Identification of a monoclonal antibody against platelet GPIIb that interacts with a calcium-binding site and induces aggregation.
We have characterized a monoclonal antibody named D33C, specific for platelet glycoprotein (GP) IIb, which induces fibrinogen binding and platelet aggregation. D33C Fab fragments interact with an average of 44,000 +/- 20,000 sites on resting platelet with a Kd value of 0.8 microM. This value decreased to 0.17 microM in the presence of 1 mM EDTA suggesting that Ca2+ chelation increases the antibody affinity. Purified IgGs and Fab fragments exhibit a similar potency and induce binding of fibrinogen and aggregation at levels comparable to those obtained with ADP. D33C-induced platelet aggregation, however, was not inhibited by 1 microM PGE1 and was not associated with a significant [14C]serotonin release, suggesting differences with ADP in the mechanism of activation. Among a large series of synthetic peptides corresponding to potential antigenic sequences within the structure of GPIIb, one peptide with the sequence DIDDNGYPDLIV was found to inhibit D33C activity. This peptide corresponds to a putative calcium-binding site whose sequence is highly homologous to similar sequences present in the alpha subunits of the fibronectin and the vitronectin receptors. Despite this homology, D33C interacts only with platelet GPIIb suggesting that the identified epitope may be differently exposed at the surface of the cells. This antibody may prove to be a valuable tool to study the induction reaction on recombinant GPIIbIIIa expressed in cells that lack the appropriate signal transduction reactions.
DRF/Laboratoire d'Hématologie, Institut National de la Santé et de la Recherche Médicale U.217, Grenoble, France.
D
D
Gulino
J J
JJ
Ryckewaert
A
A
Andrieux
M J
MJ
Rabiet
G
G
Marguerie
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000527:Alprostadil / Q000494:pharmacology; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D000918:Antibody Specificity; D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology; D005340:Fibrinogen / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D007074:Immunoglobulin G / Q000494:pharmacology; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000276:immunology* / Q000502:physiology; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000276:immunology; D019030:Receptors, Vitronectin; D012701:Serotonin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D010980:Platelet Membrane Glycoproteins; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic; D019030:Receptors, Vitronectin; D012701:Serotonin; D000244:Adenosine Diphosphate; D005340:Fibrinogen; D000527:Alprostadil; D002118:Calcium