Primary platelet aggregation requires agonist-mediated activation of membrane receptor glycoprotein (GP) IIb/IIIa, binding of fibrinogen to GpIIb/IIIa, and cellular events after fibrinogen binding. This study investigated whether fibrinogen receptor GpIIb/IIIa is also the binding site for low-density lipoprotein (LDL) in platelets by using GpIIb/IIIa-coated polystyrene microbeads incubated with various concentrations of fluorescein isothiocyanate (FITC)-labeled ligands. Binding was assayed by flow cytometry. Binding of fibrinogen (Fg)-FITC and LDL-FITC to GpIIb/IIIa coated microbeads was concentration dependent, reaching saturation. Binding of LDL-FITC to GpIIb/IIIa coated microbeads was inhibited by fibrinogen. Binding of LDL-FITC or Fg-FITC to freshly isolated platelets gave similar results as those of GpIIb/IIIa coated microbeads. Glycoprotein IIb/IIIa, the fibrinogen receptor on platelets is also one of the binding sites of LDL on platelets. This rapid and reliable flow cytometric technique using coated microbeads may be used as a first step for the study of all ligand receptor interactions.
Eksioglu-Demiralp E., Kitada S., Carson D.A method for functional evaluation of caspase activation pathways in intact lymphoid cells using electroporation-mediated protein delivery and flow cytometric analysis. J Immunol Methods . 2003;275:41-56.
2.
Ulutin ONAtherosclerosis and Hemostasis. Semin Thromb Hemost . 1986;12:156-174.
3.
Yardimci T., Yaman A., Ulutin ONCharacterization of platelet gamma glutamyltransferase and its alteration in cases of atherosclerosis. Clin Appl Thromb Hemost. 1995; 1103-1113.
Huo Y., Ley KFRole of platelets in the development of atherosclerosis. Trends Cardiovasc Med. 2004;14:18-22.
6.
Plow EF, D'Souza SE, Ginsberg MHLigand binding to GpIIb/IIIa: a status report. Semin Thromb Hemost1992; 18:324-338.
7.
Sims PJ, Ginsberg MH, Plow EF, Shattil S.Effect of platelet activation on the conformation of the plasma membrane glycoprotein IIb/IIIa complex. J Biol Chem. 1991;226:7345-7365.
8.
Ozsavcι D., Yardιmcι KT, Yanιkkaya-Demirel G., Eksioglu-Demiralp E., Uras F., Önder E. Flow cytometric assay of platelet glycoprotein receptor numbers in hypercholesterolemia. Platelets. 2002;13:223-229.
9.
Rosenson RS, Lowe GDO.Effects of lipids and lipoproteins on thrombosis and rheology. Atherosclerosis. 1998;140: 271-280.
Bruckdorfer KRLipoprotein, platelet and coagulation. In: Betteridge DJ, Illingworth DR, Shepherd J, eds. Lipoproteins in Health and Diseases. London , United Kingdom: Oxford Univ Press; 1999:608-642.
12.
Umemura K., Saniabadi ARAggregation of human blood platelets by remnant-like lipoprotein particles . Nippon Yakurigaku Zasshi. 2000;116:269-274.
Aviram M., Brook JGPlatelet enhancement of macrophage cholesterol accumulation: a novel mechanism for atherogenesis. In: Halpem MJ, ed. Molecular Biology of Atherosclerosis. Proceeding of the 57th European Atherosclerosis. Society Meeting. London, United Kingdom: John Libbey and Co Ltd; 1992;14:67-71.
15.
Woods VL, Wolff LE, Keller DMResting platelets contain a substantial centrally located pool of glycoprotein IIb-IIIa complex which may be accessible to some but not other extracellular proteins. J Biol Chem. 1986;261:15242-15247.
16.
Nofer JR, Tepel M., Kehrel B., et al. Low density lipoproteins inhibited Na+/H+ antiport in human platelets. A novel mechanism enhancing platelet activity in hypercholesterolemia . Circulation. 1997;95:1370-1377.
