Activation of proPHBSP, the Zymogen of a Plasma Hyaluronan Binding Serine Protease, by an Intermolecular Autocatalytic Mechanism

Abstract

The hyaluronic acid binding serine protease (PHBSP), an enzyme with the ability to activate the coagulation factor FVII and the plasminogen activator precursors and to inactivate factor VIII and factor V, could be isolated from human plasma in the presence of 6M urea as a single-chain zymogen, whereas under native conditions only its activated two-chain form was obtained. The total yield of proenzyme (proPHBSP) was 5-6 mg/l, corresponding to a concentration of at least 80-100nM in plasma. Upon removal of urea, even in the absence of charged surfaces a rapid development of amidolytic activity was observed that correlated with the appearance of the two-chain enzyme. The highest activation rate was observed at pH 6. ProPHBSP processing was concentration-dependent following a second order kinetic and was accelerated by catalytic amounts of active PHBSP, indicating an intermolecular autocatalytic activation. Charged macromolecules like poly-L-lysine, heparin, and dextran sulfate strongly accelerated the autoactivation, suggesting that in vivo proPHBSP activation might be a surface-bound process. The intrinsic activity of the proenzyme was determined to be 0.25-0.3%, most likely due to traces of PHBSP. The presence of physiological concentrations of known plasma inhibitors of PHBSP, like alpha2 antiplasmin and C1 esterase inhibitor, but not antithrombin III/heparin, slowed down zymogen processing. Our in vitro data suggest that the autoactivation of proPHBSP during plasma fractionation is induced by the removal of inhibitors of PHBSP and is accelerated by charged surfaces of the chromatographic resins.

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

“…Materials 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.…”

“…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.…”

The Hyaluronan-Binding Serine Protease from Human Plasma Cleaves HMW and LMW Kininogen and Releases Bradykinin

“…A total concentration of 12 µg/ml (200 nM) PHBSP in plasma was determined by quantitative ELISA (Römisch et al, 2001). The proenzyme undergoes an intermolecular autocatalytic activation by cleavage at an internal Arg 290 -Ile bond (Etscheid et al, 2000) resulting in an N-terminal 48 kDa heavy chain and a C-terminal 30 kDa light chain held together by a single disulfide bond. PHBSP activity and proenzyme activation in vitro are effectively inhibited by plasma serpins, especially α 2 -antiplasmin and C1-esterase inhibitor.…”

Inhibition of Plasma Hyaluronan-Binding Protein Autoactivation by Laccaic Acid

“…4) Although activation of pro-PHBP in vivo is observed under inflammatory conditions, 5,6) no physiologically relevant enzyme responsible for pro-PHBP has been found. Alternatively, negatively charged molecules (such as heparin and RNA) and positively charged molecules (such as polyamines) dramatically promote pro-PHBP autoactivation.…”

“…Figure 1 shows some of the results of a screening of compounds structurally related to bikaverin. In this screening, the compound to be tested and pro-PHBP (5 nM), isolated from human plasma, 4,8) were incubated with spermidine (5 mM) and 0.1 mM of the substrate SPECTROZYME Ò TH (STH) in buffer A (50 mM Tris-HCl, pH 7.4, 75 mM NaCl, 5 mM CaCl 2 , and 0.05% w/v Tween 20) during monitoring A 405 at 37 C. In this assay, lac dye showed potent inhibitory activity (IC 50 = 0.65 mg/ml), along with related anthraquinones, carminic acid (IC 50 = 7.4 mg/ml) and purpurin (IC 50 = 1.7 mg/ml). Other quinone compounds tested were inactive (Fig.…”