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An echistatin‐like Arg‐Gly‐Asp (RGD)‐containing sequence in the heavy chain CDR3 of a murine monoclonal antibody that inhibits human platelet glycoprotein IIb/IIIa function
Author(s) -
Deckmyn Hans,
Stanssens Patrick,
Hoet Bernard,
Declerck Paul J.,
Lauwereys Marc,
Gansemans Yannick,
Tornai Istvan,
Vermylen Jos
Publication year - 1994
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.1994.tb08313.x
Subject(s) - monoclonal antibody , platelet , microbiology and biotechnology , chemistry , platelet membrane glycoprotein , thrombin , glycoprotein , platelet activation , von willebrand factor , antibody , biochemistry , ristocetin , biology , immunology
Summary We describe the production and biochemical characterization of the first GPIIb/IIIa‐inhibiting monoclonal antibody that contains an RGD sequence in the CDR3 region of the heavy chain. Monoclonal antibodies obtained by immunizing mice with human platelets were screened using consecutive ELISAs based on human platelets and immunoaffinity‐purified glycoprotein (GP) IIb/IIIa coated on microtitre plates. Out of 30 monoclonal antibodies reacting with GPIIb/IIIa, one, MA‐16N7C2, potently inhibited platelet aggregation induced by ADP, thrombin, arachidonic acid, collagen, U46619, adrenaline and platelet‐activating factor, whereas ristocetin‐induced aggregation was unaffected. MA‐16N7C2 (IgG2a) bound ˜ 4 times faster to activated than to resting platelets, with a K dcalc of 6.6 n m and of 17.5 n m , respectively. Equilibrium binding studies to non‐activated platelets showed a K d of 18.2 n m with 41 x 10 3 binding sites per platelet. The antibody recognized GPIIb/IIIa only as a Ca 2+ ‐dependent complex. MA‐16N7C2 blocked fibrinogen and von Willebrand factor binding to GPIIb/IIIa in a competitive manner with a K i of 8.5 n m and 13.2 n m , respectively. Sequence analysis revealed a RGD‐containing sequence with homology to disintegrins, in the CDR3 region of the heavy chain. That this RGD‐containing sequence could be involved in the interaction of the antibody to GPIIb/IIIa was finally indicated by showing that the binding is completely and competitively inhibited by echistatin.