Further characterization of the loop structure of platelet glycoprotein IIIa: partial mapping of functionally significant glycoprotein IIIa epitopes.
Glycoprotein (GP) IIb-IIIa serves as the platelet fibrinogen receptor. Studies of the tertiary structure of GPIIIa have shown that the protein has a large loop structure of at least 325 amino acids in length. To further characterize this loop structure, intact platelets were digested with alpha-chymotrypsin. Digestion products were examined using the anti-GPIIIa monoclonal antibodies (MoAbs) AP3, D3GP3, and C5GP3, as well as the human alloantibody, anti-PLA1. AP3 recognized GPIIIa digestion products of 109, 95, and 68 Kd. D3GP3 and C5GP3 recognized an additional band of 51 Kd. Time course digestions demonstrated that the 51-Kd fragment was generated by proteolysis of the 68-Kd peptide. Sequence analysis of the reduced 51-Kd peptide showed that this fragment began at amino acid 422. The nonreduced 51-Kd peptide was reactive with antibodies directed against the first 13 amino acids of GPIIIa, demonstrating the presence of a covalently attached N-terminal peptide. These data suggest that: (1) the minimum length of the loop structure is at least 384 amino acids; (2) the AP3 epitope is formed at least in part by a determinant contained within residues 348 to 421; and (3) the D3GP3 and C5GP3 epitopes are contained within amino acids 422 to 692 of GPIIIa, a region that may be flexible and involved in conformational changes that occur after ligand binding.
Kouns
W C
WC
Department of Medicine, University of Tennessee, Memphis 38163.
Newman
P J
PJ
Puckett
K J
KJ
Miller
A A
AA
Wall
C D
CD
Fox
C F
CF
Seyer
J M
JM
Jennings
L K
LK
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000737:chemistry; D015153:Blotting, Western; D002918:Chymotrypsin / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D002918:Chymotrypsin