Activation of the fibrinogen binding site on platelets isolated from a patient with the Strasbourg I variant of Glanzmann's thrombasthenia.
One proposed ligand binding site on platelet integrin alpha IIb beta 3 is the region of the beta 3 subunit encompassing amino acids 211-221. However, we recently showed that synthetic peptides corresponding to amino acids 211-221 inhibit fibrinogen binding to alpha IIb beta 3 by binding to alpha IIb beta 3 and not to fibrinogen. In this study, we show that AP6, a monoclonal antibody (MoAb) directed against amino acids 214-221 of beta 3, bound to immobilized active alpha IIb beta 3 but did not inhibit fibrinogen binding to the complex. We then determined whether nonfunctional alpha IIb beta 3 on platelets with a beta 3 Arg-214-->Trp mutation (Strasbourg I variant of Glanzmann's thrombasthenia or GTV) could be induced to aggregate after treatment with dithiothreitol (DTT). DTT has been shown to expose the fibrinogen receptor on normal platelets. DTT treatment of GTV platelets did result in the formation of the fibrinogen binding site as indicated by the binding of pI-55, an MoAb that only binds to the activated form of alpha IIb beta 3. Furthermore, DTT-treated GTV platelets aggregated in the presence of fibrinogen and divalent cations. This aggregation was inhibited by EDTA, RGDS, and the selective alpha IIb beta 3 antagonist, Ro 43-5054. These data show that Arg-214 of beta 3 is not required for fibrinogen binding or for platelet aggregation. However, this amino acid appears to be critical for the formation and for the maintenance of the correct tertiary structure of the fibrinogen binding site on alpha IIb beta 3.
Kouns
W C
WC
F. Hoffmann-La Roche Ltd, Basel Switzerland.
Steiner
B
B
Kunicki
T J
TJ
Moog
S
S
Jutzi
J
J
Jennings
L K
LK
Cazenave
J P
JP
Lanza
F
F
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D017354:Point Mutation; D012016:Reference Values; D013915:Thrombasthenia / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D005340:Fibrinogen