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Competitive inhibition of hydrogen peroxide-induced aggregation of calf platelets by prostaglandin H2/thromboxane A2 receptor ligands

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Abstract

Hydrogen peroxide (H2O2)-induced aggregation of calf platelets and its modification by agents with specific properties were characterized employing a spectrophotometric assay. An Arrhenius activation energy of 20 ± 1 kcal/mol was found in the temperature range of 25‡-36‡C. Rate inhibition occurred on either side of this temperature range, and under anaerobic conditions. Exogenous Ca2+ ions were not required but Ca2+ ions, at 1 mM-concentration, optimally increased rates and extent of aggregation at suboptimal H2O2 concentrations but only extent of aggregation at optimal H2O2 concentrations. Ba2+, Sr2+, Cd2+, Mn2+ and Ni2+ ions (1 mM) and Zn2+, Pb2+ and Hg2+ ions (10 mM) were inhibitory. The cyclo-oxygenase inhibitor, indomethacin (10-30 mM) exerted only mild inhibition by a competitive mechanism. Another cyclo-oxygenase inhibitor, aspirin, functioned to increase aggregation. Ligands acting directly at the prostaglandin H2/thromboxane A, receptor (5Z. 9, 11, 13E, 15(S) 15-hydroxy 9(11) epoxy methano prosta 5, 13-dien-1-oic acid, pinane thromboxane A2, arachidonic acid, eicosapentaenoic acid, and N-ethylmaleimide) functioned as competitive inhibitors. Another platelet-activating sulphydryl reagent, thimerosal, also inhibited competitively while the protein kinase C inhibitor, sphingosine, and the protein kinase C modulator, Zn2+ ions, inhibited by different mechanisms. The results indicate direct action of H2O2 at the prostaglandin H2/thromboxane A2 receptor, possibly its sulphydryls, to activate the protein kinase C pathway, independently of cyclo-oxygenase products. The results underscored the power of the kinetic approach for investigating mechanisms of platelet activation.

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Abbreviations

PLC:

Phospholipase C

(Ca2+)i:

intracellular free Ca2+ ion concentration

IP3 :

inositol 1

4:

5-triphosphate

DAG:

1

2-diacylglycerol:

PKC

protein kinase C:

PLA2

phospholipase A2 :

AA

arachidonic acid:

COx

cyclo-oxygenase:

PGH2

prostaglandin H2 :

TxA2

thromboxane A2 :

MDA

malondialdehyde:

U46619

5Z:

9

11:

13E

15(S)15-hydrox 9(11) epoxy methano prosta 5:

13-dien-1-oicacid

PTxA2 :

pinane thromboxane A2

PMA:

4s-phorobol 12-myristate 13-acetate

EPA:

5

8:

11

14:

17-eicosapentaenoic acid

NEM:

N-ethylmaleimide

References

  • Burke S E, Lefer A M, Nicolaou K C and Smith J B 1983 Responsiveness of platelets and coronary arteries from different species to synthetic thromboxane and prostaglandin endoperoxide analogues;Br. J. Pharmacol. 78 287–292

    PubMed  CAS  Google Scholar 

  • Chetty N, Vicker J D, Kinlough-Rathbone R L, Packham M A and Mustard J F 1989 Eicosapentaenoic acid interfere with U-46619-stimulated formation of inositol phosphates in washed rabbit platelets;Thromb. Haemostas. 62 1116–1120

    CAS  Google Scholar 

  • di Minno G, Bertele V, Bianchi L, Barbieri B, Cerletti C, Dejana E, de Gaetano G and Silver M J 1981 Effect of an epoxymethan stable analogue of prostaglandin endoperoxide (U-46619) on human platelets;Thromb. Haemostas. 45 103–106

    CAS  Google Scholar 

  • Dorn II G W 1990 Cyclic oxidation-reduction reactions regulate thromboxane A2/prostaglandin H2 receptor number and affinity in human platelet membranes;J. Biol. Chem. 265 4240–4246

    PubMed  CAS  Google Scholar 

  • Hanasaki K and Arita H 1988 Characterization of thromboxane A2/prostaglandin H2 (TxA2/PGH2) receptors of rat platelets and their interaction with TxA2/PGH2 receptor antagonists;Biochem. Pharmacol. 37 3923–3929

