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Compartmentation and Compartment-Specific Regulation of PDE5 by Protein Kinase G Allows Selective Cgmp-Mediated Regulation of Platelet Functions

Compartmentation and Compartment-Specific Regulation of PDE5 by Protein Kinase G Allows Selective Cgmp-Mediated Regulation of Platelet Functions

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

Lindsay S. Wilson*, Hisham S. Elbatarny†, Scott W. Crawley‡, Brian M. Bennett†, and Donald H. Maurice*†§

Departments of *Pathology and Molecular Medicine, †Pharmacology and Toxicology, and ‡Biochemistry, Queen’s University, Kingston, ON, Canada K7L 3N6

Edited by Joseph A. Beavo, University of Washington School of Medicine, Seattle, WA, and approved July 1, 2008 (received for review May 16, 2008) It is generally accepted that nitric oxide (NO) donors, such as inhibitors as anti-thrombotic agents, other reports suggest that sodium nitroprusside (SNP), or phosphodiesterase 5 (PDE5) inhib- sildenafil might have proaggregatory effects (12). To date, no itors, including sildenafil, each impact human platelet function. basis for these seemingly contradictory findings were offered. Although a strong correlation exists between the actions of NO A significant literature supports the concept that cellular donors in platelets and their impact on cGMP, agents such as cyclic signaling is compartmented. Indeed, distinct sildenafil act without increasing global intra-platelet cGMP levels. macromolecular cAMP-signaling complexes have been shown to This study was undertaken to identify how PDE5 inhibitors might allow resolution of the distinct spatial and temporal effects of act without increasing cGMP. Our data identify PDE5 as an integral several cAMP-mediated actions in cells (13, 14). Although most component of a protein kinase G1␤ (PKG1␤)-containing signaling cAMP-signaling complexes contain protein kinase A (PKA), and complex, reported previously to coordinate cGMP-mediated inhi- are defined based on the identity of the A-kinase anchoring bition of inositol-1, 4, 5-trisphosphate receptor type 1 (IP3R1)- protein (AKAP) tethering the PKA, others contain the ex- mediated Ca2؉-release. PKG1␤ and PDE5 did not interact in sub- change-protein activated by cAMP (EPAC) (14–16). More cellular fractions devoid of IP3R1 and were not recruited to IP3R1- recently, a critically important role for integration of specific enriched membranes in response to cGMP-elevating agents. cyclic nucleotide phosphodiesterases (PDEs) into these com- Activation of platelet PKG promoted phosphorylation and activa- plexes has emerged as a mechanism, allowing their coordinated tion of the PDE5 fraction tethered to the IP3R1-PKG complex, an actions in cells. Although much less extensively studied, growing effect not observed for the nontethered PDE5. Based on these evidence also supports an important role for compartmentation findings, we elaborate a model in which PKG selectively activates of cGMP-based cellular signaling. Indeed, putative PKG-binding PDE5 within a defined microdomain in platelets and propose that proteins (GKAPs) have been identified in certain cells (17, 18) this mechanism allows spatial and temporal regulation of cGMP and selective subcellular distribution of cGMP-activated kinases signaling in these cells. Recent reports indicate that sildenafil might (PKG) and cGMP-hydrolyzing PDEs has also been reported prove useful in limiting in-stent and the thrombotic (19–21). For example, a model in which distinct PDEs selectively events associated with the acute coronary syndromes (ACS), situ- regulate either plasma membrane, or cytosolic, cGMP ‘‘pools,’’ ations poorly regulated with currently available therapeutics. We has emerged from recent experiments using heterologously submit that our findings may define a molecular mechanism by expressed cGMP biosensors (19, 21). Herein we report that which PDE5 inhibition can differentially impact selected cellular PDE5 forms an intrinsic and critically important regulatory functions of platelets, and perhaps of other cell types. component of a previously identified IP3R1-based, PKG- containing signaling complex in human platelets (22, 23). We calcium ͉ PKG 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 lood platelets prevent loss and promote wound healing in inhibiting platelet activation. Bby aggregating in response to injury and promoting throm- bus formation. Although these processes are of significant Results adaptive advantage, chronic platelet activation results in altered Sodium Nitroprusside and Sildenafil Synergize to Increase Platelet blood flow patterns and promotes the formation of thrombus- cGMP and Inhibit Aggregation. NO donors (i.e., sodium nitroprus- based arterial occlusions. Arterial occlusions contribute to the side, SNP) inhibit platelet activation by increasing cGMP and pathogenesis of acute coronary syndromes (ACS) (1), a spec- inhibitors of the dominant platelet cGMP phosphodiesterase, trum of conditions including unstable angina and myocardial PDE5, potentiate these effects (26–29). In our experiments, SNP infarctions. Although drug-eluting stents have largely mitigated (1–100 ␮M) inhibited platelet aggregation in a concentration- the problem of in-stent restenosis after percutaneous coronary dependent manner; a selective PDE5 inhibitor, sildenafil (100 interventions, in-stent thrombosis, which can occur anytime nM), potentiated this effect (Fig. 1A). Sildenafil alone did not after stenting, often presents catastrophically triggering death or acute myocardial infarctions (2–6). Although anti-platelet agents, including or , can reduce throm- Author contributions: L.S.W., S.W.C., and D.H.M. designed research; L.S.W. and H.S.E. bosis in ACS or at stents, their weak potencies rarely eliminate performed research; B.M.B. contributed new reagents/analytical tools; L.S.W., H.S.E., platelet-mediated re-occlusions in response to strong platelet S.W.C., B.M.B., and D.H.M. analyzed data; and L.S.W. and D.H.M. wrote the paper. activation signals, including those associated with The authors declare no conflict of interest. (7, 8). The anti-platelet actions of the selective cyclic nucleotide This article is a PNAS Direct Submission. phosphodiesterase 5 (PDE5) inhibitors, including sildenafil ci- §To whom correspondence should be addressed. E-mail: [email protected]. trate (Viagra), was suggested to represent a therapeutic option This article contains supporting information online at www.pnas.org/cgi/content/full/ in controlling ACS and in-stent thrombosis (9–11). Although the 0804738105/DCSupplemental. anti-platelet actions of sildenafil support the use of PDE5 © 2008 by The National Academy of Sciences of the USA

