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 Vllaltissue factor and factor Xa were inhibited by BSZx at heparin independent association rates (kass) of 4.5 x 103-1.3 x 105 M -l s i at 22°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 PI Arg. Activated protein C and leukocyte clastase were slowly inhibited by BSZx (kass = I-2X 102 M -l s -t) whereas factor Xlla, 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.

Proteinase inhibitors and their function in plants: A review

“…Studies of plant serpins revealed a broad spectrum of biological activities. Recombinant barley serpin (BSZx) suppresses the activity of trypsin, chymotrypsin, and cathepsin G; it also affects blood coagulation enzymes (thrombin, callicrein, and factor VII) [204,206]. Inhibition of subtilisin or endogenous plant serine proteinases has never been observed [204,207].…”

Inhibitory Serpins from Wheat Grain with Reactive Centers Resembling Glutamine-rich Repeats of Prolamin Storage Proteins

“…CaCl 2 (5 mM) was added in assays with coagulation factor VIIa/sTF. The serine proteinases and their specific substrates were as described previously (8), except that in the present study trypsin activity was measured with Ser-Pro-Arg-pnitroanilide (Bachem) as substrate. Curve fitting and data treatment have been described in detail previously (7).…”

“…Concentration of active enzyme was determined using either active site titrants or tightbinding inhibitors as described previously (8). Concentrations of purified serpins were determined by amino acid analysis (21).…”

“…motrypsin-like enzymes at the overlapping site P 2 Leu (7,8). Therefore, rWSZ2b was screened for inhibitory activity toward a panel of serine proteinases from human blood, human leukocytes, and porcine or bovine pancreas.…”

Enhancement of tomato resistance to Tuta absoluta by the expression of two barley proteinase inhibitors

“…Serpins have mixed specificities toward proteases (Al-Khunaizi et al, 2002;Hejgaard & Hauge, 2002;Huntington, 2011). Barley serpin is a potent inhibitor of trypsin and chymotrypsin (Dahl et al, 1996a), but it also inhibits thrombin, plasma, Factor VIIa and Factor Xa (Dahl et al, 1996b). Wheat serpin inhibits chymotrypsin and cathepsin G (Roberts et al, 2003).…”