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(Circulation Research. 2006;98:593.)
© 2006 American Heart Association, Inc.

Reports

De Novo Synthesis of Cyclooxygenase-1 Counteracts the Suppression of Platelet Thromboxane Biosynthesis by Aspirin

Virgilio Evangelista, Stefano Manarini, Angelo Di Santo, Marta L. Capone, Emanuela Ricciotti, Luigia Di Francesco, Stefania Tacconelli, Andrea Sacchetti, Sandra D’Angelo, Antonio Scilimati, Maria G. Sciulli, Paola Patrignani

From the Consorzio Mario Negri Sud (V.E., S.M., A.DS., S.D.), Santa Maria Imbaro, "Gabriele d’Annunzio" University and Foundation and Ce.S.I. (M.L.C., E.R., L.DF., S.T., A. Sacchetti, M.G.S., P.P.), Chieti, University of Bari (A. Scilimati), Italy.

Correspondence to Paola Patrignani, PhD, Università "G. d’Annunzio," 66013 Chieti, Italy. E-mail ppatrignani{at}unich.it

Abstract

Aspirin affords cardioprotection through the acetylation of serine⁵²⁹ in human cyclooxygenase-1 (COX-1) of anucleated platelets, inducing a permanent defect in thromboxane A₂ (TXA₂)–dependent platelet function. However, heterogeneity of COX-1 suppression by aspirin has been detected in cardiovascular disease and may contribute to failure to prevent clinical events. The recent recognized capacity of platelets to make proteins de novo paves the way to identify new mechanisms involved in the variable response to aspirin. We found that in washed human platelets, the complete suppression of TXA₂ biosynthesis by aspirin, in vitro, recovered in response to thrombin and fibrinogen in a time-dependent fashion (at 0.5 and 24 hours, TXB₂ averaged 0.1±0.03 and 3±0.8 ng/mL; in the presence of arachidonic acid [10 µmol/L], it was 2±0.7 and 25±7 ng/mL, respectively), and it was blocked by translational inhibitors, by rapamycin, and by inhibitors of phosphatidylinositol 3-kinase. The results that COX-1 mRNA was readily detected in resting platelets and that [³⁵S]-methionine was incorporated into COX-1 protein after stimulation strongly support the occurrence of de novo COX-1 synthesis in platelets. This process may interfere with the complete and persistent suppression of TXA₂ biosynthesis by aspirin necessary for cardioprotection.

Key Words: aspirin • thromboxane • platelets • protein synthesis

Aspirin affords cardioprotection inducing a complete and permanent defect in the capacity of platelets to generate thromboxane A₂ (TXA₂) through the acetylation of serine⁵²⁹ of cyclooxygenase-1 (COX-1).^1,2 Because of a nonlinear relationship of inhibition of platelet TXA₂ generation with inhibition of TXA₂-mediated platelet aggregation, an excess of 95% inhibition of COX-1 activity is required to influence platelet function.³ In fact, even tiny concentrations of TXA₂ have been shown to cause platelet activation. Thus, 10 nmol/L of the TXA₂ mimetic U46619 induces platelet adhesion and shape change,⁴ and in the presence of a subthreshold concentration of collagen, U46619 (0.5 to 10 nmol/L) causes platelet aggregation.⁵ Recently, Maree et al⁶ showed that many patients who are treated with low-dose enteric-coated aspirin (75 mg) for secondary prevention of cardiovascular events have persistent elevated serum TXB₂ levels (>2.2 ng/mL), which translates into a more frequent occurrence of arachidonic acid (AA)–induced platelet aggregation. Reduced bioavailability of aspirin⁶ and genetic variants in COX-1⁷ may participate in the intersubject variable response to aspirin. The recent recognized capacity of platelets to make proteins de novo⁸ paves the way to identify new mechanisms involved in aspirin failure to cause complete and persistent suppression of platelet COX-1 activity in some individuals.

Thus, in the present study, we assessed, in vitro, the hypothesis that de novo synthesis of COX-1 could account for TXA₂ biosynthesis in platelets in which the activity of preformed COX-1 was blocked by pretreatment with aspirin in vitro.

