Studies on the Binding of ³H-SR121566, an Inhibitor of Gp IIb-IIIa Activation

Summary

SR121566 is a new synthetic agent which inhibits the binding of fibrinogen to activated platelets, and platelet aggregation. ³H-SR121566 bound with nanomolar affinity (KD ranging from 45 to 72 nM) to Gp IIb-IIIa expressing cells only. On activated human platelets, this ligand allowed the detection of a maximal number of 100-140,000 binding sites. The binding of SR121566 to platelets, was displaced by several agents including RGD-containing peptides and synthetic RGD mimetics, but not by ReoPro^®, a humanised monoclonal antibody which inhibits the binding of fibrinogen to the Gp IIb-IIIa complex. Neither the fibrinogen dodecapeptide nor fibrinogen itself were able to compete with SR121566 whether platelets were activated or not.

Flow cytometry studies indicated that SR121566 which did not activate Gp IIb-IIIa by itself, dose-dependently prevented the detection of activation-induced binding sites on TRAP-stimulated platelets in the presence or absence of exogenous fibrinogen, indicating a direct effect on the activation state of the Gp IIb-IIIa complex. Moreover, SR121566 was able to reverse the activation of Gp IIb-IIIa and to displace the binding of fibrinogen when added up to 5 min after TRAP stimulation of platelets. When added at later times (15 to 30 min), SR121566 failed to displace fibrinogen binding, even if SR121566 binding sites were still accessible and the Gp IIb-IIIa complex not activated.

In conclusion, our study is in accordance with the finding that fibrinogen is recognised by the activated Gp IIb-IIIa complex through the dodecapeptide sequence present on its gamma chain, and that this interaction is inhibited by SR121566 by preventing and reversing the activated conformation of Gp IIb-IIIa and not by direct competition with fibrinogen.

