Heterogenous inhibition of platelet aggregation by monoclonal antibodies binding to multiple sites on GPIIIa.
Six monoclonal IgG1-k antibodies (LK2, LK3r, LK4-55, LK5, LK6-55, LK7r) were raised against platelet membrane GPIIIa in order to study the structure-function relationship of this molecule. Antibodies were selected on their ability to react with GPIIIa by ELISA on adherent platelets, by immunoblot on platelet lysates and by fluorescence flow cytometry on intact platelets. Fluorescence reactivity varied from 3- to 202-fold greater than isotype control fluorescence. Two MoAbs reacted on immunoblot under reduced conditions (LK7r and LK3r). Two reacted with a 55 kD chymotrypsin/subtilisin digest of GPIIIa which is likely to exclude amino acids 121-348 (LK4-55 and LK6-55). Four of the MoAbs (LK5, LK3r, LK2 and LK4-55) inhibited tyrosine phosphorylation of one to four distinct bands on immunoblot. LK4-55 reacted with an N-terminal 66 amino acid fusion protein of GPIIIa near the PLA epitope (Leu 33). LK7r reacted with a 212-222 peptide reported to be an RGD fibrinogen binding site. LK2 reacted near a disintegrin-RGD binding site. Except for LK5, all inhibited ADP, collagen and thrombin-induced platelet aggregation in a heterogeneous fashion. Percentage inhibition of 125I-fibrinogen binding to platelets varied from 18% to 98%. No correlation was noted between inhibition of fibrinogen binding, location of MoAb binding on GPIIIa, reactivity of MoAb binding with GPIIIa, inhibition of thrombin-induced tyrosine phosphorylation or inhibition of platelet aggregation induced by ADP, collagen or thrombin. Thus MoAbs, binding to platelet GPIIIa at different sites, inhibit platelet aggregation in a heterogeneous manner.
Liu
L X
LX
New York University Medical School, New York 10016, USA.
Nardi
M A
MA
Nierodzik
M L
ML
Karpatkin
S
S
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects / Q000276:immunology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000494:pharmacology; D014757:Viper Venoms / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D014757:Viper Venoms; C084060:eristostatin; D005340:Fibrinogen; D013917:Thrombin