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.
Veterans Affairs Medical Center and the Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38104, USA. TChiang@utmen.edu
Thomas M
TM
Chiang
Flecia
F
Cole
Virginia
V
Woo-Rasberry
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