Keywords

Human platelets - anti-Gp IIb-IIIa - Gp IIb-IIIa activation

• References

• 1 Farrell DH, Thiagarajan P, Chung DW, Davie EW. Role of fibrinogenandchain sites in platelet aggregation. Proc Natl Acad Sci USA 1992; 89: 10729-32.
• 2 Marguerie GA, Plow EF, Edgington TS. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem 1979; 254: 5357-63.
• 3 Plow EF, Pierschbacher MD, Ruoslahti E, Marguerie GA, Ginsberg MH. The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets. Proc Natl Acad Sci USA 1985; 82: 8057-61.
• 4 Ruggeri ZM, Houghten RA, Russell SR, Zimmerman TS. Inhibition of platelet function with synthetic peptides designed to be high-affinity antagonists of fibrinogen binding to platelets. Proc Natl Acad Sci USA 1986; 83: 5708-12.
• 5 D’Souza SE, Ginsberg MH, Burke TA, Lam SC, Plow EF. Localization of an Arg-Gly-Asp recognition site within an integrin adhesion receptor. Science 1988; 242: 91-3.
• 6 D’Souza SE, Ginsberg MH, Burke TA, Plow EF. The ligand binding site of the platelet integrin receptor GPIIb/IIIa is proximal to the second calcium binding domain of its alpha subunit. J Biol Chem 1990; 265: 3440-6.
• 7 Ginsberg MH, Xiaoping D, O’Toole TE, Loftus JC, Plow EF. Platelets integrins. Thromb Haemost 1993; 70: 87-93.
• 8 Mohri H, Takao O. The role of the RGD peptides and thechain peptide of fibrinogen on fibrinogen binding to activated platelets. Peptides 1993; 14: 353-7.
• 9 Hu DD, White CA, Panzer-Knodle S, Page JD, Nicholson N, Smith JW. A new model of dual interacting ligand binding sites on integrin aIIb b3. J Biol Chem 1999; 274: 4633-9.
• 10 Kouns WC, Hadvary P, Haering P, Steiner B. Conformational modulation of purified glycoprotein (GP) IIb-IIIa allows proteolytic generation of active fragments from either active or inactive GPIIb-IIIa. J Biol Chem 1992; 267: 18844-51.
• 11 Liu QD, Rooney MM, Kasirerfriede A, Brown E, Lord ST, Frojmovic MM. Role of thechain Ala-Gly-Asp-Val and achain Arg-Gly-Asp-Ser sites of fibrinogen in coaggregation of platelets and fibrinogen-coated beads. Biochim Biophys Acta-Protein Structure & Molecular Enzymolology 1998; 1385: 33-42.
• 12 Parise LV, Phillips DR. Reconstitution of the purified platelet fibrinogen receptor. J Biol Chem 1985; 260: 10698-707.
• 13 Topol EJ, Byzova TV, Plow EF. Platelet GPIIb-IIIa blockers. Lancet 1999; 353: 227-31.
• 14 Scarborough RM, Kleiman NS, Phillips DR. Platelet glycoprotein IIb/IIIa antagonists. What are the relevant issues concerning their pharmacology and clinical use? Circulation 1999; 100: 437-44.
• 15 Badorc A, Borde MF, de Cointet P, Savi P, Bernat A, Lalé A, Petitou M, Maffrand JP, Herbert JM. New orally active fibrinogen receptor (GpIIbIIIa) antagonists: Identification of ethyl 3-[N-[4-[4-[amino[(ethoxycarbonyl)imino]methyl]-phenyl]-1,3-thiazol-2-yl]-N-[1-[(ethoxycarbonyl) methyl]piperid-4-yl]amino]-propionate (SR121787) as a potent and long-acting antithrombotic agent. J Med Chem 1997; 40: 3393-401.
• 16 Savi P, Badorc A, Lalé A, Bordes MF, Bornia J, Labouret C, Bernat A, de Cointet P, Hoffmann P, Maffrand JP, Herbert JM. SR121787, a new orally active fibrinogen receptor antagonist. Thromb Haemost 1998; 80: 469-76.
• 17 Bernat A, Hoffmann P, Savi P, Lalé A, Herbert JM. Interspecies comparison of the antiplatelet, antithrombotic and haemorrhagic effects of SR121566A, a novel nonpeptide GpIIb/IIIa antagonist. J Cardiovasc Pharmacol 1999; 33: 897-904.
• 18 Savi P, Laplace MC, Maffrand JP, Herbert JM. Binding of [3H]-2-methylthio ADP to rat platelets: effect of clopidogrel and ticlopidine. J. Pharm Exp Ther 1994; 269: 772-7.
• 19 Mohri H, Takao O. “Bridged” Peptide of fibrinogen binds to platelets activated by RGD peptide as well as by ADP. Peptides 1994; 15: 683-7.
• 20 Mc Pherson A mathematical approach to receptor characterization. In: Williams, Glennon and Timmersmans, ed. Receptor pharmacology and function. New York: Marcel Dekker Inc; 1988: 47-84.
• 21 Scatchard G. The attractions of proteins for small molecules and ions. Ann N Y Acad Sci 1949; 51: 660-72.
• 22 Cheng YC, Prusoff WH. Relationship between the inhibition constant (Ki) and the concentration of inhibitors which causes 50% inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 1973; 22: 3099-108.
• 23 Pierschbacher MD, Ruoslahti E. Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion. J Biol Chem 1987; 262: 17294-8.
• 24 Shattil SJ, Brass LF. Induction of the fibrinogen receptor on human platelets by intracellular mediators. J Biol Chem 1987; 262: 992-1000.
• 25 Kouns WC, Kirchhofer D, Hadváry P, Edenhofer A, Weller T, Pfenninger G, Baumgartner HR, Jennings LK, Steiner B. Reversible conformational changes induced in glycoprotein IIb-IIIa by a potent and selective peptidomimetic inhibitor. Blood 1992; 80: 2539-47.
