Measurement of Glycoprotein llb/llla Blockade by Flow Cytometry with Fluorescein Isothiocyanate-conjugated Crotavirin, a Member of Disintegrins

Summary

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 Ilb/IIIa, remaining available. It was evidenced by the observation that the sum of glycoprotein Ilb/IIIa occupation by 7E3 and that of FITC-crotavirin approached the total amount of glycoprotein Ilb/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 Ilb/IIIa blockade by 7E3. At the saturation binding concentration (5 |xg/ml), FITC-crotavirin did not displace platelet bound 7E3. Gating the light-scattering profile for platelets, the binding of FITC-crotavirin to platelet glycoprotein Ilb/IIIa could be easily determined in diluted whole blood by direct stain method. The available unoccupied glycoprotein Ilb/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 fig/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 Ilb/IIIa by 7E3, a promising anti-thrombotic agent, thus providing a monitoring method for adjusting the therapeutic dose of 7E3 or its related derivatives.

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