Purpurin as a Specific Inhibitor of Spermidine-Induced Autoactivation of the Protease Plasma Hyaluronan-Binding Protein

Abstract

Plasma hyaluronan-binding protein (PHBP), alternatively named factor VII activating protease, is a serine protease that can activate blood coagulation factor VII and the proenzyme form of urokinase-type plasminogen activator.1-4) PHBP circulates as a single-chain proenzyme form (pro-PHBP). It is autoproteolytically converted to an active two-chain form in vitro. 5,6) No physiologically relevant enzyme that activates pro-PHBP has been found so far, while its activation in vivo is observed under inflammatory conditions. 7,8) Both negatively charged molecules (such as heparin and RNA) and positively charged molecules (such as polyamines) dramatically promote pro-PHBP autoactivation.9-11) Thus, it is postulated that such molecules contribute to physiological pro-PHBP activation. Polyamines promote the formation of pro-PHBP autoactivation complex, 10) while heparin and RNA may act as a scaffold for the accumulation of pro-PHBP. 12)These findings suggest that pro-PHBP activation can proceed through multiple mechanisms depending on the kind of pathophysiological stimuli. 10,11) Polyamine concentrations are higher in cells and tissues with inflammation and malignancy, where significant cell death is observed. Inflammatory responses are associated with an increase in vascular permeability. Thus, one of the possible sites of pro-PHBP activation is extravascular tissue with inflammation. Polyamines released from the damaged cells may play a role in the extravascular regulation of pro-PHBP activation. In this study, we attempted to identify a specific agent that can inhibit polyamine-promoted pro-PHBP autoactivation. MATERIALS AND METHODSMaterials Pro-PHBP was isolated from human plasma as described by Etscheid et al. 5) Briefly, human citrated plasma was fractionated on Q-Sepharose column chromatography in the presence of urea (6 M), and fractions rich in pro-PHBP were further purified on Mono-Q column chromatography. The purified pro-PHBP preparation was stored in 6 M urea, in which pro-PHBP autoactivation/degradation was completely inhibited. The preparation was diluted just before assays with buffer A (50 mM Tris-HCl, pH 7.4, 75 mM NaCl, 5 mM CaCl 2 , and 0.05% (w/v) Tween 20). The active form of PHBP was prepared by incubating pro-PHBP (200 nM) in buffer B (50 mM Tris-HCl, pH 6.0, 150 mM NaCl, 10 mM CaCl 2 , and 0.1% (w/v) Tween 20) for 20 min at 37°C. The following proteins and chemicals were from commercial sources: human plasmin and alizarin from Wako Chemical (Osaka, Japan); factor Xa from Haematologic Technologies (Essex Junction, VT, U.S.A.); purpurin, emodin, spermidine, heparin, human thrombin, bovine trypsin, and e-poly-L-lysine from Sigma (St. Louis, MO, U.S.A.); and SPECTROZYME ® TH (STH; H-D-hexahydrotyrosyl-Ala-Arg-p-nitroanilide) and SPECTROZYME ® FXa (methoxycarbonyl-D-cyclohxylglycyl-Gly-Arg-p-nitroanilide-acetate) from American Diagnostica (Greenwich, CT, U.S.A.). Along with the supplier's information, the authenticity and purity of purpurin, emodin, and alizarin were examined by in-house analyses (H...

Elucidation of Crucial Structures for a Catechol-Based Inhibitor of Plasma Hyaluronan-Binding Protein (Factor VII Activating Protease) Autoactivation

“…10,[14][15][16] Most of these inhibitors have a polyphenol feature. In particular, potent inhibitors have plural catechol units, such as galloyl and caffeoyl groups.…”

Inhibitors of Autoactivation of Plasma Hyaluronan-Binding Protein (Factor VII Activating Protease)

“…Anthocyanidins Along with delphinidin (2), cyanidin (21), another anthocyanidin that lacks 5Ј-OH in the B-ring of 2, showed activity comparable to that of 2 in inhibiting spermidine-induced autoactivation (IC 50 ϭ0.13Ϯ 0.02 mM) (Fig. 3A).…”

“…21) However, its action was not specific for spermidine-induced autoactivation. Bikaverin was inhibitory to noninduced autoactivation and the enzymatic activities of the active form of PHBP as well as other serine proteases (such as plasmin and coagulation factor Xa).…”

“…15 mM, respectively) as selective inhibitors. 16,18,21) In the present study, we identified several potent inhibitors (IC 50 ϭ0.02-1 mM) after comprehensive screening of natural sources including a panel of randomly selected natural compounds (n ca. 2500), ca.…”

Inhibition of Plasma Hyaluronan-Binding Protein Autoactivation by Laccaic Acid

“…15) However, bikaverin, at concentrations slightly higher than the concentration which affected pro-PHBP autoactivation, inhibited the enzymatic activities of other serine proteases. 15) To identify a more selective inhibitor, we screened a panel of compounds related to bikaverin, and identified three anthraquinone compounds, purpurin, 15) carminic acid, 8) and a lac dye (a mixture of laccaic acids). In this report, we describe the action of laccaic acid.…”