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Thromb Haemost 1996; 76(04): 585-591
DOI: 10.1055/s-0038-1650626
Original Article
Schattauer GmbH Stuttgart

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

Chao-Zong Liu
The Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
,
Buo-Tsang Hur
The Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
,
Tur-Fu Huang
The Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
› Institutsangaben
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Publikationsverlauf

Received: 20. Februar 1996

Accepted after resubmission19. Juni 1996

Publikationsdatum:
10. Juli 2018 (online)

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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.

 

  • References

  • 1 Stein B, Fuster V, Halperin JL, Chesebro JH. Antithrombotic therapy in cardiac disease: an emerging approach based on pathogenesis and risk. Circulation 1989; 80: 1501-1513
    CrossrefPubMedSuche in Google Scholar
  • 2 Coller BS. Platelets and thrombolytic therapy. N Engl J Med 1990; 322: 33-42
    CrossrefPubMedSuche in Google Scholar
  • 3 Schror K. Antiplatelet drugs: a comparative review. Drugs 1995; 50: 7-28
    CrossrefPubMedSuche in Google Scholar
  • 4 Coller BS. Antiplatelet agents in the prevention and therapy of thrombosis. Ann Rev Med 1992; 43: 171-180
    CrossrefPubMedSuche in Google Scholar
  • 5 Weller T, Alig L, Muller MH, Kouns WC, Steiner B. Fibrinogen receptor antagonists: a novel class of promising antithrombotics. Drugs Future 1994; 19: 461-476
    CrossrefPubMedSuche in Google Scholar
  • 6 Coller BS, Anderson K, Weisman HF. New antiplatelet agents: platelet GPIIb/IIIa antagonists. Thromb Haemost 1995; 74: 302-308
    Thieme ConnectPubMedSuche in Google Scholar
  • 7 Hynes RO. Integrins: a family of cell surface receptors. Cell 1987; 48: 549-554
    CrossrefPubMedSuche in Google Scholar
  • 8 Nurden AT, Caen JP. An abnormal platelet glycoprotein pattern in three cases of Glanzmann’s thrombasthenia. Br J Haematol 1974; 28: 253-260
    CrossrefPubMedSuche in Google Scholar
  • 9 Nurden AT, Caen JP. Specific roles for platelet surface glycoproteins in platelet function. Nature 1975; 255: 720-722
    CrossrefPubMedSuche in Google Scholar
  • 10 Coller BS, Scudder LE, Beer J, Gold HK, Folts JD, Cavagnaro J, Jordan R, Wagner C, Iuliucci J, Knight D, Ghrayeb J, Smith C, Weisman HF, Berger H. Monoclonal antibodies to platelet glycoprotein Ilb/IIIa as antithrombotic agents. Ann NY Acad Sci 1991; 614: 193-213
    CrossrefPubMedSuche in Google Scholar
  • 11 Coller BS. A new murine monoclonal antibody reports an activation-dependent change in the conformation and/or microenvironment of the platelet GPIIb/IIIa complex. J Clin Invest 1985; 76: 101-108
    CrossrefPubMedSuche in Google Scholar
  • 12 Yasuda T, Gold HK, Fallon JT, Leinbach RC, Guerrero JL, Scudder LE, Kanke MK, Shealy D, Ross MJ, Collen D, Coller BS. Monoclonal antibody against the platelet glycoprotein (GP) Ilb/IIIa receptor prevents coronary artery reocclusion after reperfusion with recombinant tissue-type plasminogen activator in dogs. J Clin Invest 1988; 81: 1284-1291
    CrossrefPubMedSuche in Google Scholar
