A Simple and Rapid Method to Determine GPIIb/IIIa-Receptor Inhibitor Activity in Whole Blood

A. C. Matzdorff; Gitta Kühnel

doi:10.1055/s-0038-1660401

Hämostaseologie, Table of Contents

Hamostaseologie 1999; 19(03): 134-138
DOI: 10.1055/s-0038-1660401

Übersichts- und Originalarbeiten/Review and Original Articles

F. K. Schattauer Verlagsgesellschaft

A Simple and Rapid Method to Determine GPIIb/IIIa-Receptor Inhibitor Activity in Whole Blood

A. C. Matzdorff

¹Dept. of Hematology/Oncology (Chair: Prof. Dr. H. Pralle), Center for Internal Medicine, Justus-Liebig-University, Giessen, Germany

,

Gitta Kühnel

¹Dept. of Hematology/Oncology (Chair: Prof. Dr. H. Pralle), Center for Internal Medicine, Justus-Liebig-University, Giessen, Germany

› Author Affiliations

Abstract

Summary

We studied the effect of GPIIb/IIIa-inhibitors on platelet activation with flow cytometry in vitro. Citrated whole blood was incubated with increasing concentrations of three different GPIIb/IIIa-inhibitors (c7E3, DMP728, XJ757), then thrombin or ADP were added and after 1 min the sample was fixed. Samples without c7E3 but with 0.1 U/ml thrombin had a decrease in platelet count. Samples with increasing concentrations of c7E3 had a lesser or no decrease in platelet count. The two other inhibitors (DMP 725, XJ757) gave similar results. GPIIb/IIIa-inhibitors prevent aggregate formation and more single platelets remain in the blood sample. The agonist-induced decrease in platelet count correlates closely with the concentration of the GPIIb/IIIa inhibitor and receptor occupancy. This correlation may be used as a simple measure for inhibitor activity in whole blood.