13650–13655 ͉ PNAS ͉ September 9, 2008 ͉ vol. 105 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804738105 Downloaded by guest on October 3, 2021 inhibited -induced Ca2ϩ release in human platelets (Fig. 1B). Thus, at a concentration that inhibited platelet aggre- gation by approximately 50% (10 ␮M), SNP inhibited thrombin- induced Ca2ϩ release by 39 Ϯ 4% (Fig. 1B). In marked contrast to its effect on platelet aggregation, sildenafil alone significantly inhibited thrombin-induced platelet Ca2ϩ release. In fact, silde- nafil (100 nM) had a more marked effect than SNP (10 ␮M) 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 Ca2ϩ release. Rather, these data are consistent with the concept that sildenafil acted locally to regulate this intra-platelet event. 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 Ca2ϩ release, we inhibited PKA in some experiments. Although the PKA-activator, 6BzcAMP (30 ␮M), inhibited thrombin-induced Ca2ϩ 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 Ca2ϩ release (Fig. 1B). Together these data demonstrate that the cAMP-PKA system did not coordinate the effects of SNP, or sildenafil, on Ca2ϩ transients in human platelets.

PDE5 Is Resident in an ER cGMP-Signaling Complex. Because silde- Fig. 1. Regulation of platelet functions. (A) PRP was incubated with SNP (10 nafil inhibited thrombin-induced Ca2ϩ transients without in- ␮M), sildenafil (100 nM), or both agents (30 s, 37°C while stirring at 1,000 rpm). creasing global intra-platelet cGMP, we tested the hypothesis Subsequently, platelet aggregation was promoted [ADP (2 ␮M), 3 min, 1000 that PDE5 might be acting locally at the platelet ER. More Ͻ rpm]. *, significant difference (P 0.05) against ‘‘Basal’’; **, significant precisely, we determined whether PDE5 formed a functional difference (P Ͻ 0.05) against SNP. Aggregation are expressed as mean Ϯ SEM. ␤ (n ϭ 5). (B) Fluo-4-loaded platelets were incubated with or without My-PKI (10 part of the IP3R1-IRAG-PKG1 -signaling complex (23). Dif- ␮ ␮ ␮ ferential centrifugation of platelet lysates identified PKG1␤ M, 5 min) and then with 6BzcAMP (30 M), SNP (10 M), sildenafil (100 nM) Ϸ Ϸ Ϸ either singly or in combination (3 min). Subsequently, thrombin (0.4 units/ml) ( 25%), PDE5 ( 45%), and IP3R1 ( 100%) in particulate ϩ was added and [Ca2 ] measured at 520 nm (n ϭ 4). *, significant differences cellular fractions (Fig. 2A). Interestingly, selective immunopre- ␤ (P Ͻ 0.05) between thrombin alone and thrombin with these agents; **, cipitation of PKG1 , PDE5, or IP3R1, allowed coimmunopre- significant difference (P Ͻ 0.05) between SNP and SNP/sildenafil. cipitation of all three proteins (Fig. 2C); a finding consistent with the idea that PDE5 was integral to the IP3R1-IRAG-PKG1␤ intra-platelet complex. Consistent with our finding that PKA inhibit platelet aggregation (Fig. 1A). SNP (10 ␮M) caused a inhibition did not impact