Materials and Methods

Healthy volunteers (n=9 to 22; mean age 40±5 years) who provided blood for the study did not assume aspirin or nonsteroidal anti-inflammatory drugs (NSAIDs) within 10 days before donation. Platelet-rich plasma (PRP)⁹ was incubated for 30 minutes at 37°C without or with 0.3 mmol/L of aspirin. Platelets were then isolated from plasma as described previously⁹ and resuspended in RPMI 1640 supplemented with 0.5% FCS. Platelet suspension contained <1 leukocyte per 10 000 platelets. Unless otherwise indicated, 5x10⁸ platelets/mL were used for each experimental point. Mononuclear cells were separated from whole blood containing heparin (10 IU/mL) by Ficoll-Paque and stimulated for 12 hours with lipopolysaccharide (LPS; 10 µg/mL).¹⁰ Aspirin-treated or untreated platelets were incubated in the absence or in the presence of fibrinogen (0.38 mg/mL) alone or with thrombin (1 U/mL), for different times, at 37°C. The reaction was stopped by immediate centrifugation and supernatants stored at –80°C. In some experiments, exogenous AA (10 µmol/L) was added at different times, and the incubation carried on for an additional 10 minutes. In selected experiments, the following translation or signal transduction inhibitors, cycloheximide (1 mmol/L), puromicine (500 µmol/L), rapamycin (100 nmol/L), actinomycinD (1 mmol/L), staurosporine (10 µmol/L), wortmannin (100 nmol/L), LY294002 (10 µmol/L), AG490 (10 µmol/L), or vehicle (dimethylsulfoxide 0.1%), were added to platelets 10 minutes before starting incubation with fibrinogen and thrombin. The effects of a selective and reversible COX-1 inhibitor (P6),¹¹ aspirin, and 2 selective COX-2 inhibitors, rofecoxib and etoricoxib, were studied by their addition to platelets immediately before starting the incubation with thrombin and fibrinogen. In platelet supernatants, TXB₂ was evaluated by radioimmunoassay,¹ reflecting TXA₂ generation. All reagents were from Sigma-Aldrich.

RT-PCR of COX-1 and COX-2 and radiolabeling of platelets and immunoprecipitation of COX-1 are described in the expanded Material and Methods section in the online data supplement, available at http://circres.ahajournals.org.

Statistical Analysis
Data, presented as means±SEM, were analyzed by repeated-measurement ANOVA. P<0.05 was considered statistically significant.

Results and Discussion

Aspirin (0.3 mmol/L) pretreatment of PRP of healthy individuals was associated with an almost complete suppression of TXA₂ generation in washed platelets incubated for 30 minutes with fibrinogen (0.38 mg/mL) and thrombin (1 U/mL; 162.3±30.5 versus 0.1±0.03 ng/mL; n=9). A significant increase of TXA₂ generation was detected in supernatants of aspirinated platelets cultured for 24 hours with fibrinogen and thrombin (3±0.8 ng/mL; n=22) but not in resting platelets or when they were incubated with fibrinogen alone (Figure 1A). A similar effect was detected with other platelet agonists (ADP [20 µmol/L], collagen [5 µg/mL], or thrombin receptor–activating peptide [50 µmol/L]; supplemental online Table). The generation of TXA₂ in aspirinated platelets was significantly increased in the presence of exogenous AA (10 µmol/L; at 0.5 versus 24 hours: 2±0.7 and 25±7 ng/mL [n=3]; P=0.0004 and P<0.0001 versus the same time points without exogenous AA; supplemental online Figure). These concentrations of TXA₂ are biologically active^4–6 and, in the presence of other platelet agonists, may further boost platelet responses.^4,5

View larger version (19K): [in this window] [in a new window]   Figure 1. Aspirin-treated platelets recover COX-1–dependent capacity to synthesize TXA2: Role of translational and signal transduction pathways. A, PRP from healthy donors was treated for 30 minutes at 37°C with aspirin (0.3 mmol/L), and washed platelets were incubated for 0.5 or 24 hours without or with fibrinogen (0.38 mg/mL) alone or with thrombin (1 U/mL), and TXA2 biosynthesis was assessed.1 B, Aspirin-treated platelets, 5x108/mL, activated by thrombin and fibrinogen were incubated for 24 hours without or with aspirin, P6 (a reversible and selective COX-1 inhibitor),11 and 2 selective COX-2 inhibitors, etoricoxib and rofecoxib, and with inhibitors of translation and signal transduction (C).

