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/

We describe the production and biochemical characterization of the fir st GPIIb/IIIa-inhibiting monoclonal antibody that contains an RGD sequ ence in the CDR3 region of the heavy chain. Monoclonal antibodies obta ined by immunizing mice with human platelets were screened using conse cutive ELISAs based on human platelets and immunoaffinity-purified gly coprotein (GP) IIb/IIIa coated on microtitre plates. Out of 30 monoclo nal antibodies reacting with GPIIb/IIIa, one, MA-16N7C2, potently inhi bited 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 similar to 4 times faster to activated than to resting platelets, wit h a K-dcalc of 6.6 nM and of 17.51 nM, respectively. Equilibrium bindi ng studies to nonactivated platelets showed a K-d of 18.2 nM With 41 x 10(3) binding sites per platelet. The antibody recognized GPIIb/IIIa only as a Ca2+-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 nM and 13.2 nM, respectively. Sequence analysis revealed a RGD-containing sequence with homology to disintegrins, in the CDR3 r egion of the heavy chain. That this RGD-containing sequence could be i nvolved in the interaction of the antibody to GPIIb/IIIa was finally i ndicated by showing that the binding is completely and competitively i nhibited by echistatin.