sildenafil-induced inhibition of Ca2ϩ rapid (3.3 Ϯ 0.4-fold after 1 min) and transient (1.8 Ϯ 0.4-fold transients, neither the platelet cAMP PDE (PDE3A), nor PKA PHARMACOLOGY after 3 min) increase in platelet cGMP but sildenafil (100 nM) were recovered in immune complexes containing the cGMP- did not increase platelet cGMP levels (0.96 Ϯ 0.2-fold control signaling proteins. To ensure specificity of our immunoprecipita- values after 3 min). Together, SNP (10 ␮M) and sildenafil (100 tions, all experiments contained a control immunoprecipitation nM) synergistically increased cGMP (24 Ϯ 3-fold after 3 min). with rabbit IgG (control IPs) (Fig. 2C). Also, immune complexes were routinely probed for, and found deficient in, abundant ER Sildenafil Inhibits Thrombin-Induced Ca2؉ Release. Thrombin gen- proteins such as BiP (data not shown). We were unable to secure erates intra-platelet Ca2ϩ transients by promoting opening of an aliquot of an anti-IRAG antibody and could not directly test for 2ϩ IP3R1 channels and releasing endoplasmic reticulum (ER) Ca association between IRAG and PDE5 in our studies. stores (30). PKG activation inhibits this action of thrombin (23) and PKG-mediated phosphorylation of IP3R1, and of an IP3R1- PKG Phosphorylates and Activates PKG-Associated PDE5 in Vitro. associated PKG-substrate protein (IRAG), coordinates the in- Because PDE5 was a known PKG substrate (31, 32), and hibition (22, 23). In agreement with previous reports (23), SNP phosphorylation activated this enzyme, we determined whether

Table 1. Impact of SNP and sildenafil treatment on intracellular platelet cGMP and cAMP levels cGMP measurement, pmol cGMP/mg protein cAMP measurement, pmol cGMP/mg protein

Additions Without thrombin 0.4 unit/ml thrombin Without thrombin 0.4 unit/ml thrombin

Basal 0.27 ϩ 0.03 0.19 ϩ 0.02 3.14 ϩ 0.41 2.68 ϩ 0.46 10 ␮M SNP 3 min 1.07 ϩ 0.35* 1.90 ϩ 0.21* 2.97 ϩ 0.54 2.50 ϩ 0.28 100 nM sildenafil 3 min 0.33 ϩ 0.08 0.34 ϩ 0.09 2.39 ϩ 0.32 2.00 ϩ 0.54 10 ␮M SNPϩ100 nM sildenafil 3 min 5.80 Ϯ 1.19** 5.25 Ϯ 1.3** 3.36 Ϯ 0.80 2.41 Ϯ 0.13

*P Ͻ 0.05 against basal; **, P Ͻ 0.05 against SNP.

Wilson et al. PNAS ͉ September 9, 2008 ͉ vol. 105 ͉ no. 36 ͉ 13651 Downloaded by guest on October 3, 2021 A B Unbound Bound (kDa) IP3R 170

PDE5 130 PKG IRAG

IP3R 95 PDE5 72

Immunoprecipitating Immunoprecipitating C antisera antisera

Immunoblot Immunoblot antisera IP R antisera 3 IP3R

PDE5 PDE5 PDE5

PKG PKG

PDE3A PDE3A

PKAc PKAc

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 ␮M) and ATP (250 ␮M[␥-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 ϭ 3). (C) Precleared platelet lysates (1.5 mg) were incubated with either antisera against PDE5 (1 ␮g), PKG (1 ␮g), IP3R1 (1 ␮g), or mouse IgG (1 ␮g) and Protein A/G beads (16 h, 4°C). Immune complexes and aliquots of cell lysates (30 ␮g) 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 PKAc (1:1,000).

