Thromb Haemost 1991; 65(04): 432-437
DOI: 10.1055/s-0038-1648166
Original Article
Schattauer GmbH Stuttgart

Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

A W J Stuttle
The Department of Diagnostic Radiology, Royal Postgraduate Medical School and Hammersmith Hospital, London, U. K.
,
M J Powling
**   The Department of Haematology, Institute of Child Health, Hospital for Sick Children, London, U. K.
,
J M Ritter
*   The Department of Clinical Pharmacology, Royal Postgraduate Medical School and Hammersmith Hospital, London, U. K.
,
R M Hardisty
**   The Department of Haematology, Institute of Child Health, Hospital for Sick Children, London, U. K.
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Weitere Informationen

Publikationsverlauf

Received: 25. August 1989

Accepted after revision 11. Dezember 1990

Publikationsdatum:
02. Juli 2018 (online)

Summary

The anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

 
  • References

  • 1 Bai Y, Durbin H, Hogg N. Monoclonal antibodies specific for platelet glycoproteins react with human monocytes. Blood 1984; 64: 139-6
  • 2 Nachman RL, Leung LL. Complex formation of platelet membrane glycoprotein IIb and IIIa with fibrinogen. J Clin Invest 1982; 69: 263-269
  • 3 Bennet JS, Vilaire G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. Lab Clin Invest 1979; 64: 1393-1401
  • 4 Magueric GA, Plow EF, Eddington TS. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem 1979; 254: 5357-5363
  • 5 Peerschke EI, Zucker MB, Grant RA, Egan JJ, Johnson MM. Correlation between fibrinogen binding to human platelets and platelet aggregability. Blood 1980; 55: 841-847
  • 6 Peters AM, Lavender JP, Needham SG, Loutfi I, Snook D, Epenetos AA, Lumley P, Keery RJ, Hogg N. Imaging thrombus with radiolabelled monoclonal antibody to platelets. Br Med J 1986; 293: 1525-1527
  • 7 Boucheix C, Benoit P, Knef P, Billard M, Mishel Z, Azzarone B, Rendu E, Esnouf J, Hermant L, Bredoux R, Levy-Toledano S, Soria C, Perrot JY, Mirshahi M, Giannoni F, Bemadou A, Soria J. Platelet aggregation of mabs directed against other membrane antigens. In: Leucocyte Typing III. White Cell Differentiation Antigens McMichael AJ. (ed). Oxford University Press; London: pp 780-782
  • 8 Haque S, Saizawa K, Rojo J, Janeway CA. The influence of valence on the functional activities of monoclonal anti-L3T4 antibodies. J Immunol 1987; 139: 3207-3212
  • 9 Parham P, Androlewicz MJ, Brodsky FM, Holmes NJ, Ways JP. Monoclonal antibodies: purification, fragmentation and application to structural and functional studies of class 1 MHC antigens. J Immunol Meth 1982; 53: 133-173
  • 10 Gorman RR, Bunting S, Miller OV. Modulation of human platelet adenylate cyclase by prostacyclin (PGX). Prostaglandins 1977; 13: 377-388
  • 11 Tatson JE, Moncada S, Vane JR. Effects of prostacyclin (PGX) on cyclic AMP concentrations in human platelets. Prostaglandins 1977; 13: 389-397
  • 12 Whittle BJ, Moncada S, Vane JR. Comparison of the effects of prostacyclin (PG12), prostaglandin E1 and D2 on platelet aggregation in different species. Prostaglandins 1978; 16: 373-388
  • 13 Paton WEM, Rang HP. The uptake of atropine and related drugs by intestinal smooth muscle of the guinea-pig in relation to acetylcholine receptors. Proc R Soc London 1965; 163: 1-44
  • 14 Tangen O, Berman HJ, Marfey P. Gel filtration: A new technique for separation of blood platelets from plasma. Thromb Diat Haemorrh 1971; 25: 268-278
  • 15 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
  • 16 Hallam TJ, Sanchez A, Rink TJ. Stimulus-response coupling in human platelets: changes evoked by platelet-activating factor in cytoplasmic free calcium monotoring by quin2. Biochem J 1984; 218: 819-827
  • 17 Born GVR, Cross MJ. The aggregation of blood platelets. J Physiol (London) 1963; 168: 178
  • 18 Jones D, Fritschy J, Garson J, Nokes TIC, Kemshead JT, Hardisty RM. A monoclonal antibody binding to human medulloblastoma cells and to the platelet glycoprotein IIb-IIIa complex. Br J Haematol 1984; 57: 621-631
  • 19 Powling MJ. Activation mechanisms in normal and abnormal platelets: the role of calcium influx and mobilization. PhD Thesis, University of London 1987
  • 20 Powling MJ, Hardisty RM. The dependence of platelet ATP secretion in response to various agonists on Ca2+ mobilisation and aggregation in Quin 2 loaded platelets. Thromb Haemostas 1987 58. 509 (Abstr)
  • 21 Hnatowich D, Layne WW, Childs RL, Lanteigne D, Davis MA. Radioactive labelling of antibody: a simple and efficient method. Science 1983; 220: 613-615
  • 22 Mongar JL, Winne D. Further studies on the mechanism of passive sensitization. J Physiol 1966; 182: 79-91
  • 23 Chen WJ, Chen Z, Bao CX. HIP2: a new stimulant type monoclonal antibody against human platelets. Chin J Haematol 1987; 8: 65-69
  • 24 Boucheix C, Soria C, Mirshahi M, Soria J, Perrot JY, Fournier N, Billard M, Rosenfield C. Characteristics of platelet aggregation induced by the monoclonal antibody ALB6 (acute lymphoblastic leukaemia antigen p24). Inhibition of aggregation by ALB Fab’. FEBS Lett 1983; 161: 289-295
  • 25 Higashihara M, Maeda H, Shibata Y, Kume S, Ohashi T. A monoclonal anti-human platelet antibody: A new platelet aggregatory substance. Blood 1985; 65: 382-391
  • 26 Arunlakshana O, Schild HO. Some quantitative uses of drug antagonists. Br J Pharmacol 1959; 14: 48-58
  • 27 Bennet JS, Hoxie JA, Leitman SF, Vilaire G, Cines DB. Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody. Proc Natl Acad Sci USA 1983; 80: 2417-2421
  • 28 Coller BS, Peerschke LE, Scudder LE, Sullivan CA. A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/ or IIIa. J Clin Invest 1983; 72: 325-338
  • 29 McEver RP, Bennett EM, Martin MN. Identification of two structurally and functionally distinct sites on human platelet membrane glycoprotein IIb-IIIa using monoclonal antibodies. J Biol Chem 1983; 258: 5269-5275
  • 30 Pidard D, Montgomery RR, Bennett JS, Kunicki TJ. Interaction of AP2, a monoclonal antibody specific for the human platelet glycoprotein IIb-IIIa complex, with intact platelets. J Biol Chem 1983; 258: 12582-12586
  • 31 Lombardo VT, Hodson E, Roberts JR, Kunicki TJ, Zimmerman TS, Ruggeri ZM. Independent modulation of von Willebrand factor and fibrinogen binding to the platelet membrane glycoprotein IIb/IIIa complex as demonstrated by monoclonal antibody. J Clin Invest 1985; 76: 1950-1958