Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen

• Published in: The Journal of clinical investigation Vol. 100; no. 3; pp. 514 - 521
• Main Authors: Chiang, T M, Rinaldy, A, Kang, A H
• Format: Journal Article
• Language: English
• Published: United States 01.08.1997
• Subjects: Amino Acid Sequence; Base Sequence; Blood Platelets - metabolism; Cloning, Molecular; Collagen - metabolism; Humans; Integrins - genetics; Integrins - metabolism; Molecular Sequence Data; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Receptors, Collagen; Sequence Analysis
• ISSN: 0021-9738
• DOI: 10.1172/JCI119560

A cDNA (1.6 kb) encoding a platelet protein receptor that binds type I collagen has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide probe derived from the amino acid sequence of a CNBr fragment of the purified receptor. Computer search revealed that this cDNA represents the coding sequence of a unique protein. Using the prokaryotic expression system pKK 223-3-65 cDNA, a 54-kD recombinant protein was obtained and purified to apparent homogeneity. In an eukaryotic expression vector (pcDNA3-65 cDNA), a 65-kD protein was identified that was recognized by monoclonal anti-65 kD antibody (anti-65m). The recombinant protein binds to type I, but not to type III collagen by affinity column chromatography. The binding of the recombinant protein to type I collagen-coated Petri dishes is inhibited by anti-65m in a dose-dependent manner. The pcDNA3-65 cDNA-transfected nonadherent T cells express the protein, allowing them to attach to a type I collagen matrix, and are inhibited by anti-65m in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type I collagen-induced platelet aggregation and the adhesion of [14C]serotonin-labeled platelets to type I collagen in a dose-dependent manner. The recombinant protein neither binds to type III collagen-coated Petri dishes nor inhibits type III collagen and ADP-induced platelet aggregation, indicating specificity for type I collagen.