    Article  PubMed  CAS  Google Scholar 

  • Hannun Y A and Bell R M 1989 Function of sphingolipids and sphingolipid breakdown products in cellular regulation;Science 243 500–507

    Article  PubMed  CAS  Google Scholar 

  • Hannun Y A, Greenberg C S and Bell R M 1987 Sphingosine inhibition of agonist-induced secretion and activation of human platelets implies that protein kinase C is a necessary and common event of the signal transduction;J. Biol. Chem. 262 13620–13626

    PubMed  CAS  Google Scholar 

  • Hecker M, Brune B, Decker K and Ullrich V 1989 The sulphydryl reagent thimerosal elicits human platelet aggregation by mobilization of intracellular calcium and secondary prostaglandin endoperoxide formation;Biochem. Biophys. Res. Commun. 159 961–968

    Article  PubMed  CAS  Google Scholar 

  • Hirata M, Hayashi Y, Ushikubi F, Yokota Y, Kageyama R, Nakanishi S and Narumiya S 1991 Cloning and expression of cDNA for human thromboxane A2 receptor;Nature (London)349 6170–620

    Article  Google Scholar 

  • Hornberger W and Patscheke H 1989 Hydrogen peroxide and methyl mercury are primary stimuli of eicosanoid release in human platelets;J. Clin. Chem. Clin. Biochem. 27 567–575

    PubMed  CAS  Google Scholar 

  • Huang E M and Detwiler T C 1982 Arachidonate is an antagonist of platelet activation by the endoperoxide analogue U-46619;Biochim. Biophys. Acta 715 246–249

    PubMed  CAS  Google Scholar 

  • Jamaluddin M 1991 New perspectives in blood platelet aggregation;Curr. Sci. 61 526–533

    Google Scholar 

  • Jamaluddin M and Krishnan L K 1987a A spectrophotometric method for following initial rate kinetics of blood platelet aggregation;J. Biochem. Biophys. Methods 14 191–200

    Article  PubMed  CAS  Google Scholar 

  • Jamaluddin M and Krishnan L K 1987b A rate equation for blood platelet aggregation;J. Theor. Biol. 129 257–261

    Article  PubMed  CAS  Google Scholar 

  • Jamaluddin M and Krishnan L K 1990 Adenosine and ATP: mutually exclusive modifiers of ADP-induced aggregation of calf platelets;J. Biosci. 15 389–396

    Article  CAS  Google Scholar 

  • Jamaluddin M, Krishnan L K and Thomas A 1988 Ajoene inhibition of platelet aggregation: possible mediation by a haemoprotein;Biochem. Biophys. Res. Commun. 153 479–486

    Article  PubMed  CAS  Google Scholar 

  • Khan W A, Doborowsky R, El Touny S and Hannun Y A 1990 Protein kinase C and platelet inhibition by D-erythrosphingosine, Comarison with N, W-dimethyl sphingosine and commercial preparation;Biochem. Biophys. Res. Commun. 172 683–691

    Article  PubMed  CAS  Google Scholar 

  • Kroll M FI and Schafer AI1989 Biochemical mechanism of platelet activation;Blood 74 1181–1195

    PubMed  CAS  Google Scholar 

  • Lapetina E G, Chandrabose K A and Cuatrecasas P 1978 Ionophore A23187- and thrombin-induced platelet aggregation: independence from cyclo-oxygenase products;Proc. Natl. Acad. Sci. USA 75 812–822

    Article  Google Scholar 

  • Leone G, Schintu S, Porfiri R, Randolfi R and Bizzi B 1979 Platelet aggregation by thimerosal: Role of ADP and SH groups;Haemostasis 8 390–399

    PubMed  CAS  Google Scholar 

  • Luksiewicz H, Peng M L, Morinelli T A, Eckardt A, Kirby E and Niewiarowski S 1989 Separation of different receptor-mediated effects of prostaglandins H2 analogue (U-46619) on human platelets by means of human granulocytic elastase and chymotripsin;Biochem. Pharmacol. 38 3213–3217

    Article  Google Scholar 

  • Mamsten C 1975 A stable endoperoxide analogue activates platelets similarly to the natural endoperoxides;Life Sci. 18 169–176

    Article  Google Scholar 

  • McClay D R, Wessel G M and Marchase R B 1981 Intercellular recognition: Quantitation of initial binding events;Proc. Nat!. Acad. Sci. USA 78 4975–4979

