these fractions (Fig. S1). Of note, we did observe that cGMP-elevating agents increased the fraction of IP₃R1 and PKG that could be coimmunoprecipitated (Fig. S1). Although it is likely that this effect will impact the dynamics of PKG-mediated phosphorylation of proteins in this signaling complex, and perhaps more broadly in the ER, further studies will be required to directly test this hypothesis and to fully elucidate its molecular basis.

Discussion

In this article, we confirm that NO donors inhibit ER Ca²⁺ release (23), that this effect correlates positively with their ability to increase intra-platelet cGMP, that human platelets contain a protein complex consisting of IP₃R1, IRAG, and PKG1β, and show that the activation of PKG in this complex promotes IP₃R1 phosphorylation. In addition, our data identify a potentially important role for cGMP hydrolysis by PDE5 in coordinating this event, a step that had previously been ignored (23). Thus, we report that PDE5 resides in the IP₃R1-based complex and show that PDE5 inhibition can play a determinant role in cGMP-based control of Ca²⁺ release in platelets without increasing global intra-platelet cGMP. We suggest that our data are inconsistent with the idea that global levels of cGMP are an estimate of the inhibition of Ca²⁺ release caused by agents acting through cGMP, but rather with the idea that localized changes in cGMP, perhaps at the ER, may more closely correlate with their effects. In addition to showing that sildenafil impacts platelet function without increasing platelet cGMP, we identify a scaffold on which this effect is coordinated. We propose that the presence of PDE5 within this complex likely allows local actions of cGMP to be regulated in a more dynamic manner; a proposition consistent with the potent inhibition of thrombin-induced release of Ca²⁺ transients induced by sildenafil in the absence of significant changes in platelet cGMP.

Of critical importance and described in this report was the demonstration that activation of platelet PKG selectively promoted the phosphorylation/activation of the IP₃R1-IRAG-PKG1β complex-associated PDE5. Indeed, no evidence of PKG-mediated phosphorylation/activation of the bulk PDE5 in the cytosol was observed. Although unexpected, our data are consistent with the idea that only PKG-associated PDE5 is subject to this mode of regulation in cells. Indeed, we report that PKG and PDE5 could only be coimmunoprecipitated from platelet fractions enriched in IP₃R1, but not from the bulk cytosol, the fraction in which PDE5 and PKG were most abundant. In addition to reporting that PKG and PDE5 are only ever in complex at membranes enriched with IP₃R1, we also found no evidence to support the idea that PKG, or PDE5, could be trafficked to the IP₃R1-enriched membranes in response to increases in cellular cGMP, or activation of PKG.

As presented in this manuscript, we found little evidence that PDE5 could be activated directly after binding of cGMP to this enzyme in cells, or in PKG-based immunoprecipitates. However, previous reports have elegantly shown that the rate of PDE5 hydrolysis of cGMP renders a direct test of this hypothesis virtually impossible in intact cells, or in concentrated PDE5-containing immune complexes (31–34). We submit that the discovery of GAF-selective cGMP analogues will likely be necessary before this hypothesis can be rigorously tested in a cellular context.

This study shows that the differential regulation of the activities of compartmented vs. noncompartmented PDEs has the potential to allow selective effects in cells. Thus, we propose a model (Fig. S2) in which compartmented PDE5 exhibits low catalytic activity and that this enzyme is only fully active subsequent to its phosphorylation by PKG. In contrast, we propose that the noncompartmented platelet PDE5 is constitutively more active than its compartmented counterpart and that this enzyme is not further activated by PKG-mediated phosphorylation. We propose that this model, combined with recent finding in which PDE2, or PDE5, were each shown to selectively regulate plasma membrane or cytosolic, pools of cGMP, respectively (21), should spur further investigations aimed at achieving the greatest degree of selectivity possible with PDE inhibition.