PKG-associated PDE5 was a PKG substrate. Thus, in vitro kinase originally to isolate and characterize the IP3R1/IRAG/PKG1␤ assays with PKG1␤-immunoprecipitates allowed phosphoryla- complex in platelets (Fig. 5). Taken together, these data were tion of several proteins with electrophoretic mobilities consistent consistent with the novel idea that only PKG-associated PDE5 with IP3R1 (Ϸ250 kDa), IRAG (Ϸ120 kDa), and PDE5 (Ϸ95 kDa) (Fig. 2B). The approximate 250-kDa and 95-kDa phos- phoproteins were recognized by IP3R1- or PDE5-selective an- Total tisera, respectively (Fig. 2C). Also, in vitro kinase assays with A Lysate PKG IP PDE5 IP µ - - PKG1␤-immunoprecipitates showed that cGMP (50 ␮M) and 250µM ATP + + 50µM cGMP + + + + ATP (250 ␮M) resulted in robust phosphorylation of PDE5 at S102 (Fig. 3A), and a significant level of activation of the tethered (P)S102-PDE5 PDE5 (Ϸ3-fold, Fig. 3B). Addition of cGMP alone to PKG- immune complexes did not cause PDE5-S102 phosphorylation or PDE5 PDE5 activation (Fig. 3 A and B). In marked contrast, in vitro kinase assays of anti-PDE5 immunoprecipitates did not result PKG in PDE5 phosphorylation at S102, nor PDE5 activation (Fig. 3 A PKG IP and B). B PDE5 IP 300 18 * 16 PKG-Associated PDE5 Is Selectively Activated by PKG in Platelets. 250 Although previous work indicated that PDE5 was activated upon 14 200 n cGMP-binding to a PDE5 GAF-A domain, or PKG phosphor- n 12 102 10 ylation of PDE5 at S (31–33), these studies were silent on the 150 8 l/mg/mi relative importance of these mechanisms in cells. To address this ol/mg/mi o 100 m 6 p

issue, we compared the phosphorylation and activation of the pm 4 PKG-associated and non-PKG-associated forms of PDE5 in 50 8BrcGMP (1 mM, 15 min)-treated platelets. Strikingly, 2 0 102 0 8BrcGMP treatment of platelets markedly increased the S DMSO SildenafilDMSO Sildenafil DMSO SildenafilDMSO Sildenafil 50µM cGMP 50µM cGMP+ 50µM cGMP 50µM cGMP+ phosphorylation status and activity of the PKG-associated form 250µM ATP 250µM ATP of PDE5, but not that which was not associated (Fig. 4 and Table 2). Consistent with the idea that the phosphorylated PDE5 was Fig. 3. In vitro phosphorylation of PKG-bound PDE5. Anti-PKG or anti-PDE5 resident within the IP R1/IRAG/PKG1␤ complex, IP R1 was immunoprecipitates were incubated with either cGMP (50 ␮M) or both cGMP 3 3 ␮ ␮ recovered in the anti-PKG immune complexes but not in those (50 M) and ATP (250 M) (30 min, 30°C). (A) Immune complexes were resolved by SDS-PAGE and immunoblotted with a S102-specific phospho-PDE5 representing bulk PDE5 (Fig. 4). Similarly, when anti-IP3R1 antibody (1:1,000). (B) Effect of treatments in (A) on PDE5 activities in the immune complexes were obtained from control or 8BrcGMP- immunoprecipitates. *, significant difference (P Ͻ 0.05) between PKG- treated platelets, only the IP3R1-associated PDE5 was activated immunoprecipitate treated with or without ATP. Immunoblots and PDE5 by 8BrcGMP (Fig. 5). An identical pattern of PDE5 activation activity values are from the same experiment and are representative of three was obtained when PDE5 was isolated using the method used experiments.

13652 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804738105 Wilson et al. Downloaded by guest on October 3, 2021 Lysate PKG IP PDE5 IP 8BrcGMP (1mM) --+++- (P)S102-PDE5

PDE5

PKG

IP3R

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.

