Alternative splicing of platelet cyclooxygenase-2 mRNA in patients after coronary artery bypass grafting

Petra Censarek; Gerhard Steger; Carla Paolini; Thomas Hohlfeld; Tilo Grosser; Norbert Zimmermann; Diana Fleckenstein; Karsten Schrör; Artur-Aron Weber

doi:10.1160/TH07-05-0346

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2007; 98(06): 1309-1315
DOI: 10.1160/TH07-05-0346

Platelets and Blood Cells

Schattauer GmbH

Alternative splicing of platelet cyclooxygenase-2 mRNA in patients after coronary artery bypass grafting

Petra Censarek

¹Institut fr Pharmakologie und Klinische Pharmakologie Universittsklinikum Duesseldorf, Germany

,

Gerhard Steger

²Institut für Physikalische Biologie, Heinrich-Heine-Universität Duesseldorf, Germany

,

Carla Paolini

¹Institut fr Pharmakologie und Klinische Pharmakologie Universittsklinikum Duesseldorf, Germany

,

Thomas Hohlfeld

¹Institut fr Pharmakologie und Klinische Pharmakologie Universittsklinikum Duesseldorf, Germany

,

Tilo Grosser

³Institute for Translational Medicine and Therapeutics and Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, USA

,

Norbert Zimmermann

⁴Bundesinstitut für Arzneimittel und Medizinprodukte, Bonn, Germany

,

Diana Fleckenstein

⁵Institut für Pharmakologie, Universität Duisburg-Essen, Universitätsklinikum Essen, Germany

,

Karsten Schrör

¹Institut fr Pharmakologie und Klinische Pharmakologie Universittsklinikum Duesseldorf, Germany

,

Artur-Aron Weber

⁵Institut für Pharmakologie, Universität Duisburg-Essen, Universitätsklinikum Essen, Germany

› Author Affiliations

Abstract

Summary

Recently, we cloned from platelet mRNA a novel cyclooxygenase (COX)-2 splice variant, designated COX-2a, which is characterized by a partial deletion of exon 5. Preliminary studies of mRNA distribution of COX-2 isoforms in platelets from coronary artery bypass grafting (CABG) patients showed a variable increase in COX-2a mRNA expression after cardiac surgery. Thus, we assessed whether this variant may play a functional role in these patients. We report a marked (about 20346-fold) increase in the expression of COX-2a mRNA after CABG. Evidence is presented that ribosomal frame-shifting may correct the coding sequence resulting in the expression of a full-length COX-2a pro-tein. In addition, a reading frame-corrected COX-2a mutant (COX-2aΔG) was generated by site-directed mutagenesis and expressed in COS-7 cells using an adenoviral expression system. However, COX-2a protein was not active in terms of prostaglandin formation. Thus, alternative mRNA splicing might represent an intriguing posttranscriptional mechanism to oppose a transcriptional activation of the COX-2 gene. Evolutionary, this mechanism may prevent COX-2-dependent thromboxane synthesis in the platelet, which would potentiate the likelihood of thrombosis; pharmacologically, this mechanism would prevent an aspirin-insensitive pathway of thromboxane formation.