    Article  PubMed  CAS  Google Scholar 

  • Morinelli T A, Niewiarowski S, Daniel J L and Smith J B 1987 Receptor-mediated effects of a PGH2 analog (U-46619) on human platelets;Am J. Physiol. 253 H1035-H1043

    PubMed  CAS  Google Scholar 

  • Nakano T, Hanasaki K and Arita H 1989 Role of protein kinase C in U-46619-induced shape-change, aggregation and secretion;Thromh. Res. 56 299–306

    Article  CAS  Google Scholar 

  • Nicolaou K C, Magolda R L, Smith J B, Aharony D, Smith E F III and Lefer A M 1979 Synthesis and biological properties of pinane thromboxane A2, a selective inhibitor of coronary artery constriction, platelet aggregation and thromboxane formation;Proc. Natl. Acad. Sci. USA 76 2566–2570

    Article  PubMed  CAS  Google Scholar 

  • Nishizuka Y 1988 The molecular heterogeneity of protein kinase C and its implication for cellular regulation;Nature (London) 334 661–665

    Article  CAS  Google Scholar 

  • Sato T, Hazhizume T, Nakao K, Akiba S and Fujii T 1989 Platelet desensitization by arachidonic acid is associated with the suppression of endoperoxide/thromboxane A2 binding;Biochem. Biophys. Acta 992 168–173

    PubMed  CAS  Google Scholar 

  • Seiss W 1989 Molecular mechanisms of platelet activation;Physiol Rev. 69 58–178

    Google Scholar 

  • Seiss W, Boehlig B, Weber P C and Lapetina E G 1985 Prostaglandin endoperoxide analogues stimulate phospholipase C and protein phosphorylation during platelet shape-change;Blood 65 1141–1148

    Google Scholar 

  • Sinko Z and Caen J P 1967 Platelet aggregation in mammalians (human, rat, rabbit, guinea pig, horse, dog). A comparative study;Thromh. Diath. Haemorrh. 17 99–111

    Google Scholar 

  • Stuart M J Murphy S and Oski F A 1975 A simple non-radioisotope technique for the determination of platelet life-span;New Engl. J. Med. 292 1310–1313

    Article  PubMed  CAS  Google Scholar 

  • Swann P G, Parent C A, Croset M, Fonlupt P P, Lagarde M, Venton D L and Le Brenton G C 1990 Enrichment of platelet phospholipids with eicosapentaenoic acid and docosa hexa enoic acid inhibits thromboxane A2/prostaglandin H2, receptor function;J. Biol. Chem. 265 21692–21697

    PubMed  CAS  Google Scholar 

  • Takahara K, Murray R, Fitz Gerald G, Garnet A and Fitzgerald DJ 1990 The response to thromboxane A2 analogues in human platelets. Discrimination of two binding sites linked to distinct effector systems;J. Biol. Chem. 265 6836–6844

    PubMed  CAS  Google Scholar 

  • White J G and Krumbwiede M 1973 Influence of cytochalasin B on the shape-change induced in platelets by cold;Blood 41 823–832

    PubMed  CAS  Google Scholar 

  • Yamada K. Shuko K and Nakamizo N 1983Thromb. Res. 29 197–206

    Article  PubMed  CAS  Google Scholar 

  • Zatta A and Prosduocimi M 1989 Platelet activation induced by a stable analogue of endoperoxides (U-46619);Thromb. Haemostas. 61 328–329

    CAS  Google Scholar 

  • Zenian A 1981 Attachment ofLeishmania tropica to macrophages is inhibited by both low temperature and metabolic inhibitors;Exp. Parasitol. 51 175–187

    Article  PubMed  CAS  Google Scholar 

Download references

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  1. Thrombosis Research Unit, Sree Chitra Tirunal Institute for Medical Sciences and Technology, 695 012, Thiruvananthapuram, India

    M. Jamalucdin & A. Thomas

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  1. M. Jamalucdin
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  2. A. Thomas
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Jamalucdin, M., Thomas, A. Competitive inhibition of hydrogen peroxide-induced aggregation of calf platelets by prostaglandin H2/thromboxane A2 receptor ligands. J Biosci 17, 129–140 (1992). https://doi.org/10.1007/BF02703498

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  • DOI: https://doi.org/10.1007/BF02703498

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