Cloning, characterization, and functional studies of a 47-kDa platelet receptor for type III collagen.
A 1.2-kb cDNA fragment encoding a platelet 47-kDa protein has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide of the sequenced amino terminus of the purified platelet protein with a poly(dT)(12).(dG) by polymerase chain reaction. A computer search revealed that the cDNA represents the coding sequence of a protein with a fragmentary homology to several proteins. Using a prokaryotic expression system, pBad TOPO-47 cDNA, a 47-kDa recombinant protein was obtained and purified to apparent homogeneity by nickel-nitrilotriacetic acid resin and collagen affinity column. The recombinant protein binds to type III but not type I collagen-Sepharose 2B affinity columns. Anti-47-kDa but not anti-65-kDa antibody inhibits the binding of the recombinant protein to the type III collagen-coated micro titer wells in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type III collagen-induced platelet aggregation also in a dose-dependent manner. We have defined two active peptides from the cloned deduced amino acid sequence. Both peptides inhibit type III but not type I collagen-induced platelet aggregation in a dose-dependent fashion. These results suggest that the active binding site of the platelet receptor to type III collagen resides in these portions of the protein.
Chiang
Thomas M
TM
Veterans Affairs Medical Center and the Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38104, USA. TChiang@utmen.edu
Cole
Flecia
F
Woo-Rasberry
Virginia
V
D000595:Amino Acid Sequence; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism*; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D024061:Collagen Type III / Q000378:metabolism*; D018076:DNA, Complementary; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D038981:Receptors, Collagen; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001798:Blood Proteins; D024061:Collagen Type III; D018076:DNA, Complementary; D016023:Integrins; D010750:Phosphoproteins; D038981:Receptors, Collagen; D011994:Recombinant Proteins; C039077:platelet protein P47