Keywords

Platelet COX-2 - COX-2a - aspirin - thromboxane - CABG - qPCR

Full Text

References

References
1 Antithrombotic Trialists’ Collaboration.. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. Br Med J 2002; 324: 71-86.
2 Patrono C, Garcia Rodriguez LA, Landolfi R. et al. Low-dose aspirin for the prevention of atherothrombosis. N Engl J Med 2005; 353: 2373-2383.
3 FitzGerald GA. Mechanisms of platelet activation: thromboxane A2 as an amplifying signal for other agonists. Am J Cardiol 1991; 68: 11B-15B.
4 Grosser T, Zucker TP, Weber A-A. et al. Thromboxane A2 induces cell signaling but requires platelet-derived growth factor to act as a mitogen. Eur J Pharmacol 1997; 319: 327-332.
5 Cheng Y, Austin SC, Rocca B. et al. Role of prostacyclin in the cardiovascular response to thromboxane A2. Science 2002; 296: 539-541.
6 Di Minno G, Silver MJ, Murphy S. Monitoring the entry of new platelets into the circulation after ingestion of aspirin. Blood 1983; 61: 1081-1085.
7 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.
8 Patrono C, Coller B, FitzGerald GA. et al. Plateletactive drugs: the relationships among dose, effectiveness, and side effects: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004; 126 (Suppl. 03) 234S-264S.
9 McKee SA, Sane DC, Deliargyris EN. Aspirin resistance in cardiovascular disease: a review of prevalence, mechanisms, and clinical significance. Thromb Haemost 2002; 88: 711-715.
10 Maree AO, Fitzgerald DJ. Aspirin and coronary artery disease. Thromb Haemost 2004; 92: 1175-1181.
11 Payne DA, Jones CI, Hayes PD. et al. Platelet inhibition by aspirin is diminished in patients during carotid surgery: a form of transient aspirin resistance?. Thromb Haemost 2004; 92: 89-96.
12 Hankey GJ, Eikelboom JW. Aspirin resistance. Lancet 2006; 367: 606-617.
13 Schrör K, Hohlfeld T, Weber A-A. Aspirin resistance – does it clincally matter?. Clin Res Cardiol 2006; 95: 1-6.
14 Hohlfeld T, Zimmermann N, Weber A-A. et al. Pyrazole analgesics prevent the antiaggregatory effect of aspirin and preserve platelet thromboxane synthesis. J Thromb Haemost 2007; in press.
15 Lepantalo A, Mikkelsson J, Resendiz JC. et al. Polymorphisms of COX-1 and GPVI associate with the antiplatelet effect of aspirin in coronary artery disease patients. Thromb Haemost 2006; 95: 253-259.
16 Zimmermann N, Kurt M, Wenk A. et al. Is cardiopulmonary bypass a reason for aspirin resistance after coronary artery bypass grafting?. Eur J Cardiothorac Surg 2005; 27: 606-610.
17 Weber A-A, Przytulski B, Schanz A. et al. Towards a definition of aspirin resistance: a typological approach. Platelets 2002; 13: 37-40.
18 Zimmermann N, Kienzle P, Weber A-A. et al. Aspirin resistance after coronary artery bypass grafting. J Thorac Cardiovasc Surg 2001; 121: 982-984.
19 Weber A-A, Zimmermann KC, Meyer-Kirchrath J. et al. Cyclooxygenase-2 in human platelets as a possible factor in aspirin resistance. Lancet 1999; 353: 900.
20 Zimmermann N, Wenk A, Kim U. et al. Functional and biochemical evaluation of platelet aspirin resistance after coronary artery bypass surgery. Circulation 2003; 108: 542-547.
21 Weber A-A, Przytulski B, Schumacher M. et al. Flow cytometry analysis of platelet cyclooxygenase-2 expression. Induction of platelet cyclooxygenase-2 in patients undergoing coronary artery bypass grafting. Br J Haematol 2002; 117: 424-426.
22 Rocca B, Secchiero P, Ciabattoni G. et al. Cyclooxygenase- 2 expression is induced during human megakaryopoiesis and characterizes newly formed platelets. Proc Natl Acad Sci USA 2002; 99: 7634-7639.
23 Schrör K, Weber A-A. COX-2 inhibitors and the thrombotic risk. Thromb Haemost 2006; 96: 391-392.
24 Censarek P, Freidel K, Udelhoven M. et al. Cyclooxygenase COX-2a, a novel COX-2 mRNA variant, in platelets from patients after coronary artery bypass grafting. Thromb Haemost 2004; 92: 925-928.
25 Weber A-A, Reimann S, Schrör K. Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro. Br J Pharmacol 1999; 126: 415-420.
26 Hermann A, Rauch BH, Braun M. et al. Platelet CD40 ligand (CD40L)--subcellular localization, regulation of expression, and inhibition by clopidogrel. Platelets 2001; 12: 74-82.
27 Schrör K, Seidel H. Blood-vessel wall arachidonate metabolism and its pharmacological modification in a new in vitro assay system. Naunyn Schmiedeberg’s Arch Pharmacol 1988; 337: 177-182.
28 Hofacker IL. Vienna RNA secondary structure server. Nucleic Acids Res 2003; 31: 3429-3431.
29 Reeder J, Giegerich R. Design, implementation and evaluation of a practical pseudoknot algorithm based on thermodynamics. BMC Bioinformatics 2004; 5: 104.
30 Atkins JF, Elseviers D, Gorini L. Low activity of b-galactosidase in frameshift mutants of Escherichia coli. Proc Natl Acad Sci 1972; 69: 1192-1195.
31 Giedroc DP, Theimer CA, Nixon PL. Structure, stability and function of RNA pseudoknots involved in stimulating ribosomal frameshifting. J Mol Biol 2000; 298: 167-185.
32 Matsufuji S, Matsufuji T, Miyazaki Y. et al. Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell 1995; 80: 51-60.
33 Patrignani P, Sciulli MG, Manarini S. et al. COX-2 is not involved in thromboxane biosynthesis by activated human platelets. J Physiol Pharmacol 1999; 50: 661-667.
34 Reiter R, Resch U, Sinzinger H. Do human platelets express COX-2?. Prostaglandins Leukot Essent Fatty Acids 2001; 64: 299-305.
35 Chandrasekharan NV, Dai H, Roos LT. et al. COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proc Natl Acad Sci USA 2002; 99: 13926-13931.
36 Censarek P, Freidel K, Hohlfeld T. et al. Human cyclooxygenase-1b is not the elusive target of acetaminophen. Eur J Pharmacol 2006; 551: 50-53.
37 Grosser T, Fries S, FitzGerald G. Biological basis for the cardiovascular consequences of COX-2 inhibition: therapeutic challenges and opportunities. J Clin Invest 2006; 116: 4-15.
38 Grosser T. The pharmacology of selective inhibition of COX-2. Thromb Haemost 2006; 96: 393-400.
39 Weber A-A, Hohlfeld T, Harder S. Does a coxib-associated thrombotic risk limit the clinical use of the compounds as analgesic, antiinflammatory drugs? – Arguments against. Thromb Haemost 2006; 96: 413-416.