A new method for the assay of exposed platelet fibrinogen receptor using a chemiluminescent label.
Assay of the platelet fibrinogen-binding receptor glycoprotein (GP) IIb/IIIa is widely performed using 125I-labeled fibrinogen (125I-fibrinogen). We successfully devised a receptor binding assay system with high selectivity and sensitivity using a stable chemiluminescent acridinium derivative-I-labeled fibrinogen (acridinium-fibrinogen). Human fibrinogen is saline was labeled with equimolar acridinium dissolved in dimethylformamide, and allowed to react with gel-filtered human platelets in the presence of ADP. Acridinium-fibrinogen binding to GPIIb/IIIa 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 binding to human platelets was rapid and reversible, specific and saturable, and dependent on ADP concentrations. Scatchard plot analysis revealed one class of binding sites with Kd of 326 nM and Bmax of 7.8 pmol/10(8) platelets. These values were comparable to the data obtained by using 125I-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 microM and 31.3 microM, respectively. Thus, this binding assay system may be useful in measuring the binding between platelet GPIIb/IIIa and fibrinogen without using a radioisotope.
Katoh
M
M
Pharmaceutical Development, Research Laboratories, Tanabe Seiyaku Co., Ltd, Saitama, Japan.
Chishima
S
S
Kiuchi
N
N
Ikeo
T
T
Sasaki
Y
Y
D000166:Acridines; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D006801:Humans; D007118:Immunoassay; D008163:Luminescent Measurements; D008297:Male; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013223:Statistics as Topic; D013388:Succinimides
D000166:Acridines; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D013388:Succinimides; C032193:glycoprotein receptor GPIb-IX; D000244:Adenosine Diphosphate; C053333:4-(2-succinimidyloxycarbonylethyl)phenyl-10-methylacridinium-9-carboxylate