Skip to main content
Log in

Human whole blood assays for inhibition of prostaglandin G/H synthases-1 and-2 using A23187 and lipopolysaccharide stimulation of thromboxane B2 production

  • Original Research Papers
  • Published:
Inflammation Research Aims and scope Submit manuscript


When freshly drawn, heparinized human whole blood is incubated with 50μM calcium ionophore A23187, platelets are stimulated to produce thromboxane B2 (TxB2) by activation of prostaglandin G/H synthase-1 (PGHS-1). TxB2 concentration, as measured by immunoassay, is maximal at 20–30 min and declines thereafter. Addition of acetylsalicylic acid (IC50=2.8μM) or other nonsteroidal antiinflammatory drugs (NSAIDs) 15 min or 4.5 h prior to 30 min stimulation with ionophore results in concentration dependent inhibition of TxB2 production. When blood is incubated with 0.01–10 μg/ml E. coli lipopolysaccharide (LPS), PGHS-2 is induced and TxB2 levels become detectable at 3h and continue to increase through 24 h. Using a 5 h incubation with 10 μg/ml LPS, aspirin (10 μM added at 0 h), which is rapidly metabolized to salicylic acid, had no effect on 10 μg/ml LPS-induced TxB2, but inhibited TxB2 production by ionophore A23187 added at 4.5 h, through acetylation of preexisting PGHS-1. In a 5 h assay, NSAIDs added at 0 h were compared for inhibition of TxB2 production stimulated by addition of ionophore A23187 at 4.5 h (PGHS-1), or by addition of LPS at 0 h (PGHS-2). Most NSAIDs were more potent against PGHS-1 than PGHS-2. Diclofenac, naproxen and flufenamic acid were equipotent or slightly selective for PGHS-2. Diffunisal and nimesulide were >4-fold selective for PGHS-2, and NS-398 was >30-fold selective for PGHS-2.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others


PGHS-1 and-2:

prostaglandin G/H synthase-1 and-2 TxB2, thromboxane B2




nonsteroidal antiinflammatory drug

PGE2 :

prostaglandin E2


  1. Fu JY, Masferrer JL, Seibert K, Raz A, Needleman P. The induction and suppression of prostaglandin H2 synthase (cyclooxygenase) in human monocytes. J Biol Chem 1990; 265:16737–40.

    Google Scholar 

  2. Xie W, Chipman JG, Robertson DL, Erikson RL, Simmons DL. Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by m-RNA splicing. Proc Natl Acad Sci USA 1991;88:2692–6.

    PubMed  Google Scholar 

  3. Kujubu DA, Fletcher BS, Varnum BC, Lim RW, Herschman HR. TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/ cyclooxygenase homologue. J Biol Chem 1991;266:12866–72.

    PubMed  Google Scholar 

  4. O'Banion MK, Sadowski HB, Winn V, Young DA. A serumand glucocorticoid-regulated 4-kilobase mRNA encodes a cyclooxygenase related protein. J Biol Chem 1991;266:23261–7.

    PubMed  Google Scholar 

  5. Sirois J, Richards JS. Purification and characterization of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles. J Biol Chem 1992;267:6382–8.

    PubMed  Google Scholar 

  6. Hla T, Neilson K. Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci USA 1992;89:7384–8.

    PubMed  Google Scholar 

  7. Fletcher BS, Kujubu DA, Perrin DM, Herschman HR. Structure of the mitogen-inducible TIS10 gene and demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase. J Biol Chem 1992;267:4338–44.

    PubMed  Google Scholar 

  8. Holtzman MJ, Turk J, Shornick LP. Identification of a pharmacologically distinct prostaglandin H synthase in cultured epithelial cells. J Biol Chem 1992;267:21438–45.

    PubMed  Google Scholar 

  9. Feng L, Sun W, Xia Y, Tang WW, Chanmugam P, Soyoola E, et al. Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression. Arch Biochem Biophys 1993; 307:361–8.

    Article  PubMed  Google Scholar 

  10. Meade EA, Smith WL, DeWitt DL. Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin and other non-steroidal anti-inflammatory drugs. J Biol Chem 1993;268:6610–4.

    PubMed  Google Scholar 

  11. Mitchell JA, Akarasereenont P, Thiemermann C, Flower RJ, Vane JR. Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase. Proc Natl Acad Sci USA 1993;90:11693–7.