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 dephos- phorylated and further reduced their PDE5 activity. In contrast, phosphatase treatment was without effect on the PDE5 activity present in anti-PDE5 immunoprecipitates (Table 2). In addition to being selectively activated by PKG in cells, our data indicate that the IP3R1-IRAG-PKG1␤-associated PDE5 had a lower specific activity than that isolated from the bulk cytosol (Fig. 6). Indeed, the IP3R1-IRAG-PKG1␤-associated PDE5 was only approximately 14% as active as that isolated in anti-PDE5 immunoprecipitates (i.e., Ϸ21 vs. Ϸ140 pmol/mg/ min, respectively). Because we routinely assessed PDE5 activity Fig. 5. Particulate, IP3R1-associated PDE5, is selectively activated by PKG. in our studies under nonsaturating substrate conditions, further Control or 8BrcGMP-treated human platelet lysates were fractionated and studies will be required to assess the kinetic basis of the lower processed as described in the legend to Fig. 2. Data are cGMP PDE activities specific activity of the tethered PDE5 in platelets. obtained in representative IP3R1-immunoprecipitates or cGMP-agarose pre- cipitates from three experiments. *, significant difference (P Ͻ 0.05) between Impact of PKG-Activation on IP3R1-IRAG-PKG-PDE5 Complex Dynamics cGMP PDE activity in IP3R1-immunoprecipitates, or cGMP-agarose precipi- in Platelets. To determine whether PDE5 and PKG formed a tates, derived from the 100,000 ϫ g pellet of 8BrcGMP-treated platelets stable complex in platelets, or only interacted within the ER- compared with this fraction in control platelets. based IP3R1-signaling complex, we assessed whether these pro- teins could be coimmunoprecipitated in the IP3R1-devoid cyto- Discussion solic fractions. Our data are inconsistent with the idea that PDE5 2ϩ and PKG form a stable complex in platelet cellular fractions In this article, we confirm that NO donors inhibit ER Ca release (23), that this effect correlates positively with their ability devoid of IP3R1. Indeed, although PKG, or PDE5, were each individually immunoprecipitated from platelet supernatants, in to increase intra-platelet cGMP, that human platelets contain a ␤ the four separate experiments in which it was tested, these protein complex consisting of IP3R1, IRAG, and PKG1 , and proteins never coimmunoprecipitated from this fraction [sup- show that the activation of PKG in this complex promotes IP3R1 phosphorylation. In addition, our data identify a potentially

porting information (SI) Fig. S1]. Because PDE5, PKG, or IP3R1 PHARMACOLOGY was not enriched in IP3R1-containing subcellular fraction in cells important role for cGMP hydrolysis by PDE5 in coordinating incubated with a cGMP analogue, or with an NO donor, our data this event, a step that had previously been ignored (23). Thus, we are inconsistent with the idea that cGMP elevation triggers report that PDE5 resides in the IP3R1-based complex and show movement of these proteins between these fractions (Fig. S1). Of that PDE5 inhibition can play a determinant role in cGMP-based ϩ note, we did observe that cGMP-elevating agents increased the control of Ca2 release in platelets without increasing global fraction of IP3R1 and PKG that could be coimmunoprecipitated intra-platelet cGMP. We suggest that our data are inconsistent (Fig. S1). Although it is likely that this effect will impact the with the idea that global levels of cGMP are an estimate of the dynamics of PKG-mediated phosphorylation of proteins in this inhibition of Ca2ϩ release caused by agents acting through signaling complex, and perhaps more broadly in the ER, further cGMP, but rather with the idea that localized changes in cGMP, studies will be required to directly test this hypothesis and to fully perhaps at the ER, may more closely correlate with their effects. elucidate its molecular basis. In addition to showing that sildenafil impacts platelet function

Table 2. Impact of 8BrcGMP treatment of human platelets on PKG- and PDE5-immunoprecipitated PDE5 activities cGMP PDE activity (Ϫsildenafil), cGMP PDE activity (ϩsildenafil), cGMP PDE activity (ϩ1 unit Sample Addition pmol⅐mgϪ1⅐minϪ1 pmol⅐mgϪ1⅐minϪ1 CIAP), pmol⅐mgϪ1⅐minϪ1

Total lysate None 31.0 ϩ 1.1 3.3 ϩ 0.8 Total lysate 8BrcGMP 40.0 ϩ 6.2 1.2 ϩ 0.6 PKG IP None 4.6 ϩ 0.7 0.9 ϩ 0.8 0.9 ϩ 0.7# PKG IP 8BrcGMP 7.6 ϩ 0.9* Ϫ0.5 ϩ 0.8 0.0 ϩ 0.7# PDE5 IP None 626.3 ϩ 13.4 25.9 ϩ 1.1 572.7 ϩ 22.4 PDE5 IP 8BrcGMP 660.9 ϩ 21.6 80.8 ϩ 5.9 567.4 ϩ 27.1

*, P Ͻ 0.05 against basal PKG-IP; #, P Ͻ 0.05 against basal cGMP PDE activities.

