Detection of intermediary Clr with complete active site, using a synthetic proteinase inhibitor

Abstract

The synthetic proteinase inhibitor, FUT-175 (6-amidino-2-naphthyl-4-guanidinobenzoate), strongly suppressed activation of Clr at 37"C, causing 50% inhibition at 0.03 mM. To clarify whether the inhibitor was incorporated into the active site of intermediary Clr formed during the incubation, determination of the active site was tried using this inhibitor. Consequently, release of amidinonaphthol equimolar with the amount of Clr used was observed in the early period of incubation, in which the activation to Clrwas about 5%. These results indicate that intermediary Clr already has a complete active site.

Inhibitory Effect of a New Synthetic Protease Inhibitor (FUT-175) on the Coagulation System

“…However, the IC50 values of FUT-175 on the fibrinolytic activity and amidolytic activity of plasmin were different for some unknown reason. These effects were almost the same as those of the co-guanidinino acid ester deriva tives reported by Ohno et al [10], and also they could not clear, for some unknown rea son, FUT-175 blockade of the active center of the protease [12]. This difference may pos sibly be due to the different amounts of en zymes in both assay systems, stability of FUT-175 at low concentration in the reac tion medium of the fibrin-plate method, and the different affinities of synthetic substrate or fibrin for plasmin.…”

In Vitro and In Vivo Gene Delivery by Recombinant Baculoviruses

“…FUT-175 specifically binds the Bb fragment of factor B, an important enzyme in the alternative complement pathway. FUT-175 is also incorporated into the active site of the intermediary C1r form, inhibiting both the alterna- tive and classical pathways of the complement system (13,26). This compound also protects retroviral vectors against serum inactivation (23).…”

“…To determine the effects of serum complement on the inactivation of baculovirus, 10 l of either AcVSVG-CAluc or AcGFP-CAluc (2 ϫ 10 9 PFU/ml) was incubated with 90 l of either untreated or heat-inactivated serum for 1 h at 37°C. AcGFP-CAluc was also incubated for 1 h at 37°C in the presence of rat or human serum with various concentrations of FUT-175 (6-amidino-2-naphthyl 4-guanidinobenzoate; Torii & Co., Ltd., Tokyo, Japan), a synthetic protease inhibitor that inhibits C1r or C1 esterase (26,40) and C3 convertase (13). Residual infectivity was determined by inoculation into HepG2 cells.…”

Protective Effect of Nafamostat Mesilate on Injury of Porcine Hepatocytes by Human Plasma

“…tection of porcine hepatocytes. NM is a synthetic inhibi tor of serine protease that has several beneficial effects for cells and tissues (5,10,17).…”