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Thromb Haemost 1995; 74(06): 1546-1550
DOI: 10.1055/s-0038-1649980
Original Articles
Platelets
Schattauer GmbH Stuttgart

A New Method for the Assay of Exposed Platelet Fibrinogen Receptor Using a Chemiluminescent Label

Makoto Katoh
The Pharmaceutical Development, Tanabe Seiyaku Co., Ltd., Toda, Saitama, Japan
,
Susumu Chishima
The Pharmaceutical Development, Tanabe Seiyaku Co., Ltd., Toda, Saitama, Japan
,
Nobukazu Kiuchi
1   The Lead Optimization Research Laboratories, Tanabe Seiyaku Co., Ltd., Toda, Saitama, Japan
,
Tomihiro Ikeo
1   The Lead Optimization Research Laboratories, Tanabe Seiyaku Co., Ltd., Toda, Saitama, Japan
,
yasuhiko Sasaki
1   The Lead Optimization Research Laboratories, Tanabe Seiyaku Co., Ltd., Toda, Saitama, Japan
› Author Affiliations
Further Information

Publication History

Received 05 May 1995

Accepted after resubmission 21 August 1995

Publication Date:
10 July 2018 (online)

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Summary

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 in 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% H202 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 a Kd of 326 nM and Bmax of 7.8 pmol/108 platelets. These values were comparable to the data obtained by using 125I-fibrinogen. Unlabeled fibrinogen, RGDS, and HHLGGAKQAGDV (fibrinogen γ-chain 400-411) displaced acridinium-fibrinogen from its binding site with Ki values of 322 nM, 9.2 μM and 31.3μM, respectively. Thus, this binding assay system may be useful in measuring the binding between platelet GPIIb/IIIa and fibrinogen without using a radioisotop.

 

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