Wilson et al. PNAS ͉ September 9, 2008 ͉ vol. 105 ͉ no. 36 ͉ 13653 Downloaded by guest on October 3, 2021 A Total Total catalytic activity and that this enzyme is only fully active subse- Lysate IP: PDE5 Lysate IP: PKG quent to its phosphorylation by PKG. In contrast, we propose that the noncompartmented platelet PDE5 is constitutively more IB: PDE5 active than its compartmented counterpart and that this enzyme is not further activated by PKG-mediated phosphorylation. We IB: PKG 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 B spur further investigations aimed at achieving the greatest 140 degree of selectivity possible with PDE inhibition. ) 120 The catastrophic consequences of coronary artery disease- associated ACS, and the recent reports identifying very signif- 100 icant rates of ‘‘late’’ and ‘‘very late’’ in-stent thrombotic events

mol/min/mg 80 in otherwise healthy individuals have each spurred efforts to identify more effective agents than those anti-thrombotic agents 60 currently available. Indeed, although it is generally acknowl-

E activity (p E activity 40 * edged that drugs such as aspirin and the thienopyridines can

PD effectively reduce thrombosis in ACS, they are significantly less 20 effective at inhibiting the strong platelet activations associated

0 with thrombolysis or in-stent thrombosis (7, 8); the latter rep- DMSO sildenafil DMSO sildenafil resent a significant cause of acute myocardial infarctions and PDE5 ip PKG ip sudden cardiac death (2–5). In this context, recent attention has shifted to using selective PDE5 inhibitors such as sildenafil Fig. 6. Low specific activity of compartmented PDE5. Precleared platelet citrate (Viagra) for prevention of thrombosis (10, 11). Although lysates were processed as described in the legend to Fig. 2. Immunoblotting the use of PDE5 inhibitors for anti-platelet therapeutics is ␮ data (A) and cGMP PDE activities with cGMP (1 M) (B) are representative of consistent with the widely accepted idea that increases in seven experiments. *, significant difference (P Ͻ 0.05) between the cGMP PDE activity in anti-PKG and anti-PDE5-derived immune complexes. intracellular cGMP result in inhibition of platelet aggregation, scattered recent reports have suggested that sildenafil might have proaggregatory effects and that these prothrombotic effects without increasing platelet cGMP, we identify a scaffold on could limit their utility (12). Based on the findings reported here, which this effect is coordinated. We propose that the presence we suggest that further analysis of the effects of selective of PDE5 within this complex likely allows local actions of cGMP inhibition of the IP3R1-IRAG-PKG1␤-associated PDE5, and of to be regulated in a more dynamic manner; a proposition the fraction not associated with this complex, may illuminate consistent with the potent inhibition of thrombin-induced re- these conflicting reports. In conclusion, our study is consistent lease of Ca2ϩ transients induced by sildenafil in the absence of with an important role for PDE5 in shaping and maintaining significant changes in platelet cGMP. distinct cGMP pools in platelets and demonstrates that cGMP Of critical importance and described in this report was the compartmentation in platelets depends on both selective target- demonstration that activation of platelet PKG selectively pro- ing and the differential regulation of this enzyme. Our data shed moted the phosphorylation/activation of the IP3R1-IRAG- light on the molecular mechanism by which sildenafil, and perhaps PKG1␤ complex-associated PDE5. Indeed, no evidence of PKG- other potent and selective PDE5 inhibitors, could reduce human mediated phosphorylation/activation of the bulk PDE5 in the platelet activation and support the notion that they may prove cytosol was observed. Although unexpected, our data are con- useful in reducing unwanted thrombotic episodes. sistent with the idea that only PKG-associated PDE5 is subject to this mode of regulation in cells. Indeed, we report that PKG Experimental Procedures and PDE5 could only be coimmunoprecipitated