17.
Leake DSDoes an acidic pH explain why low density lipoprotein is oxidised in atherosclerotic lesions? Atherosclerosis. 1997;129:149-157.
18.
Wang JS, Chow SE, Chen JK, Wong MKEffects of exercise training on oxidized LDL-mediated platelet function in rats. Thromb Haemost. 2000;83:503-508.
19.
Ardlie NGLipoproteins, platelet function and blood coagulation. In: Miller NE, Lewis B, eds. Lipoproteins, Atherosclerosis and Coronary Heart Disease. Amsterdam, the Netherlands: Elsevier/North Holland Biomedical Press; 1994:107-125.
20.
Zieseniss S. , Zahler S., Muller I., Hermetter A., Engelmann B.Modified phosphatidylethanolamine as the active component of oxidized low density lipoprotein promoting platelet prothrombinase activity. J Biol Chem. 2001 ;276:19828-19835.
21.
Volf E., Koller E., Bielek E., Koller F.Colocalization of gold-labelled LDL and fibrinogen on platelets. Enhanced fibrinogen binding induced by LDL. Am J Physiol. 1997;273:C118-C129.
22.
Zhao B., Dierichs R., Lio B., Berkes P.Gold-labelled low density lipoproteins bind to washed human platelets . Platelets. 1994;5:113-120.
23.
Pedreno J., Fernandez R., Cullane C.Platelet integrin alpha IIb/beta3 (GpIIb/IIIa) is not implicated in the binding of LDL to intact resting platelets. Arterioscler Thromb Vasc Biol. 1997;17:156-163.
Endeman G., Stanton L., Madden K., Bryant K., While RT, Protter A.CD36 is a receptor for oxidized low density lipoproteins. J Biol Chem. 1993;268:1181-1186.
26.
Hartwitch J. , Dembinska-Kiec A., Gruca A., et al. Regulation of platelet adhesion by oxidized lipoproteins and oxidized phospholipids. Platelets . 2000;13:141-151.
27.
Pedreno J., Hurt-Camejo E., Wiklund O., Masana L.CD36 is a specific receptor for native and modified low density lipoproteins . Atherosclerosis. 1997;138-184.
28.
Pytela R., Pierschbacher MD, Ginsberg MH, Plow EF, Rouslahti E.Membrane glycoprotein IIb/IIIa member of a family of Arg-Gly-Asp-specific adhesion receptors. Science. 1986;231:1559-1562.
29.
Tetik S., Uras F., Yardimci KTPurification and characterization of human platelet fibrinogen receptor the GpIIb/IIIa complex. J Pharm Sci. 2005;2:155-162.
30.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJProtein measurement with the folin phenol reagentJ Biol Chem.1951;193:265-275.
31.
Laemnli UKCleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685.
32.
Tetik S., Yardιmcι KT, Eksioglu-Demiralp E.A flow cytometric method of ligand binding to isolated receptors using microbeads . In: Linkesch W, ed. Proceedings of the XVIIth Meeting of the International Society of Haemotology . 2003;210-213 (ISBN 88-323-3148-9).
33.
Xia Z., Wong T., Liu Q., Kasirer-Freide A., Brown E., Frojmovic MMOptimally functional fluorescein isothiocyanate-labelled fibrinogen for quantative studies of binding to activated platelets and platelet aggregation . J Haematol. 1996;93:204-221.
34.
Aviram M., Brook JGPlatelet interaction with high and low density lipoproteins. Atherosclerosis. 1983;46:259-268.
35.
Mazurov AV, Preobrazhensky SN, Leytin VL, Repin VS, Smirnov VNStudy of low density lipoprotein interaction with platelets by flow cytofluorometry . FEBS Lett. 1982;137:319-322.
36.
Koller E., Koller F., Binder BRPurification and identification of the lipoprotein-binding proteins from human blood platelet membrane. J Biol Chem. 1989 ;264: 12412-12418.