As shown in Figure 1B, TXA₂ generation was depressed in a concentration-dependent fashion by P6, a COX-1 inhibitor,¹¹ and by aspirin (added de novo immediately before starting incubation) but not by etoricoxib or rofecoxib, 2 COX-2 inhibitors. The potency of aspirin to inhibit the recovery of TXA₂ synthesis in aspirinated platelets at 24 hours of incubation (IC₅₀, 22 µmol/L; 95% CI, 9 to 56) was significantly lower than that found at 30 minutes (IC₅₀, 4.5 µmol/L; 95% CI, 3.8 to 5.5; data not shown). This led us to hypothesize that time-dependent de novo synthesis of COX-1 protein could account for recovered COX-1 activity. Consistently, we demonstrated that TXA₂ biosynthesis in aspirinated platelets stimulated with fibrinogen and thrombin occurred by a regulated pathway for protein translation. In fact, it was prevented by puromycin, which causes the premature release of nascent polypeptide chains, cycloheximide, which blocks the translocation reaction on ribosomes and rapamycin, a bacterially derived immunosuppressant that inhibits the translation of a specific subset of mRNAs. In contrast, actinomycin D, a transcriptional inhibitor, did not affect the biosynthesis of this prostanoid. In addition, we found that inhibitors of phosphatidylinositol 3-kinase, LY294002 and wortmannin, also blocked TXA₂ recovery, indicating that this response is phosphatidylinositol 3-kinase dependent. In contrast, AG-490, a tyrosine kinase inhibitor, and staurosporine, a protein kinase C inhibitor, did not significantly affect TXA₂ generation in this setting (Figure 1C). The apparent reduction obtained by staurosporine may be attributable to its possible interaction with phosphatidylinositol 3-kinase.

Thus, aspirin-treated platelets recovered their ability to produce TXA₂ via a signal-dependent de novo protein synthesis. We hypothesized that this protein could be COX-1 itself. First, we explored whether platelets contained mRNA for COX isozymes. As shown in Figure 2A, COX-1, but not COX-2, mRNA can be detected by RT-PCR in resting platelets (lanes 1 and 3) as well as in platelets incubated for 3 hours in the presence of both fibrinogen and thrombin (lanes 2 and 4). As positive control, COX-2 mRNA was detected in purified human mononuclear cells activated with LPS. To explore platelet ability to translate mRNA in new proteins, they were loaded with [³⁵S]-methionine and then cultured in the absence or presence of both fibrinogen and thrombin. As shown in Figure 2B, several [³⁵S]-methionine–labeled proteins were synthesized in activated platelets. Immunoprecipitation with an anti–COX-1–specific antibody yielded a [³⁵S]-methionine–labeled protein corresponding to the exact COX-1 size.

View larger version (37K): [in this window] [in a new window]   Figure 2. Platelets synthesize de novo COX-1 from pre-existing mRNA. A, Detection of COX-1 but not COX-2 mRNA in resting or activated platelets. Total RNA was extracted from 2–3x106 mononuclear cells, stimulated for 12 hours with LPS (10 µg/mL) or from 8x109 platelets, from 2 donors (lanes 1 and 2, donor 1; lanes 3 and 4, donor 2), before (lanes 1 and 3) or after 3 hours of incubation with thrombin and fibrinogen (lanes 2 and 4). One microgram of total RNA served as template for RT-PCR. This figure is representative of 3 different experiments. B, Radiolabeled incorporation of [35S]-methionine into COX-1 in platelets activated with thrombin and fibrinogen. Platelets (1x109/mL) were incubated with 50 µCi [35S]-methionine in the absence or in the presence of both fibrinogen and thrombin for 0.5 or 24 hours. Total cell lysates and immunoprecipitates, with a goat anti-human COX-1 antibody, were subjected to SDS-PAGE.10 The figure is representative of 5 different experiments.

In conclusion, signal-dependent de novo synthesis of COX-1 occurring in aspirin-treated platelets after activation may represent a mechanism involved in the interference of complete and persistent suppression of TXA₂ biosynthesis by aspirin necessary for cardioprotection. The occurrence of this phenomenon in vivo in patients with cardiovascular disease treated with low-dose aspirin remains to be verified. Our results may contribute to shed some light on the rather nebulous concept of aspirin resistance.

Acknowledgments

This work was supported by a grant from EICOSANOX (LSHM-CT-2004-0050333 to P.P.) and Fondazione Carichieti-Fondazione Negri Sud Onlus (to V.E. and S.D.).

Footnotes

Original received December 22, 2005; revision received February 3, 2006; accepted February 8, 2006.

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This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 98/5/593    most recent 01.RES.0000214553.37930.3ev1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Evangelista, V. Articles by Patrignani, P. Search for Related Content PubMed PubMed Citation Articles by Evangelista, V. Articles by Patrignani, P. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB ACETYLSALICYLIC ACID Related Collections Cardiovascular Pharmacology Platelet function inhibitors Platelets