Keywords

Platelet activation - GPIIb/IIIa-inhibitors - flow cytometry

Full Text

References

References
1 Chesebro J.H, Rauch U, Fuster V, Badimon J.J. Pathogenesis of thrombosis in coronary artery disease. Haemostasis 1997; 27 Suppl. I 12-18.
2 The PRISM-PLUS Study Investigators. Inhibition of the platelet glycoprotein IIb/IIIa receptor with tirofiban in unstable angina and non-q-wave myocardial infarction. N Engl J Med 1998; 338: 1488-97.
3 The PURSUIT Trial Investigators. Inhibition of platelet glycoprotein IIb/IIIa with eptifibatide in patients with acute coronary syndromes. N Engl J Med 1998; 339: 436-43.
4 The EPIC Ivestigators. Use of a monoclonal antibody directed against the platelet glycoprotein IIb/IIIa receptor in high-risk coronary angioplasty. N Engl J Med 1994; 330: 956-61.
5 Simpfendorfer C, Kottke-Marchant K, Lowrie M, Anders R.J, Burns D.M, Miller D.P, Cove C.S, DeFranco A.C, Ellis S.G, Moliterno D.J, Raymond R.E, Sutton J.M, Topol E.J. First chronic platelet glycoprotein IIb/IIIa integrin blockade. Circulation 1997; 96: 76-81.
6 Coller B.S. A new murine monoclonal antibody reports an activation-depndent change in the conformation and/or microenviron-ment of the platelet glycoprotein IIb/IIIa complex. J Clin Invest 1985; 76: 101-8.
7 Mousa S, Bozarth J.M, Forsythe M.S, Lorelli W, Thoolen M.J, Ramachandran N, Jackson S, DeGrado W, Reilly T.M. Antiplatelet efficacy and specificity of DMP728, a novel platelet GPIIb/IIIa receptor antagonist. Cardiology 1993; 83: 374-82.
8 Mousa S.A, DeGrado W, Dun-Xu M, Kapil R.P, Lucchesi B.R, Reilly T.M. Oral antiplatelet, antithrombotic efficacy of DMP 728, a novel platelet GPIIb/IIIa antagonist. Circulation 1996; 93: 537-43.
9 Matzdorff A.C, Kiihnel G, Kemkes-Matthes B, Pralle H. Quantitative assessment of platelets, platelet microparticles, and platelet aggregates with flow cytometry. J Lab Clin Med 1998; 131: 507-17.
10 Cannon C.P, McCabe C.H, Borzak S, Henry T.D, Tischler M.D, Mueller H.S, Feldman R, Palmeri S.T, Aull K, Hamilton S.A, Rothman J.M, Novotny W.F, Braunwald E. Randomized trial of an oral platelet glycoprotein IIb/IIIa antagonist, sibrafiban, in patients after acute coronary syndrome. Circulation 1998; 97: 340-9.
11 Tscheng J.E, Ellis S.G, George B.S, Kereiakes D.J, Kleiman N.S, Talley D, Wang A.L, Weisman H.F, Califf R.M, Topol E.J. Pharmacodynamics of chimeric glycoprotein IIb/IIIa integrin antiplatelet antibodiy fab 7E3 in high-risk coronary angioplasty. Circulation 1994; 90: 1757-64.
12 Phillips D.R, Teng W, Arfsten A, Nannizzi-Alaimo L, White M.M, Longhurst C, Shattil S, Randolph A, Jakubowski J.A, Jennings L.K, Scarborough R.M. Effect of Ca²⁺ on G PI lb-11 la interactions with integrum: enhanced GPIIb-IIIa binding and inhibition of platelet aggregation by reductions in the concentration of ionized calcium in plasma anticoagulated with citrate. Circulation 1997; 96: 1488-94.
13 Gold H.K, Coller B.S, Yasuda T, Saito T, Fallon J.T, Guerrero J.L, Leinbach R.C, Ziskind A.A, 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: 670-7.
14 Yasuda T, Gold H.K, Fallon J.T, Leinbach R.C, Guerrero J.L, Scudder L.E, Kankc M, Shealy D, Ross M.J, Collen D, Coller B.S. Monoclonal antibody against the platelet glycoprotein (GP) IIb/III a receptor prevents coronary artery reocclusion after reperfusion with recombinant tissue-type plasminogen activator in dogs. J Clin Invest 1988; 81: 1284-91.
15 Winterhagen A, Krzywanek H.J. Time and temperature dependent changes of ADP-and collagen induced “spontaneous” aggregation. Thromb Res 1980; 19: 621-38.
16 Breddin K, Ziemen M, Bauer O, Herrmann W, Schaudinn L, Schlooser U, Winterhagen A, Krzywanek H.J. Time and temperature dependent changes of ADP - and collagen induced “spontaneous” aggregation. Thromb Res 1980; 19: 621-38.
17 Coller B.S, Lang D, Scudder L.E. Rapid and simple platelet function assay to assess glycoprotein IIb/IIIa receptor blockade. Circulation 1997; 95: 860-7.
18 Dcering A, Fitzgerald D.J, Cox D.M. A novel method for the study of GPIIb/IIIa receptor occupancy. Circulation 1997; 96 (Suppl. 01) 168.
19 Gawaz M, Neumann F.J, Achömig A. Evaluation of platelet membrane glycoproteins in coronary artery disease. Circulation 1999; 99: e1-e11 (circulation electronic pages).
20 Greilich P.E, Alving B.M, O’Neill K.L, Chang A.S, Reid T.J. Modified thromboelastogra-phy: a rapid, simple method for monitoring c7E3 Fab in heparinized patient. Blood 1995; 86: 545a.
21 Kundu S, Heilmann E, Sio R, Garcia C, Ostgaard R. Characterization of the platelet function analyzer, PFA-100™. Thromb Haemost 1995; 73: 1061.
22 Mascelli M.A, Worley S, Veriabo N.J, Lance E.T, Mack S, Schaible T, Weisman H.F, Jordan R.E. Rapid assessment of platelet function with a modified whole-blood aggre-gometer in percutaneous transluminal coronary angioplasty patients receiving anti-GPI-Ib/IIIa therapy. Circulation 1997; 96: 3860-6.
23 Nunn B, White J.P. Inhibitors of platelet aggregation have different activity in blood than in platelet rich plasma. Brit J Pharmacol 1980; 70: 163P-4P.
24 Kleiman N.S, Raizner A.E, Jordan R, Wang A.L, Norton D, Mace K.F, Joshi A, Coller B.S, Weisman H.F. Differential inhibition of platelet aggregation induced by adenosine diphosphate or a thrombin receptor-activating peptide in patients treated with bolus chimeric 7E3 Fab: implications for inhibition of the internal pool of GPIIb/IIIa receptors. J Am Coll Cardiol 1995; 26: 1665-71.
25 Born G.V.R, Richardson P.D. Activation time of blood platelets. J Membr Biol 1980; 57: 87-90.
26 Gear A.R.L. Platelet adhesion, shape change, and aggregation: rapid initiation and signal transduction events. Can J Physiol Pharmacol 1994; 72: 285-94.
27 Gear A.R.L. Preaggregation reactions of platelets. Blood 1981; 58: 477-80.
28 Nurden P, Poujol C, Durrieu-Jais C, Winckler J, Combrié R, Macchi L, Bihour C, Wagner C, Jordan R, Nurden A.T. Labeling of the internal pool of GPIIb/IIIa in platelets by c7E3 Fab fragments (abciximab): flow and endocytic mechanisms contribute to the transport. Blood 1999; 93: 1622-33.
29 Aoki T, Tomiyama Y, Honda S, Senzaki K, Tanaka A, Okubo M, Takahashi F, Takasugi H, Seki J. Difference of (Ca²⁺)¡ movements in platelets stimulated by thrombin and TRAP: the involvement of a_nß₃-mediated TXA₂-synthesis. Thromb Haemost 1998; 79: 1184-90.