The catastrophic consequences of coronary artery disease-associated ACS, and the recent reports identifying very significant rates of “late” and “very late” in-stent thrombotic events in otherwise healthy individuals have each spurred efforts to identify more effective agents than those anti-thrombotic agents currently available. Indeed, although it is generally acknowledged that drugs such as aspirin and the thienopyridines can effectively reduce thrombosis in ACS, they are significantly less effective at inhibiting the strong platelet activations associated with thrombolysis or in-stent thrombosis (7, 8); the latter represent a significant cause of acute myocardial infarctions and sudden cardiac death (2–5). In this context, recent attention has shifted to using selective PDE5 inhibitors such as sildenafil citrate (Viagra) for prevention of thrombosis (10, 11). Although the use of PDE5 inhibitors for anti-platelet therapeutics is consistent with the widely accepted idea that increases in intracellular cGMP result in inhibition of platelet aggregation, scattered recent reports have suggested that sildenafil might have proaggregatory effects and that these prothrombotic effects could limit their utility (12). Based on the findings reported here, we suggest that further analysis of the effects of selective inhibition of the IP₃R1-IRAG-PKG1β-associated PDE5, and of the fraction not associated with this complex, may illuminate these conflicting reports. In conclusion, our study is consistent with an important role for PDE5 in shaping and maintaining distinct cGMP pools in platelets and demonstrates that cGMP compartmentation in platelets depends on both selective targeting and the differential regulation of this enzyme. Our data shed light on the molecular mechanism by which sildenafil, and perhaps other potent and selective PDE5 inhibitors, could reduce human platelet activation and support the notion that they may prove useful in reducing unwanted thrombotic episodes.

Experimental Procedures

Materials.

Pharmacological agents were from Sigma–Aldrich, Biolog, Calbiochem, or Fisher Scientific. Radiolabeled nucleotides were from Perkin Elmer. Sildenafil was isolated as described in ref. 24.

Platelet Function Studies.

Platelet-rich plasma (PRP) was prepared by centrifugation of heparinized (15 units/ml) blood (284 × g, 15 min at 25°C) and used in aggregation studies as described in ref. 25. Platelets were washed in a Ca²⁺-free buffer (0.35% BSA, 137 mM NaCl, 2.7 mM KCl, 11.9 mM NaHCO₃, 1 mM MgCl₂, 0.26 mM EGTA, 3 mg/ml apyrase, and 5 mM Pipes, pH 6.5) and then in this buffer containing 2 mM CaCl₂ and 5 mM Hepes (pH7.4) before being loaded with Fluo-4 A.M. (3 μM, 30 min) for use in Ca²⁺-release studies. Drug-induced Ca²⁺ transients were measured with a spectrophotometer. Cyclic nucleotides were measured by RIA from trichloroacetic acid (6% vol/vol) precipitates of platelets (10⁸) after incubation with pharmacological agents as described in ref. 26.

Protein–Protein Interaction Studies.

Platelets were lysed in a Tris-based buffer (pH 7.4) [1 mM EDTA, 100 mM NaCl, 5 mM MgCl₂, 5 mM benzamidine, 1 μl/ml aprotinin, 5 μl/ml bestatin, 2 μg/ml leupeptin, 10 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM sodium β-glycerophosphate, 10 mM sodium pyrophosphate, 10 mM NaF, and 10 mM sodium vanadate], and precleared with rabbit IgG (1 μg)/protein A/G-agarose (3 h, 4°C). Precipitates were generated from precleared platelet lysates (1.5 mg), or isolated subcellular fraction prepared by centrifugation, using PKG (Stressgen), PDE5 [a gift from S. S. Visweswariah (Indian Institute of Science, Bangalore, India)], IP₃R1 (Neuro-Mab), phospho-S¹⁰² PDE5 (Fabgennix) or control rabbit-antisera (1 μg), and protein A/G 40 μl or 8-AET-cGMP agarose (25 μl). Immune complex-associated proteins were detected by SDS/PAGE/immunoblot analysis and cGMP PDE activities were measured using a fixed concentration of cGMP (1 μM), as described in ref. 14. For in vitro PKG kinase assays, immune complexes were incubated in a buffer (50 μM cGMP, 20 mM TES, 2 mM MgCl₂, 10 mM NaF, and 10 mM Na₂VO₄) supplemented with 250 μM ATP or [γ-³²P ATP] (30 min, 30°C). Reaction products were analyzed by immunoblot or PDE assays as described above.

Statistical Analysis.

Values are presented as Mean ± SEM from at least three independent experiments. Effect of agents on aggregation, cGMP or cAMP levels, Ca²⁺ transients, or PDE activities were independently tested for significance using a two-tailed Student's t test with P < 0.05 considered significant.