Evidence that the central region of glycoprotein IIIa participates in integrin receptor function.
We have obtained evidence that the ligand-recognition region of the integrin beta-subunit, platelet glycoprotein IIIa (GPIIIa), is discontinuous. Receptor function can be localized to residues near the N-terminus and to the central region of the polypeptide chain. The epitope recognized by our monoclonal antibody, CS-1, which substantially inhibits fibrin(ogen) binding to ADP- and thrombin-stimulated platelets [Ramsamooj, Doellgast & Hantgan (1990) Thromb. Res. 58, 577-592], is contained within residues 349-422 of GPIIIa. This sequence is adjacent to a proteinase-resistant domain of GPIIIa which is linked by disulphide bond(s) to an N-terminal segment near to the putative Arg-Gly-Asp recognition site [D'Souza, Ginsberg, Burke, Lam & Plow (1988) Science 242, 91-93]. Limited trypsin digestion of purified platelet GPIIIa yielded a mixture of two-chain molecules comprised of an N-terminal fragment disulphide-bonded to one of four fragments, which began at residues 299, 303, 353 or 423. Tryptic cleavage of the 300-422 segment correlated with loss of immunoreactivity with anti-GPIIIa monoclonal antibody, CS-1. Chymotrypsin cleavage of GPIIIa resulted in an N-terminal 19 kDa fragment joined by at least one intrachain cystine residue to a 46 kDa polypeptide beginning at residue 349. Partial reduction with dithiothreitol released the larger chymotryptic fragment with its epitope for CS-1 intact. These results have enabled us to localize the epitope recognized by our inhibitory monoclonal antibody, CS-1, to residues 349-422 of GPIIIa. Our data are consistent with a structure in which both the N-terminal and central regions of GPIIIa, which may be in close proximity in the functional GPIIb-IIIa complex, participate in ligand binding.
Ramsamooj
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Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27103.
Lively
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MO
Hantgan
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002918:Chymotrypsin; D005340:Fibrinogen / Q000502:physiology; D006868:Hydrolysis; D016023:Integrins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000302:isolation & purification / Q000502:physiology*; D016029:Receptors, Very Late Antigen / Q000502:physiology*; D012697:Serine Endopeptidases; D014357:Trypsin
D000911:Antibodies, Monoclonal; D016023:Integrins; D010980:Platelet Membrane Glycoproteins; D016029:Receptors, Very Late Antigen; D005340:Fibrinogen; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D014357:Trypsin