Role of Extracellular Ionized Calcium in the In Vitro Assessment of GPIIb/IIIa Receptor Antagonists
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Several preclinical studies have found a poor correlation between the ex vivo platelet inhibitory potency and the in vivo antithrombotic efficacy of GPIIb/IIIa receptor antagonists. The present study was designed to examine the differential in vitro potencies of c7E3, MK-383, DMP-728, and SM-20302 in inhibiting ex vivo platelet aggregation under normocalcemic and hypocalcemic conditions. Human blood was collected in either trisodium citrate (0.37%) or PPACK (20 µg/mL). Platelet aggregation assays were performed in platelet-rich plasma from citrate-anticoagulated blood (cPRP) and PPACK-anticoagulated blood (pPRP) using ADP (20 µM) and TRAP (10 µM) as agonists in the presence of c7E3, MK-383, DMP-728, or SM-20302. The concentration of ionized calcium in cPRP was 16–19 times lower than that in pPRP. The IC50 of c7E3 for inhibiting ADP-induced platelet aggregation in cPRP (2.76 ± 0.11 µg/mL) was 1.6 times lower than that in pPRP (4.46 ± 0.48 µg/mL; P < 0.05). Similarly, the IC50 for c7E3 for inhibiting TRAP-induced platelet aggregation in cPRP (4.52 ± 0.34 µg/mL) was 1.7 times lower than that in pPRP (7.69 ± 0.43 µg/mL; P < 0.05). MK-383, DMP-728, and SM-20302 also demonstrated 1.96-, 1.15-, and 1.43-fold lower IC50 values, respectively, in cPRP as compared with pPRP. Chelation of ionized calcium in pPRP led to a progressive increase in platelet inhibition by all the antagonists. These results suggest that the observed in vitro inhibitory potency of a GPIIb/IIIa receptor antagonist is markedly enhanced when trisodium citrate is used as an anticoagulant to collect blood for ex vivo assay. These findings indicate that dosing regimens for GPIIb/IIIa receptor antagonists based on the platelet inhibition profile in citrate may provide misleading information with respect to their true in vivo antithrombotic efficacy.
- White JG. Effects of ethylenediamine tetracetic acid (EDTA) on platelet structure. Scand J Haematol 1968;5: 241–254.
- Pidard D, Didry D, Kunicki TJ, Nurden AT. Temperature-dependent effects of EDTA on the membrane glycoprotein IIb-IIIa complex and platelet aggregability. Blood 1986;67: 604–611.
- Shattil SJ, Brass LF, Bennett JS, Pandhi P. Biochemical and functional consequences of dissociation of the platelet membrane glycoprotein IIb-IIIa complex. Blood 1985;66:92–98.
- Fujimura K, Philips DR. Calcium cation regulation of glycoprotein IIb-IIIa complex formation in platelet plasma membranes. J Biol Chem 1983;258:10247–10252.
- Steiner B, Cousot D, Trzeciak A, Gillessen D, Hadvary P. Ca+2;-dependent binding of a synthetic Arg-Gly-Asp (RGD) peptide to a single site on the purified platelet glycoprotein IIb-IIIa complex. J Biol Chem 1989;264:13102–13108.
- 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;255:154–161.
- Dandona P, Thusu K, Khurana U, Love J, Aljada A, Mousa S. Calcium, calmodulin and protein kinase C dependence of platlet shape change. Thromb Res 1996;81:163–175.
- Lefkovitz J, Plow EF, Topol EJ. Platelet glycopotein IIb/IIIa receptors in cardiovascular medicine. N Engl J Med 1995;332:1553–1559.
- Lanza F, Stierle A, Gachet C, Cazenave JP. Differential effects of extra and intracellular calcium chelation on human platelet function and glycoprotein IIb/IIIa complex stability. Nouv Rev Fr Hematol 1992;34:123–131.
- Heidenreich R, Eisman R, Delgrosso K, Surrey S, Bennett JS, Schwartz E, Poncz M. Organization of the gene for platelet glycoprotein IIb. Biochemistry 1990;29:1232–1244.
- Kieffer N, Phillips DR. Platelet membrane glycoproteins: Functions in cellular interactions. Annu Rev Cell Biol 1990;6:329–357.
- Lee JO, Rieu P, Arnaout MA, Liddington R. Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell 1995;80:631–638.
- Loftus JC, O'Toole TE, Plow EF, Glass A, Frelinger AL, Ginsberg MH. A beta 3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation. Science 1990;249:915–918.
- Tozer EC, Liddington RC, Sutcliffe MJ, Smeeton AH, Loftus JC. Ligand binding to integrin alphaII beta3 is dependent on a MIDAS-like domain in the beta3 subunit. J Biol Chem 1996;271:21978–21984.
