title: Inhibition of coagulation factors by recombinant barley serpin BSZx.
abstract: Barley serpin BSZx is a potent inhibitor of trypsin and chymotrypsin at overlapping reactive sites (Dahl, S.W., Rasmussen, S.K. and Hejgaard, J. (1996) J. Biol. Chem., in press). We have now investigated the interactions of BSZx with a range of serine proteinases from human plasma, pancreas and leukocytes, a fungal trypsin and three subtilisins. Thrombin, plasma kallikrein, factor VIIa/tissue factor and factor Xa were inhibited by BSZx at heparin independent association rates (k(ass)) of 4.5 X 10(3)-1.3 x 10(5) M(-1) s(-1) at 22 degrees C. Only factor Xa turned a significant fraction of BSZx over as substrate. Complexes of these proteinase with BSZx resisted boiling in SDS, and amino acid sequencing showed that cleavage in the reactive center loop only occurred after P1 Arg. Activated protein C and leukocyte elastase were slowly inhibited by BSZx (k(ass)=1-2 x 10(2) M(-1) s(-1)) whereas factor XIIa, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein and elastase were not or only weakly affected. The inhibition pattern with mammalian proteinases reveal a specificity of BSZx similar to that of antithrombin III. Trypsin from Fusarium was not inhibited while interaction with subtilisin Carlsberg and Novo was rapid but most BSZx was cleaved as a substrate. Identification of a monoclonal antibody specific for native BSZx indicate that complex formation and loop cleavage result in similar conformational changes.
authors.affiliation: Department of Biochemistry and Nutrition, Technical University of Denmark, Lyngby.
authors.forename: S W
authors.initials: SW
authors.lastname: Dahl
authors.forename: S K
authors.initials: SK
authors.lastname: Rasmussen
authors.forename: L C
authors.initials: LC
authors.lastname: Petersen
authors.forename: J
authors.initials: J
authors.lastname: Hejgaard
mesh_terms: D001779:Blood Coagulation Factors / Q000037:antagonists & inhibitors*; D015153:Blotting, Western; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D001467:Hordeum / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D010940:Plant Proteins / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D015843:Serpins / Q000494:pharmacology*; D013381:Subtilisins / Q000378:metabolism
chemical_list: C101795:BSZx protein, Hordeum vulgare; D001779:Blood Coagulation Factors; D010940:Plant Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D010450:Endopeptidases; D012697:Serine Endopeptidases; D013381:Subtilisins; D002918:Chymotrypsin