  • 13 Gold HK, Coller BS, Yasuda T, Saito T, Fallon JT, Guerrero JL, Leinbach RC, Ziskind AA, Collen D. Rapid and sustained coronary artery recanalization with combined bolus injection of recombinant tissue-type plasminogen activator and monoclonal antiplatelet GPIIb/IIIa antibody in a canine preparation. Circulation 1988; 77: 670-677
    CrossrefPubMedSuche in Google Scholar
  • 14 Gold HK, Gimple LW, Yasuda T, Leinbach RC, Werner W, Holt R, Jordan R, Berger H, Collen D, Coller BS. Pharmacodynamic study of F(ab)2 fragments of murine monoclonal antibody 7E3 directed against human platelet glycoprotein Ilb/IIIa in patients with unstable angina pectoris. J Clin Invest 1990; 86: 651-659
    CrossrefPubMedSuche in Google Scholar
  • 15 Kohmura C, Gold HK, Yasuda T, Holt R, Nedelman MA, Guerrero JL, Weisman HF, Collen D. A chimeric murine/human antibody Fab fragment directed against the platelet GPIIb/IIIa receptor enhances and sustains arterial thrombolysis with recombinant tissue-type plasminogen activator in baboons. Arterioscler Thromb 1993; 13: 1837-1842
    CrossrefPubMedSuche in Google Scholar
  • 16 Kleiman NS, Ohman EM, Califf RM, George BS, Kereiakes D, Aguirre FV, Weisman H, Schaible T, Topol EJ. Profound inhibition of platelet aggregation with monoclonal antibody 7E3 Fab after thrombolytic therapy: results of the thrombolysis and angioplasty in myocardial infarction (TAMI) 8 pilot study. J Am Coll Cardiol 1993; 22: 381-389
    CrossrefPubMedSuche in Google Scholar
  • 17 Jordan RE, Knight DM, Wagner C, McAleer MF, McDonough M, Mattis JA, Coller BS, Weisman HF, Ghrayeb J. A dramatic reduction of the immunogenicity of the anti-GPIIb/IIIa monoclonal antibody, 7E3 Fab, by humanization of the murine constant domains (abstr.). Circulation 1992; 86 (Suppl. 01) Suppl1 1-141
    CrossrefPubMedSuche in Google Scholar
  • 18 Bates ER, McGillem MJ, Michelson JK, Pitt B, Mancini GBJ. A monoclonal antibody against the platelet glycoprotein Ilb/IIIa receptor complex prevents platelet aggregation and thrombosis in a canine model of coronary angioplasty. Circulation 1991; 84: 2463-2469
    CrossrefPubMedSuche in Google Scholar
  • 19 Tcheng JE, Ellis SG, George BS, Kereiakes DJ, Kleiman NS, Talley JD, Wang AL, Weisman HF, Califf RM, Topol EJ. Pharmacodynamics of chimeric glycoprotein Ilb/IIIa integrin antiplatelet antibody Fab 7E3 in high-risk coronary angioplasty. Circulation 1994; 90: 1757-1764
    CrossrefPubMedSuche in Google Scholar
  • 20 The EPIC investigators. Use of a monoclonal antibody directed against the platelet glycoprotein Ilb/IIIa receptor in high-risk coronary angioplasty N Eng J Med 1994 330. 956-961
    PubMedSuche in Google Scholar
  • 21 Topol EJ, Califf RM, Weisman HF, Ellis SG, Tcheng JE, Worley S, Ivanhoe R, George BS, Fintel D, Weston M, Sigmon K, Anderson KM, Lee KL, Willerson JT. on behalf of the EPIC investigators. Randomised trial of coronary intervention with antibody against platelet Ilb/IIIa integrin for reduction of clinical restenosis: results at six months. Lancet 1994; 343: 881-886
    CrossrefPubMedSuche in Google Scholar
  • 22 Gould RJ, Polokoff MA, Friedman PA, Huang TF, Holt JC, Cook JJ, Niewiarowski S. Disintegrins: a family of integrin inhibitory proteins from viper venoms. Proc Soc Exp Biol Med 1990; 195: 168-171
    CrossrefPubMedSuche in Google Scholar
  • 23 Huang TF, Holt JC, Lukasiewicz H, Niewiarowski S. Trigramin: a low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein Ilb-IIIa complex. J Biol Chem 1987; 262: 16157-16163
