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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.

 

  • References

  • 1 Siess W. Molecular mechanisms of platelet activation. Physiol Rev 1989; 69: 58-178
    CrossrefPubMedGoogle Scholar
  • 2 Doolittle RF, Watt KW K, Cottrell BA, Strong DD, Riley M. The amino acid sequence of the a-chain of human fibrinogen. Nature 1979; 280: 464-468
    CrossrefPubMedGoogle Scholar
  • 3 Smith JW, Ruggeri ZM, Kunicki TJ, Cheresh DA. Interaction of integrins avb3 and glycoprotein IIb-IIIa with fibrinogen. J Biol Chem 1990; 265: 12267-12271
    PubMedGoogle Scholar
  • 4 Calvete JJ, Arias J, Alvarez MV, Lopez MM, Henschen A, González-rodoríguez J. Further studies on the topography of the N-terminal region of human platelet glycoprotein IIIa. Biochem J 1991; 274: 457-463
    CrossrefPubMedGoogle Scholar
  • 5 Kloczewiak M, Timmons S, Lukas TJ, Hawiger J. Platelet receptor recognition site on human fibrinogen. Biochemistry 1984; 23: 1767-1774
    CrossrefPubMedGoogle Scholar
  • 6 D’Souza SE, Ginsberg MH, Matsueda GR, Plow EF. A discrete sequence in a platelet integrin is involved in ligand recognition. Nature 1991; 350: 66-68
    CrossrefPubMedGoogle Scholar
  • 7 Hawiger J, Kloczewiak M, Bednarek MA, Timmons S. Platelet receptor recognition domains on the α chain of human fibrinogen. Biochemistry 1989; 28: 2909-2914
    CrossrefPubMedGoogle Scholar
  • 8 Kloczewiak M, Timmons S, Bednarek MA, Sakon M, Hawiger J. Platelet receptor recognition domain on the γ chain of human fibrinogen and its synthetic peptide analogues. Biochemistry 1989; 28: 2915-2919
    CrossrefPubMedGoogle Scholar
  • 9 Timmons S, Bednarek MA, Kloczewiak M, Hawiger J. Antiplatelet “Hybrid” peptides analogous to receptor recognition domains on γ and α chains of human fibrinogen. Biochemistry 1989; 28: 2919-2923
    CrossrefPubMedGoogle Scholar
  • 10 Cox D, Aoki T, Seki J, Motoyama Y, Toshida K. The pharmacology of the integrins. Med Res Rev 1994; 14: 195-228
    CrossrefPubMedGoogle Scholar
  • 11 Minno GD, Cerbone AM, Mattioli PL, Turco S, Iovine C, Mancini M. Functionally thrombasthenic state in normal platelets following the administration on ticlopidine. J Clin Invest 1985; 75: 328-338
    CrossrefPubMedGoogle Scholar
  • 12 Peerschke El, Zucker MB, Grant RA, Egan JJ, Johnson MM. Correlation between fibrinogen binding to human platelets and platelet aggregability. Blood 1980; 55: 841-847
    PubMedGoogle Scholar
  • 13 Bennett JS, Vilare G. Exposure of platelet fibrinogen receptor by ADP and epinephrine. J Clin Invest 1979; 64: 1393-401
    CrossrefPubMedGoogle Scholar
  • 14 Marguerie GA, Plow EF, Edgington TS. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem 1979; 254: 5357-5363
    PubMedGoogle Scholar
  • 15 Hawiger J, Parkinson S, Timmons S. Prostacyclin inhibits mobilisation of fibrinogen-binding sites on human ADP-and thrombin-treated platelets. Nature 1980; 283: 195-197
    CrossrefPubMedGoogle Scholar
  • 16 Ruggeri ZM, Houghten RA, Russell SR, Zimmerman TS. Inhibition of platelet function with synthetic peptides designed to be high-affinity antagonists of fibrinogen binding to platelets. Proc Natl Acad Sci USA 1986; 83: 5708-5712
    CrossrefPubMedGoogle Scholar
  • 17 Marguerie GA, Edgington TS, Plow EF. Interaction of fibrinogen with its platelet receptor as part of a multistep reaction in ADP-induced platelet aggregation. J Biol Chem 1980; 255: 154-161
    PubMedGoogle Scholar
  • 18 Samanen J, Ali F, Romoff T, Calvo R, Sorenson E, Vasko J, Storer B, Berry D, Bennett D, Strohsacker M, Powers D, Stadel J, Nichols A. Development of a small RGD peptide fibrinogen receptor antagonist with potent antiaggregatory activity in vitro. J Med Chem 1991; 34: 3114-3125
    CrossrefPubMedGoogle Scholar
  • 19 Gartner TK, Bennett JS. The tetrapeptide analogue of the cell attachment site of fibronectin inhibits platelet aggregation and fibrinogen binding to activated platelets. J Biol Chem 1985; 260: 11891-11894
    PubMedGoogle Scholar
  • 20 Bennett JS, Shattil SJ, Power JW, Gartner TK. Interaction of fibrinogen with its platelet receptor. J Biol Chem 1988; 263: 12948-12953
    PubMedGoogle Scholar
  • 21 Stabler TV, Siegel AL. Chemiluminescence immunoassay of cyclosporine in whole blood. Clin Chem 1990; 36: 906-908
    PubMedGoogle Scholar
  • 22 Boever JD, Kohen F, Vandekerckhove D. Solid-phase chemiluminescence immunoassay for plasma estradiol-17 (3 during gonadotropin therapy compared with two radioimmunoassays. Clin Chem 1983; 29: 2068-2072
    PubMedGoogle Scholar
  • 23 Weeks I, Beheshti I, McCapra F, Campbell AK, Woodhead JS. Acridinium esters as high-specific-activity labels in immunoassay. Clin Chem 1983; 29: 1474-1479
    PubMedGoogle Scholar
  • 24 Richardson AP, Kim JB, Barnard GJ, Collins WP, McCapra F. Chemiluminescence immunoassay of plasma progesterone, with progesterone-acri-dinium ester used as the labeled antigen. Clin Chem 1985; 31: 1664-1668
    PubMedGoogle Scholar
  • 25 Jim RT S. A study of the plasma thrombin time. J Lab Clin Med 1957; 50: 45-60
    PubMedGoogle Scholar
  • 26 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
    CrossrefPubMedGoogle Scholar
  • 27 Scatchard G. The attraction of proteins for small molecules and ions. Ann NY Acad Sci 1949; 51: 660-672
    CrossrefPubMedGoogle Scholar
  • 28 Cheng YC, Prusoff WH. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (150) of an enzymatic reaction. Biochem Pharmacol 1973; 22: 3094-3108
    PubMedGoogle Scholar