- D'souza SE, Haas TA, Piotrowicz RS, et al. Ligand and cation binding are dual functions of a discrete segment of the integrin b3 subunit: Cation displacement is involved in ligand binding. Cell 1994;79:659–667.
- Hu DD, Barbas CF, Smith JW. An allosteric Ca21 binding site on the beta3-integrins that regulates the dissociation rate for RGD ligands. J Biol Chem 1996;271:21745–21751.
- Rebello SS, Driscoll EM, Lucchesi BR. TP-9201, a glycoprotein IIb/IIIa platelet receptor antagonist, prevents rethrombosis after successful arterial thrombolysis in the dog. Stroke 1997;28:1–8.
- Collen D, Lu HR, Stassen JM, Vreys I, Yasuda T, Bunting S, Gold HK. Antithrombotic effects and bleeding time prolongation with synthetic platelet GPIIb/IIIa inhibitors in animal models of platelet-mediated thrombosis. Thromb Haemost 1994;71:95–102.
- Philips DR, Charo IF, Parise LV, Fitzgerald LA. The platelet membrane glycoprotein IIb/IIIa complex. Blood 1988;71: 831–843.
- Marguerie GA, Plow EF, Edgington TS. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem 1979;254:5357–5363.
- Mustard JF, Packham MA, Kinlough-Rathbone RL, Perry DW, Regoeczi E. Fibrinogen and ADP-induced platelet aggregation. Blood 1978;52:453–466.
- Peerschke EI, Zucker MB, Grant RA, Egan JJ, Johnson MM. Correlation between fibrinogen binding to human platelets and platelet aggregability. Blood 1980;55:841–847.
- Lam SC. Isolation and characterization of a chymotryptic fragment of platelet glycoprotein IIb/IIIa retaining Arg-Gly-Asp binding activity. J Biol Chem 1992;267:5649–5655.
- Zucker MB, Grant RA. Nonreversible loss of platelet aggregability induced by calcium deprivation. Blood 1978;52: 505–514.
- Winters KJ, Eisenberg PR, Jaffe AS, Santoro SA. Dependence of plasmin-mediated degragdation of platelet adhesive receptors on temperature and Ca2+. Blood 1990;76: 1546–1557.
- Rote WE, Nedelman MA, Mu DX, Manley PJ, Weisman H, Cunningham MR, Lucchesi BR. Chimeric 7E3 prevents carotid artery thrombosis in cynomolgus monkeys. Stroke 1994;25:1223–1233.
- Lynch JJ, Cook JJ, Sitko GR, et al. Nonpeptide glycoprotein IIb/IIIa inhibitors. 5. Antithrombotic effects of MK-0383. J Pharmacol Exp Ther 1995;272:20–32.
- Cook JJ, Sitko GR, Holahan MA, et al. Nonpeptide glycoprotein IIb/IIIa inhibitors. 15. Antithrombotic efficacy of L-738,167, a long-acting GPIIb/IIIa antagonist, correlates with inhibition of adenosine diphosphate-induced platelet aggregation but not with bleeding time prolongation. J Pharmacol Exp Ther 1997;281:677–689.
- Rote WE, Werns SW, Davis JH, Feigen LP, Kilgore KS, Lucchesi BR. Platelet GPIIb/IIIa receptor inhibition by SC-49992 prevents thrombosis and rethrombosis in the canine carotid artery. Cardiovascular Res 1993;27:500–507.
- Rebello SS, Huang J, Saito K, Lucchesi BR. The in vivo efficacy of SM-20302, a GPIIb/IIIa receptor antagonist, correlates with ex vivo platelet inhibition in heparinized blood but not in citrated blood. Arterioscl Thromb Vasc Biol 1998, accepted.
- Ohman EM, Harrington RA, Lincoff AM, Kitt MM, Kleiman NS, Tcheng JE. Early clinical experience with integrilin, an inhibitor of the platelet glycoprotein IIb/IIIa integrin receptor. Eur Heart J 1995;16(Suppl. L):50–55.
- Resar JR, Brinker JA, Gerstenblith G, Blumenthal RS, Dudek A, Coombs VJ, Goldschmidt-Clermont PJ. Disparity of Integrilin inhibition of platelet aggregation and GP IIb/IIIa fibrinogen binding in angioplasty patients. Circulation 1996;94(Suppl. I):1–98.
- Phillips DR, Teng W, Arfsten A, et al. Effect of Ca2+ on GPIIb-IIIa interactions with Integrilin. Circulation 1997; 96:1488–1494.
- Role of Extracellular Ionized Calcium in the In Vitro Assessment of GPIIb/IIIa Receptor Antagonists
Journal of Thrombosis and Thrombolysis
Volume 9, Issue 1 , pp 23-28
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- Kluwer Academic Publishers
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- trisodium citrate
- PPACK (Phe-Pro-Arg chloromethyl ketone)
- Platelet aggregation
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