title: Role of extracellular ionized calcium in the in vitro assessment of GPIIb/IIIa receptor antagonists.
abstract: 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 microg/mL). Platelet aggregation assays were performed in platelet-rich plasma from citrate-anticoagulated blood (cPRP) and PPACK-anticoagulated blood (pPRP) using ADP (20 microM) and TRAP (10 microM) 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 IC(50) of c7E3 for inhibiting ADP-induced platelet aggregation in cPRP (2.76 +/- 0.11 microg/mL) was 1.6 times lower than that in pPRP (4.46 +/- 0.48 microg/mL; P < 0.05). Similarly, the IC(50) for c7E3 for inhibiting TRAP-induced platelet aggregation in cPRP (4.52 +/- 0.34 microg/mL) was 1.7 times lower than that in pPRP (7.69 +/- 0.43 microg/mL; P < 0.05). MK-383, DMP-728, and SM-20302 also demonstrated 1.96-, 1.15-, and 1.43-fold lower IC(50) 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.
authors.affiliation: Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632, USA.
authors.forename: S S
authors.initials: SS
authors.lastname: Rebello
authors.forename: J
authors.initials: J
authors.lastname: Huang
authors.forename: J D
authors.initials: JD
authors.lastname: Faul
authors.forename: B R
authors.initials: BR
authors.lastname: Lucchesi
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000925:Anticoagulants / Q000494:pharmacology; D000991:Antithrombins / Q000494:pharmacology; D002118:Calcium / Q000097:blood / Q000494:pharmacology*; D002614:Chelating Agents / Q000494:pharmacology; D002951:Citrates / Q000494:pharmacology; D006801:Humans; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors* / Q000187:drug effects; D000077559:Sodium Citrate
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000925:Anticoagulants; D000991:Antithrombins; D002614:Chelating Agents; D002951:Citrates; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D000077559:Sodium Citrate; D000244:Adenosine Diphosphate; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone; D002118:Calcium