• 26 Nurden P, Humbert M, Piotrowicz RS, Bihour C, Poujol C, Nurden AT, Kunicki TJ. Distribution of ligand-occupied alpha IIb beta 3 in resting and activated human platelets determined by expression of a novel class of ligand-induced binding site recognized by monoclonal antibody AP6. Blood 1996; 88: 887-99.
• 27 Woods Jr VL, Wolff LE, Keller DM. Resting 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: 15242-51.
• 28 Nurden P, Poujol C, Durrieu-Jais C, Winckler J, Combrié R, Macchi L, Bihour C, Wagner C, Jordan R, Nurden AT. Labeling of the internal pool of GP IIb-IIIa in platelets by c7E3 Fab fragments (abciximab): flow and endocytic mechanisms contribute to the transport. Blood 1999; 93: 1622-33.
• 29 Wagner CL, Mascelli MA, Neblock DS, Weisman HF, Coller BS, Jordan RE. Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets. Blood 1996; 88: 907-14.
• 30 Marguerie GA, Edgington TS, Plow EF. Interaction of fibrinogen with its platelet receptor as part of a multistep reaction in ADP-induced platelet aggregation. J Biol Chem 1980; 255: 154-61.
• 31 Newman PJ, Allen RW, Kahn RA, Kunicki TJ. Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3. Blood 1985; 65: 227-32.
• 32 Tomiyama Y, Tsubakio T, Piotrowicz RS, Kurata Y, Loftus JC, Kunicki TJ. The Arg-Gly-Asp (RGD) recognition site of platelet glycoprotein IIb-IIIa on nonactivated platelets is accessible to high-affinity macromolecules. Blood 1992; 79: 2303-12.
• 33 Wencel-Drake JD, Plow EF, Kunicki TJ, Woods VL, Keller DM, Ginsberg MH. Localization of internal pools of membrane glycoproteins involved in platelet adhesive responses. Am J Pathol 1986; 124: 324-34.
• 34 Niiya K, Hodson E, Bader R, Byers-Ward V, Koziol JA, Plow EF, Ruggeri ZM. Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation. Blood 1987; 70: 475-83.
• 35 Lam SC-T, Plow EF, Smith MA, Andrieux A, Ryckwaert JJ, Marguerie G, Ginsberg MH. Evidence that arginyl-glycyl-aspartate peptides and fibrinogenchain peptides share a common binding site on platelets. J Biol Chem 1987; 262: 947-50.
• 36 Andrieux A, Hudry-Clergeon G, Ryckewaert JJ, Chapel A, Ginsberg MH, Plow EF, Marguerie G. Amino acid sequences in fibrinogen mediating its interaction with its platelet receptor, Gp IIb-IIIa. J Biol Chem 1989; 264: 9258-65.
• 37 Karczewski J, Connolly TM. The interaction of disagregin with the platelet fibrinogen receptor, glycoprotein IIb-IIIa. Biochem Biophys Res Commun 1997; 241: 744-8.
• 38 Wong A, Hwang SM, Johanson K, Samanen J, Bennett D, Landwatter SW, Chen W, Heys JR, Ali FE, Ku TW, Bondinell W, Nichols AJ, Powers DA, Stadel JM. Binding of [³H]-SK&F 107260 and [³H]-SB214857 to purified integrin IIb III: evidence for a common binding site for cyclic arginylglycinyl-aspartic acid peptides and nonpeptides. J Pharmacol Exp Ther 1998; 285: 228-35.
• 39 Bennett JS, Shattil SJ, Power JW, Gartner TK. Interaction of fibrinogen with its platelet receptor. J Biol Chem 1988; 263: 12948-53.
• 40 Du XP, Plow EF, Frelinger 3d AL, O’Toole TE, Loftus JC, Ginsberg MH. Ligands “activate” integrin αIIbβ3 (platelet GP IIb IIIa). Cell 1991; 65: 409-16.
• 41 Mayo KH, Fan F, Beavers MP, Eckardt A, Keane P, Hoekstra WJ, Andrade-Gordon P. RGD induces conformational transition in purified platelet integrin GPIIb-IIIa-SDS system yielding multiple binding states for fibrinogen γ-chain C-terminal peptide. FEBS Letters 1996; 378: 79-82.
• 42 Peter K, Schwarz M, Ylänne J, Kohler B, Moser M, Nordt T, Salbach P, Kübler W, Bode C. Induction of fibrinogen binding and platelet aggregation as a potential intrinsic property of various glycoprotein IIb/IIIa (αIIbβ3) inhibitors. Blood 1998; 92: 3240-9.
• 43 Coller BS. GPIIb/IIIa antagonists: pathophysiologic and therapeutic insights from studies of c7E3 Fab. Thromb Haemost 1997; 78: 730-5.
• 44 Harrison P, Cramer EM. Plateletgranules. Blood Reviews 1993; 7: 52-62.
• 45 Peerschke EIB. Irreversible platelet fibrinogen interactions occur independently of fibrinogen alpha chain degradation and are not mediated by intact platelet membrane glycoprotein IIb-IIIa complexes. J Lab Clin Med 1988; 111: 84-92.
• 46 Wencel-Drake JD, Boudignon-Proudhon C, Dieter MG, Criss AB, Parise LV. Internalization of bound fibrinogen modulates platelet aggregation. Blood 1996; 87: 602-12.
• 47 Wolff R, Plow EF, Ginsberg MH. Interaction of thrombospondin with resting and stimulated human platelets. J Biol Chem 1986; 261: 6840-6.
• 48 Bacon-Baguley T, Ogilvie ML, Gartner TK, Walz DA. Thrombospondin binding to specific sequences within the Aα- and Bβ-chains of fibrinogen. J Biol Chem 1990; 265: 2317-23.
• 49 Huber W, Hurst J, Schlatter D, Barner R, Hübscher J, Kouns WC, Steiner B. Determination of kinetic constants for the interaction between the platelet glycoprotein IIb-IIIa and fibrinogen by means of surface plasmon resonance. Eur J Biochem 1995; 227: 647-56.
• 50 Müller B, Zerwes H-G, Tangemann K, Peter J, Engel J. Two-step binding mechanism of fibrinogen to αIIbβα3 integrin reconstituted into planar lipid balayers. J Biol Chem 1993; 268: 6800-8.