A NEW METHOD FOR THE ASSAY OF EXPOSED PLATELET FIBRINOGEN RECEPTOR USING A CHEMILUMINESCENT LABEL

Assay of the platelet fibrinogen-binding receptor glycoprotein (GP) II b/IIIa is widely performed using I-125-labeled fibrinogen (I-125-fibri nogen). We successfully devised a receptor binding assay system with h igh selectivity and sensitivity using a stable chemiluminescent acridi nium derivative I-labeled fibrinogen (acridinium-fibrinogen). Human fi brinogen in saline was labeled with equimolar acridinium dissolved in dimethylformamide, and allowed to react with gel-filtered human platel ets in the presence of ADP. Acridinium-fibrinogen binding to GPIIb/III a was assayed by measuring chemiluminescence emitted on addition of 0. 1 N NaOH containing 0.06% H2O2 in a luminometer. Non-specific binding was measured in the presence of 10 mM EDTA. Acridinium-fibrinogen bind ing to human platelets was rapid and reversible, specific and saturabl e, and dependent on ADP concentrations. Scatchard plot analysis reveal ed one class of binding sites with a Kd of 326 nM and Bmax of 7.8 pmol /10(8) platelets. These values were comparable to the data obtained by using I-125-fibrinogen, Unlabeled fibrinogen, RGDS, and HHLGGAKQAGDV (fibrinogen gamma-chain 400-411) displaced acridinium-fibrinogen from its binding site with Ki values of 322 nM, 9.2 mu M and 31.3 mu M, res pectively. Thus, this binding assay system may be useful in measuring the binding between platelet GPIIb/IIIa and fibrinogen without using a radioisotope.