37.
Lewing LLk , Kinoshita T., Nachman KLIsolation, purification and partial characterization of platelet membrane glycoproteins IIb and IIIa . J Biol Chem . 1980;256:1994-1997.
38.
Jenning, LKPhillips DRPurification of glycoproteins IIb and IIIa from human plasma membranes and characterization of a calcium-dependent glycoprotein IIb/IIIa complex. J Biol Chem. 1982;257:10458-10466.
39.
Phillip DR, Fitzgerald L., Paris L., Steiner B.Platelet receptors: assays and purification. Methods Enzymol . 1992;215:244-263.
40.
Lefkovits J. , Plow EF, Topol EJPlatelet glycoprotein IIb/IIIa receptors in cardiovascular medicine. N Engl J Med. 1995 ;332:1553-1559.
41.
Koller E., Koller F., Doleschel W.Specific binding sites on human blood platelets for plasma lipoproteins . Hoppe-Seyler's Z Physiol Chem. 1982;363:395-405.
42.
Curtiss LK, Plow EFInteraction of plasma lipoproteins with human platelets. Blood. 1984;64:365-374.
43.
Ozsavci D., Yardimci T., Demirel GY, Uras F., Hekim N., Ulutin ONApo A-I binding to platelets detected by flow cytometry. Thromb Res. 2001;103:117-122.
44.
Ault K., Mitchell J.Analysis of platelets by flow cytometry. In: Darzynkiewicz Z, Robinson JP, Crissman HA, eds. Methods in Cell Biology: Flow Cytometry. San Diego, Calif: Academic Press Inc;1994:275-294.
45.
Shattil SJ, Cunningham M., Hoxie JADetection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood. 1987; 70:307-315.
46.
Ginsberg MH, Frelinger AL, Lam S., et al. Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein IIb-IIIa with conformation specific monoclonal antibodies. Blood. 1990;76: 2017-2023.
47.
Sener A., Ozsavci D., Oba R., Yanιkkaya-Demirel G., Uras F., Yardimci KTDo platelet apoptosis, activation, aggregation, lipid peroxidation and platelet-leucocyte aggregate formation occur simultaneously in hyperlipidemia ? Clin Biochem. 2005;38:1081-1087.
48.
Tetik S., Yardιmcι KT, Ozsavcι D., Uras F., Demiralp-Eksioglu E.HDL binding to human platelets and purified GpIIb/IIIa. In: Linkesch W, ed. Proceedings of the XVIIth Meeting of the International Society of Haemotology. 2003;205-209 (ISBN 88-323-3148-9).
49.
Tetik S., Eksioglu-Demiralp E., Yardimci KTEffect of imatinib mesylate on platelet aggregation and fibrinogen binding to isolated platelets. Adv Mol Med. 2005;1: 165-170.
50.
Van Willigen G., Gorter G., Akkerman JWLDLs increase the exposure of fibrinogen binding sites on platelets and secretion of dense granules. J Arterioscler Thromb. 1994; 14:41-46.
51.
Sachais BS, Kuo A., Nassar T., et al. Platelet factor 4 binds to low-density lipoprotein rec eptors and disturbs the endocytic machinery, resulting in retention of low-density lipoprotein on the cell surface. Blood. 2002 ;15: 3613-3622.
52.
Bochkov VN, Matchin YG, Fuk IV, Lyahishev AA, Tkachuk VAPlatelets in patients with homozygous familial hypercholesterolemia are sensitive to Ca (2+)-mobilizing activity of low-density lipoproteins. Atherosclerosis. 1992;96:119-124.
53.
Tetik S.Evaluation of Relationship Between Platelet Fibrinogen Receptor GpIIb/IIIa (α ııb/β ııı) and Platelet LDL Receptor [PhD thesis]. Istanbul, Turkey: Institution of Health Scientific; 2003.