    PubMed  Google Scholar 

  12. Klein T, Nusing RM, Pfeilschifter J, Ullrich V. Selective inhibition of cyclooxygenase 2. Biochem Pharmacol 1994;48: 1605–10.

    Article  PubMed  Google Scholar 

  13. Seibert K, Masferrer J, Zhang Y, Leahy K, Hauser S, Gierse J, et al. Expression and selective inhibition of constitutive and inducible forms of cyclooxygenase. Adv in Prostagland, Throbm Leukotr Res 1995;23:125–7.

    Google Scholar 

  14. Barnett J, Chow J, Ives D, Chiou M, MacKenzie R, Osen E, et al. Purification, characterization and selective inhibition of human prostaglandin G/H synthase 1 and 2 expressed in the baculovirus system. Biochim Biophys Acta 1994;1209:120–9.

    Google Scholar 

  15. Laneuville O, Breuer DK, DeWitt DL, Hla T, Funk CD, Smith WL. Differential inhibition of human prostaglandin endoperoxide H synthases-1 and-2 by nonsteroidal antiinflammatory drugs. J Pharmacol Exptl Therap 1994;271:927–34.

    Google Scholar 

  16. Grossman CJ, Wiseman J, Lucas FS, Trevethick MA, Birch PJ. Inhibition of constitutive and inducible cyclooxygenase activity in human platelets and mononuclear cells by NSAIDs and Cox2 inhibitors. Inflamm Res 1995;44:253–7.

    Article  PubMed  Google Scholar 

  17. Glaser K, Sung ML, O'Neill K, Belfast M, Hartman D, Carlson R, et al. Etodolac selectively inhibits human prostaglandin G/H synthase 2 (PGHS-2) versus human PGHS-1. Eur J Pharmacol 1995;281:107–11.

    Article  PubMed  Google Scholar 

  18. Glaser KB, Lock YW, Sung ML, Belfast M, Hartman D, Zimmerman J, et al. Evaluation of marketed NSAIDs for cyclooxygenase (COX)-1 and COX-2 activity using recombinant human enzymes and human whole blood. Inflamm Res 1995;44 Suppl 3:S276.

    Google Scholar 

  19. Kulmacz RJ, Lands WEM. Stoichiometry and kinetics of the interaction of prostaglandin H synthase with anti-inflammatory agents. J Biol Chem 1985;260:12572–8.

    PubMed  Google Scholar 

  20. Walenga RW, Wall SF, Setty BNY, Stuart MJ: Time-dependent inhibition of platelet cyclooxygenase by indomethacin is slowly reversible. Prostaglandins 1986;31:625–37.

    Article  PubMed  Google Scholar 

  21. Sweeney FJ, Eskra JD, Ernest MJ, Carty TJ. Spectrophotometric monitoring of lipoxygenase and cyclooxygenase pathway activity using ionophore stimulated whole blood. Agents Actions 1987;21:393–6.

    Article  PubMed  Google Scholar 

  22. Nusing R, Ullrich V. Regulation of cyclooxygenase and thromboxane synthase in human monocytes. Eur J Biochem 1992;206:131–6.

    Article  PubMed  Google Scholar 

  23. Hempel SL, Monick MM, Hunninghake GW. Lipopolysaccharide induces prostaglandin H synthase-2 prortein and m-RNA in human alveolar macrophages and blood monocytes. J Clin Invest 1994;93:391–6.

    PubMed  Google Scholar 

  24. Patrignani P, Panara MR, Greco A, Fusco O, Natoli C, Iacobelli S, et al. Biochemical and pharmacological characterization of the cycloxygenase activity of human blood prostaglandin endoperoxide synthases. J Pharmacol Exptl Ther 1994; 271:1705–12.

    Google Scholar 

  25. Futaki N, Takahishi S, Yokoyama M, Arai I, Higuchi S, Otomo S. NS-398, a novel anti-inflammatory agent, selectively inhibits prostaglandinG/H synthase/cyclooxygenase (COX-2) activity in vitro. Prostaglandins 1994;47:55–9.

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations


Additional information

acceped by G. W. Carter

Rights and permissions

Reprints and permissions

About this article

Cite this article

Young, J.M., Panah, S., Satchawatcharaphong, C. et al. Human whole blood assays for inhibition of prostaglandin G/H synthases-1 and-2 using A23187 and lipopolysaccharide stimulation of thromboxane B2 production. Inflamm Res 45, 246–253 (1996).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI:

Key words