    PubMedSuche in Google Scholar
  • 24 Huang TF, Liu CZ, Ouyang C, Teng CM. Halysin, an antiplatelet Arg-Gly-Asp-containing snake venom peptide, as fibrinogen receptor antagonist. Biochem Pharmacol 1991; 42: 1209-1219
    CrossrefPubMedSuche in Google Scholar
  • 25 Huang TF, Wang WJ, Teng CM, Ouyang C. Mechanism of action of the antiplatelet peptide, arietin, from Bitis arietans venom. Biochim Biophys Acta 1991; 1074: 144-150
    CrossrefPubMedSuche in Google Scholar
  • 26 Liu CZ, Peng HC, Huang TF. Crotavirin, a potent platelet aggregation inhibitor purified from the venom of the snake Crotalus viridis. Toxicon 1995; 33: 1289-1298
    CrossrefPubMedSuche in Google Scholar
  • 27 Liu CZ, Wang YW, Shen MC, Huang TF. Analysis of human platelet glycoprotein Ilb-IIIa by fluorescein isothiocyanate-conjugated disintegrins with flow cytometry. Thromb Haemost 1994; 72: 919-925
    Thieme ConnectPubMedSuche in Google Scholar
  • 28 Mustard JP, Perry DW, Ardlie NG, Packham MA. Preparation of suspensions of washed platelets from humans. Br J Haematol 1972; 22: 193-204
    CrossrefPubMedSuche in Google Scholar
  • 29 Bom GVR, Cross MJ. The aggregation of blood platelets. J Physiol 1963; 168: 178-195
    CrossrefPubMedSuche in Google Scholar
  • 30 Jennings LK, Ashman RA, Wang WC, Dockter ME. Analysis of human platelet glycoprotein Ilb-IIIa and Glanzmann’s thrombasthenia in whole blood by flow cytometry. Blood 1986; 68: 173-179
    CrossrefPubMedSuche in Google Scholar
  • 31 Coller BS, Scudder LE, Lang D. A rapid, whole blood, bedside assay to monitor platelet glycoprotein (GP) IMIIa blockade (abstr.). Blood 1994; 84: 474a
    PubMedSuche in Google Scholar
  • 32 Ault KA, Mitchell J. Analysis of platelets by flow cytometry. In: Methods in cell biology: Flow cytometry. Edited by Darzynkiewicz Z, Robinson JP, Crissman HA. Academic Press Inc. San Diego, California 1994 42. 275-294
    PubMedSuche in Google Scholar
  • 33 Ginsberg MH, Frelinger AL, Lam SC-T, Forsyth J, McMillan R, Plow EF, Shattil SJ. Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein IIb-IIIa with conformation-specific monoclonal antibodies. Blood 1990; 76: 2017-2023
    PubMedSuche in Google Scholar
  • 34 Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987; 70: 307-315
    PubMedSuche in Google Scholar
  • 35 Johnston GI, Heptinstall S, Robins RA, Price MR. The expression of glycoproteins on single blood platelets from healthy individuals and from patients with congenital bleeding disorders. Biochem Biophy Res Commun 1984; 123: 1091-1098
    CrossrefPubMedSuche in Google Scholar
  • 36 Tschoepe D, Spangenberg P, Esser J, Schwippert B, Kehrel B, Boesen P, Gries FA. Flow-cytometric detection of surface membrane alternations and concomitant changes in the cytoskeletal actin status of activated platelets. Cytometry 1990; 11: 652-656
    CrossrefPubMedSuche in Google Scholar
  • 37 Coller BS, Folts JD, Smith SR, Scudder LE, Jordan J. Abolition of in vivo platelet thrombus formation in primates with monoclonal antibodies to the platelet GPIMIIa receptor. Circulation 1989; 80: 1766-1774
    CrossrefPubMedSuche in Google Scholar
  • 39 Coller BS, Folts JD, Scudder LE, Smith SR. Antithrombotic effect of monoclonal antibody to the platelet glycoprotein IMIIa receptor in an experimental animal model. Blood 1986; 68: 783-786
    PubMedSuche in Google Scholar