Further studies on the topography of the N-terminal region of human platelet glycoprotein IIIa. Localization of monoclonal antibody epitopes and the putative fibrinogen-binding sites.
The precise localization of the epitopes for six monoclonal antibodies specific for the N-terminal region of human platelet glycoprotein IIIa (GPIIIa) was determined. The epitope for P37, a monoclonal antibody that inhibits platelet aggregation, was found at GPIIIa 101-109, flanked by the epitopes for P23-3 (GPIIIa 16-28), P23-4 (GPIIIa 83-91), P23-5 (GPIIIa 67-73), P23-7 (GPIIIa 114-122) and P40 (GPIIIa 262-302), and very close to the early chymotryptic cleavage site of GPIIIa in whole platelets (Phe-100). When the amino acid sequence of GPIIIa was searched for peptide sequences hydropathically complementary to the fibrinogen gamma-chain C-terminal (gamma 400-411) and A alpha-chain RGD-containing peptides, none was found for the gamma 400-411, two (GPIIIa 128-132 and 380-384) were found complementary to fibrinogen A alpha 571-575 and two (GPIIIa 109-113 and 129-133) were found for A alpha 94-99. Two of these putative fibrinogen-binding sites overlap with each other, and a third one overlaps with the epitope for P37. These findings reinforce the earlier suggestion that the N-terminal region of GPIIIa is involved in fibrinogen binding, and suggest the existence in GPIIIa of either multiple or alternative RGD-binding sites or one RGD-binding domain with several moieties. Finally, early chymotryptic cleavage of GPIIIa in whole platelets liberates to the soluble fraction the peptide stretch Ser-101-Tyr-348, which carries the epitope for P37 and the putative binding sites for fibrinogen. The rest of the molecule, together with the GPIIb-resistant moiety, remains membrane-bound. This leads us to propose that the fibrinogen-binding domain of GPIIIa is not involved in the binding to GPIIb to form the Ca2(+)-dependent GPIIb-GPIIIa complex.
Calvete
J J
JJ
Instituto de Química Física, C.S.I.C., Madrid, Spain.
Arias
J
J
Alvarez
M V
MV
Lopez
M M
MM
Henschen
A
A
González-Rodríguez
J
J
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001792:Blood Platelets / Q000737:chemistry; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D000939:Epitopes / Q000032:analysis*; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011487:Protein Conformation; D014357:Trypsin
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; D005340:Fibrinogen; D002918:Chymotrypsin; D014357:Trypsin; D003488:Cyanogen Bromide