Identification of a PAI-1 binding site in vitronectin.
Active PAI-1 (plasminogen activator inhibitor 1) is bound to vitronectin in plasma and in the extracellular matrix. In this study we aimed at identifying the PAI-1 binding site in vitronectin, which at present is a matter of dispute. Vitronectin was cleaved with trypsin and the fragments were tested for inhibitory effect on the PAI-1/vitronectin interaction using vitronectin-coated microtiter plates. Intact vitronectin and the tryptic digest of vitronectin both caused a 50% reduction in PAI-1 binding at a concentration of about 2 nmol/I. Gel-filtration on Sephadex G-50 superfine of the tryptic peptides resulted in one main peak of inhibitory activity. The elution volume, Kav, was 0.55 indicating (a) medium-size peptide(s). The peptide was further purified by reverse-phase HPLC. Structural analysis revealed that it constituted the 45 NH2-terminal amino-acid residues in vitronectin. The NH2-terminal vitronectin peptide caused a 50% decrease in PAI-1 binding to the vitronectin-coated microtiter plates at a concentration of about 13 nmol/l. Thus, the peptide is a little less effective in this respect than intact vitronectin. Reduced and S-carboxymethylated peptide had no effect on the interaction. The NH2-terminal vitronectin fragment increased the stability of active PAI-1 by about 60%, which is a little less than with intact vitronectin. The peptide also prevented PAI-1 from oxidation with chloramine T. The half-life was prolonged about 4-fold as compared to about 30-fold with intact vitronectin.
Sigurdardottir
O
O
Department of Clinical Chemistry, Karolinska Hospital, Stockholm, Sweden.
Wiman
B
B
D000595:Amino Acid Sequence; D001665:Binding Sites; D002700:Chloramines / Q000494:pharmacology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D014105:Tosyl Compounds / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism; D019096:Vitronectin
D002700:Chloramines; D006023:Glycoproteins; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D014105:Tosyl Compounds; D019096:Vitronectin; C016300:chloramine-T; D014357:Trypsin