Endothelial injury in scleroderma. A protease mechanism.
The mechanism of action of the previously reported serum ECA in patients with scleroderma has been studied. Gel filtration (Sephadex G-200) followed by ion exchange chromatography (DEAE-Sephadex A-50, Tris buffer, pH 8, 1.5M NaCl) yielded a 13-fold enriched fraction. ECA biological activity was abolished by preincubating the active fraction with several protease inhibitors (STI, aprotinin, TLCK, and PMSF); exposure of target cells to protease inhibitors did not inhibit ECA. ECA-rich fractions have esterolytic activity (3H-TAME), which was irreversibly inhibited by TLCK and PMSF in a time-, temperature-, concentration-, and pH-dependent fashion. In separate studies, functional protease inhibitor activity in scleroderma sera was determined by the capacity of serum to inhibit BAPNA hydrolysis by trypsin (TIC). TIC was 1.32 +/- 0.15 (milligrams of trypsin inhibited by 1 ml of serum +/- S.D.) in 20 control sera, and 0.54 +/- 0.18 (p less than 0.001) in 38 scleroderma sera. A positive correlation was found between TIC and percent inhibition of endothelial cell 3HTdR uptake induced by the same serum (r = 0.94, p less than 0.001). These studies suggest that ECA is mediated in vitro via a protease mechanism associated with a functional deficiency of protease inhibitors in scleroderma sera.
D002478:Cells, Cultured; D004727:Endothelium / Q000187:drug effects* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D010447:Peptide Hydrolases / Q000097:blood* / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011480:Protease Inhibitors / Q000097:blood; D012595:Scleroderma, Systemic / Q000097:blood*; D013936:Thymidine / Q000378:metabolism; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000494:pharmacology
D011480:Protease Inhibitors; D014107:Tosyllysine Chloromethyl Ketone; D010664:Phenylmethylsulfonyl Fluoride; D014360:Trypsin Inhibitor, Kunitz Soybean; D010447:Peptide Hydrolases; D013936:Thymidine