from platelet Materials. Pharmacological agents were from Sigma–Aldrich, Biolog, Calbio- chem, or Fisher Scientific. Radiolabeled were from Perkin Elmer. fractions enriched in IP3R1, but not from the bulk cytosol, the fraction in which PDE5 and PKG were most abundant. In Sildenafil was isolated as described in ref. 24. addition to reporting that PKG and PDE5 are only ever in Platelet Function Studies. Platelet-rich plasma (PRP) was prepared by centrif- complex at membranes enriched with IP3R1, we also found no ϫ evidence to support the idea that PKG, or PDE5, could be ugation 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 trafficked to the IP3R1-enriched membranes in response to 2ϩ Ca -free buffer (0.35% BSA, 137 mM NaCl, 2.7 mM KCl, 11.9 mM NaHCO3,1 increases in cellular cGMP, or activation of PKG. mM MgCl2, 0.26 mM EGTA, 3 mg/ml apyrase ,and 5 mM Pipes, pH 6.5) and then As presented in this manuscript, we found little evidence that in this buffer containing 2 mM CaCl2 and 5 mM Hepes (pH7.4) before being PDE5 could be activated directly after binding of cGMP to this loaded with Fluo-4 A.M. (3 ␮M, 30 min) for use in Ca2ϩ-release studies. enzyme in cells, or in PKG-based immunoprecipitates. However, Drug-induced Ca2ϩ transients were measured with a spectrophotometer. previous reports have elegantly shown that the rate of PDE5 Cyclic nucleotides were measured by RIA from trichloroacetic acid (6% vol/vol) hydrolysis of cGMP renders a direct test of this hypothesis precipitates of platelets (108) after incubation with pharmacological agents as virtually impossible in intact cells, or in concentrated PDE5- described in ref. 26. containing immune complexes (31–34). We submit that the Protein–Protein Interaction Studies. Platelets were lysed in a Tris-based buffer discovery of GAF-selective cGMP analogues will likely be ␮ necessary before this hypothesis can be rigorously tested in a (pH 7.4) [1 mM EDTA, 100 mM NaCl, 5 mM MgCl2, 5 mM benzamidine, 1 l/ml aprotinin, 5 ␮l/ml bestatin, 2 ␮g/ml leupeptin, 10 mM phenylmethylsulfonyl cellular context. fluoride (PMSF), 10 mM sodium ␤-glycerophosphate, 10 mM sodium pyro- This study shows that the differential regulation of the activ- phosphate, 10 mM NaF, and 10 mM sodium vanadate], and precleared with ities of compartmented vs. noncompartmented PDEs has the rabbit IgG (1 ␮g)/protein A/G-agarose (3 h, 4°C). Precipitates were generated potential to allow selective effects in cells. Thus, we propose a from precleared platelet lysates (1.5 mg), or isolated subcellular fraction model (Fig. S2) in which compartmented PDE5 exhibits low prepared by centrifugation, using PKG (Stressgen), PDE5 [a gift from S. S.

13654 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804738105 Wilson et al. Downloaded by guest on October 3, 2021 Visweswariah (Indian Institute of Science, Bangalore, India)], IP3R1 (Neuro- Statistical Analysis. Values are presented as Mean Ϯ SEM from at least three Mab), phospho-S102 PDE5 (Fabgennix) or control rabbit-antisera (1 ␮g), and independent experiments. Effect of agents on aggregation, cGMP or cAMP protein A/G 40 ␮l or 8-AET-cGMP agarose (25 ␮l). Immune complex-associated levels, Ca2ϩ transients, or PDE activities were independently tested for signif- proteins were detected by SDS/PAGE/immunoblot analysis and cGMP PDE icance using a two-tailed Student’s t test with P Ͻ 0.05 considered significant. activities were measured using a fixed concentration of cGMP (1 ␮M), as described in ref. 14. For in vitro PKG kinase assays, immune complexes were ␮ incubated in a buffer (50 M cGMP, 20 mM TES, 2 mM MgCl2, 10 mM NaF, and ACKNOWLEDGMENTS. This work was supported by Heart and Stroke Foun- 32 10 mM Na2VO4) supplemented with 250 ␮M ATP or [␥- P ATP] (30 min, 30°C). dation of Ontario and Canadian Institute of Health Research Grants (to D.H.M. Reaction products were analyzed by immunoblot or PDE assays as described and B.M.B.). D.H.M. is a Career Investigator with the Heart and Stroke Foun- above. dation of Ontario.

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