Measurement of glycoprotein IIb/IIIa blockade by flow cytometry with fluorescein isothiocyanate-conjugated crotavirin, a member of disintegrins.
The blockade of platelet membrane glycoprotein IIb/IIIa by a monoclonal antibody, 7E3, was measured by flow cytometry using a fluorescein isothiocyanate-conjugated disintegrin, FITC-crotavirin, as the probe. After treatment of platelets with 7E3 or 7E3 F(ab')2, there is a good correlation between the inhibition of platelet aggregation and the blockade of FITC-crotavirin's binding to platelets. The content of glycoprotein IIb/IIIa for the subsequent binding of FITC-crotavirin to the 7E3-pretreated platelets highly correlated to the extent of glycoprotein IIb/IIIa, remaining available. It was evidenced by the observation that the sum of glycoprotein IIb/IIIa occupation by 7E3 and that of FITC-crotavirin approached the total amount of glycoprotein IIb/IIIa expressed on the platelet membrane. This indicates that the percentage inhibition of FITC-crotavirin's binding at the saturation dose reflects the extent of glycoprotein IIb/IIIa blockade by 7E3. At the saturation binding concentration (5 micrograms/ml), FITC-crotavirin did not displace platelet bound 7E3. Gating the light-scattering profile for platelets, the binding of FITC-crotavirin to platelet glycoprotein IIb/IIIa could be easily determined in diluted whole blood by direct stain method. The available unoccupied glycoprotein IIb/IIIa of platelets in the 7E3 or 7E3 F(ab')2-pretreated whole blood were measured by flow cytometry at the saturation binding dose of FITC-crotavirin (4 micrograms/ml) and the data showed that the higher deconcentration of antibody added into whole blood, the lower debinding of FITC-crotavirin to platelets. This technique may provide an alternative rapid method for measuring the blockade of glycoprotein IIb/IIIa by 7E3, a promising anti-thrombotic agent, thus providing a monitoring method for adjusting the therapeutic dose of 7E3 or its related derivatives.
Liu
C Z
CZ
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei.
Hur
B T
BT
Huang
T F
TF
D000911:Antibodies, Monoclonal; D003094:Collagen / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D010975:Platelet Aggregation Inhibitors; D010976:Platelet Count; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors*; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C098620:crotavirin; D003094:Collagen; D016650:Fluorescein-5-isothiocyanate