De Novo Synthesis of Cyclooxygenase-1 Counteracts the Suppression of Platelet Thromboxane Biosynthesis by Aspirin
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Virgilio Evangelista, Stefano Manarini,
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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.
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 active4–6 and, in the presence of other platelet agonists, may further boost platelet responses.4,5
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.
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.
References
Patrignani P, Filabozzi P, Patrono C. Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects. J Clin Invest. 1982; 69: 1366–1372. [Order article via Infotrieve]
Patrono C, Coller B, FitzGerald GA, Hirsh J, Roth G. Platelet-active drugs: the relationships among dose, effectiveness, and side effects: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest. 2004; 126: 234S–264S.
Reilly IA, FitzGerald GA. Inhibition of thromboxane formation in vivo and ex vivo: implications for therapy with platelet inhibitory drugs. Blood. 1987; 69: 180–186.
Minuz P, Gaino S, Zuliani V, Tommasoli RM, Benati D, Ortolani R, Zancanaro C, Berton G, Santonastaso CL. Functional role of p38 mitogen activated protein kinase in platelet activation induced by a thromboxane A2 analogue and by 8-iso-prostaglandin F2alpha. Thromb Haemost. 2002; 87: 888–898. [Order article via Infotrieve]
Pulcinelli FM, Riondino S, Celestini P, Pignatelli E, Tifirò E, Di Renzo R, Violi F. Persistent production of thromboxane A2 in patients chronically treated with aspirin. J Thromb Haem. 2005; 3: 2784–2789.
Maree AO, Curtin RJ, Dooley M, Conroy RM, Crean P, Cox D, Fitzgerald DJ. Platelet response to low-dose enteric-coated aspirin in patients with stable cardiovascular disease. J Am Coll Cardiol. 2005; 46: 1258–1263.
Maree AO, Curtin RJ, Chubb A, Dolan C, Cox D, O’Brien J, Crean P, Shields DC, Fitzgerald DJ. COX-1 haplotype modulates platelet response to aspirin. J Thromb Haemost. 2005; 3: 2340–2345. [Order article via Infotrieve]
Denis MM, Tolley ND, Bunting M, Schwertz H, Jiang H, Lindemann S, Yost CC, Rubner FJ, Albertine KH, Swoboda KJ, Fratto CM, Tolley E, Kraiss LW, McIntyre TM, Zimmerman GA, Weyrich AS. Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets. Cell. 2005; 122: 379–391. [Order article via Infotrieve]
Patrignani P, Sciulli MG, Manarini S, Santini G, Cerletti C, Evangelista V. COX-2 is not involved in thromboxane biosynthesis by activated human platelets. J Physiol Pharmacol. 1999; 50: 661–667. [Order article via Infotrieve]
Patrignani P, Panara MR, Greco A, Fusco O, Natoli C, Iacobelli S, Cipolline F, Ganci A, Creminon C, Maclouf J, Patrono C. Biochemical and pharmacological characterization of the COX activity of human blood prostaglandin endoperoxide synthases. J Pharmacol Exp Ther. 1994; 271: 1705–1712.
Di Nunno L, Vitale P, Scilimati A, Tacconelli S, Patrignani P. Novel synthesis of 3,4-diarylisoxazole analogues of valdecoxib: reversal COX-2 selectivity by sulfonamide group removal. J Med Chem. 2004; 47: 4881–4890. [Order article via Infotrieve]
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.
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 active4–6 and, in the presence of other platelet agonists, may further boost platelet responses.4,5
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.
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.
References
Patrignani P, Filabozzi P, Patrono C. Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects. J Clin Invest. 1982; 69: 1366–1372. [Order article via Infotrieve]
Patrono C, Coller B, FitzGerald GA, Hirsh J, Roth G. Platelet-active drugs: the relationships among dose, effectiveness, and side effects: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest. 2004; 126: 234S–264S.
Reilly IA, FitzGerald GA. Inhibition of thromboxane formation in vivo and ex vivo: implications for therapy with platelet inhibitory drugs. Blood. 1987; 69: 180–186.
Minuz P, Gaino S, Zuliani V, Tommasoli RM, Benati D, Ortolani R, Zancanaro C, Berton G, Santonastaso CL. Functional role of p38 mitogen activated protein kinase in platelet activation induced by a thromboxane A2 analogue and by 8-iso-prostaglandin F2alpha. Thromb Haemost. 2002; 87: 888–898. [Order article via Infotrieve]
Pulcinelli FM, Riondino S, Celestini P, Pignatelli E, Tifirò E, Di Renzo R, Violi F. Persistent production of thromboxane A2 in patients chronically treated with aspirin. J Thromb Haem. 2005; 3: 2784–2789.
Maree AO, Curtin RJ, Dooley M, Conroy RM, Crean P, Cox D, Fitzgerald DJ. Platelet response to low-dose enteric-coated aspirin in patients with stable cardiovascular disease. J Am Coll Cardiol. 2005; 46: 1258–1263.
Maree AO, Curtin RJ, Chubb A, Dolan C, Cox D, O’Brien J, Crean P, Shields DC, Fitzgerald DJ. COX-1 haplotype modulates platelet response to aspirin. J Thromb Haemost. 2005; 3: 2340–2345. [Order article via Infotrieve]
Denis MM, Tolley ND, Bunting M, Schwertz H, Jiang H, Lindemann S, Yost CC, Rubner FJ, Albertine KH, Swoboda KJ, Fratto CM, Tolley E, Kraiss LW, McIntyre TM, Zimmerman GA, Weyrich AS. Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets. Cell. 2005; 122: 379–391. [Order article via Infotrieve]
Patrignani P, Sciulli MG, Manarini S, Santini G, Cerletti C, Evangelista V. COX-2 is not involved in thromboxane biosynthesis by activated human platelets. J Physiol Pharmacol. 1999; 50: 661–667. [Order article via Infotrieve]
Patrignani P, Panara MR, Greco A, Fusco O, Natoli C, Iacobelli S, Cipolline F, Ganci A, Creminon C, Maclouf J, Patrono C. Biochemical and pharmacological characterization of the COX activity of human blood prostaglandin endoperoxide synthases. J Pharmacol Exp Ther. 1994; 271: 1705–1712.
Di Nunno L, Vitale P, Scilimati A, Tacconelli S, Patrignani P. Novel synthesis of 3,4-diarylisoxazole analogues of valdecoxib: reversal COX-2 selectivity by sulfonamide group removal. J Med Chem. 2004; 47: 4881–4890. [Order article via Infotrieve]
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