Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen.
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.
Chiang
T M
TM
Department of Medicine, University of Tennessee at Memphis, Memphis, Tennessee 38104, USA. tchiang@utmem2.utmen.edu
Rinaldy
A
A
Kang
A H
AH
D000595:Amino Acid Sequence; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism*; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D038981:Receptors, Collagen; D017421:Sequence Analysis
D016023:Integrins; D011956:Receptors, Cell Surface; D038981:Receptors, Collagen; D003094:Collagen