Effect of Ca2+ on GP IIb-IIIa interactions with integrilin: enhanced GP IIb-IIIa binding and inhibition of platelet aggregation by reductions in the concentration of ionized calcium in plasma anticoagulated with citrate.
BACKGROUND
Integrilin (eptifibatide), a potent inhibitor of the fibrinogen binding function of GP IIb-llla, has been shown to reduce the thrombotic complications of angioplasty and of acute coronary syndromes. The present study was designed to determine whether the reduced Ca2+ concentrations in plasma anticoagulated with citrate affect Integrilin binding to GP IIb-IIIa and the ex vivo pharmacodynamic measurements for this drug.


RESULTS
Lower concentrations of Integrilin were found to inhibit platelet aggregation in plasma anticoagulated with citrate (for ADP, mean+/-SD IC(50)=140+/-40 nmol/L, n=6; Ca2+ =40 to 50 micromol/L) than with PPACK (IC(50)=570+/-70 nmol/L, P<.0001, n=6; Ca2+ approximately 1 mmol/L). Chelation of Ca2+ with EDTA or citrate caused a similar degree of enhancement in the inhibitory activity of Integrilin. Measurements of D3 LIBS epitope expression showed that the enhanced inhibitory activity was caused by enhanced GP IIb-IIIa occupancy by Integrilin. Citrate anticoagulation decreased the amounts of Integrilin required to inhibit the binding of PAC1, a monoclonal antibody that mimics the GP IIb-IIIa binding activity of fibrinogen. Reduced Ca2+ also increased Integrilin inhibition of the binding of biotinylated fibrinogen to purified, immobilized GP IIb-IIIa.


CONCLUSIONS
These data suggest that citrate anticoagulation removes Ca2+ from GP IIb-IIIa and enhances the apparent inhibitory activity of Integrilin. This finding indicates that the inhibitory activity of Integrilin is overestimated in blood samples collected with citrate, suggesting that it may be possible to achieve greater antithrombotic efficacy beyond that observed in clinical trials to date with Integrilin.
Phillips
D R
DR
COR Therapeutics, South San Francisco, Calif 94080, USA. david_phillips@corr.com
Teng
W
W
Arfsten
A
A
Nannizzi-Alaimo
L
L
White
M M
MM
Longhurst
C
C
Shattil
S J
SJ
Randolph
A
A
Jakubowski
J A
JA
Jennings
L K
LK
Scarborough
R M
RM
D000911:Antibodies, Monoclonal; D000925:Anticoagulants / Q000494:pharmacology*; D002118:Calcium / Q000097:blood / Q000502:physiology*; D019343:Citric Acid / Q000494:pharmacology*; D000939:Epitopes; D000077542:Eptifibatide; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D009994:Osmolar Concentration; D010455:Peptides / Q000494:pharmacology*; D010949:Plasma / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D000939:Epitopes; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; D019343:Citric Acid; D005340:Fibrinogen; D000077542:Eptifibatide; D002118:Calcium