Inactivation of human kininogen-derived antimicrobial peptides by secreted aspartic proteases produced by the pathogenic yeast Candida albicans.

Ten secreted aspartic proteases (Saps) of Candida albicans cleave numerous peptides and proteins in the host organism and deregulate its homeostasis. Human kininogens contain two internal antimicrobial peptide sequences, designated NAT26 and HKH20. In our current study, we characterized a Sap-catalyzed cleavage of kininogen-derived antimicrobial peptides that results in the loss of the anticandidal activity of these peptides. The NAT26 peptide was effectively inactivated by all Saps, except Sap10, whereas HKH20 was completely degraded only by Sap9. Proteolytic deactivation of the antifungal potential of human kininogens can help the pathogens to modulate or evade the innate immunity of the host.

Anticoagulation beyond direct thrombin and factor Xa inhibitors: indications for targeting the intrinsic pathway?

Antithrombotic drugs like vitamin K antagonists and heparin have been the gold standard for the treatment and prevention of thromboembolic disease for many years. Unfortunately, there are several disadvantages of these antithrombotic drugs: they are accompanied by serious bleeding problems, it is necessary to monitor the therapeutic window, and there are various interactions with food and other drugs. This has led to the development of new oral anticoagulants, specifically inhibiting either thrombin or factor Xa. In terms of effectiveness, these drugs are comparable to the currently available anticoagulants; however, they are still associated with issues such as bleeding, reversal of the drug and complicated laboratory monitoring. Vitamin K antagonists, heparin, direct thrombin and factor Xa inhibitors have in common that they target key proteins of the haemostatic system. In an attempt to overcome these difficulties we investigated whether the intrinsic coagulation factors (VIII, IX, XI, XII, prekallikrein and high-molecular-weight kininogen) are superior targets for anticoagulation. We analysed epidemiological data concerning thrombosis and bleeding in patients deficient in one of the intrinsic pathway proteins. Furthermore, we discuss several thrombotic models in intrinsic coagulation factor-deficient animals. The combined results suggest that intrinsic coagulation factors could be suitable targets for anticoagulant drugs.

Activation of the plasma kallikrein-kinin system on human lung epithelial cells.

Activation of the tissue kallikrein-kinin system (KKS) plays a major inflammatory role in the lung, but the contribution of the plasma KKS remains unclear. Plasma KKS involves assembly and activation of high molecular weight kininogen (HK) and plasma prekallikrein (PPK) on cell surfaces, resulting in the liberation of the inflammatory peptide, bradykinin (BK), from HK by plasma kallikrein (PK). To this end, we determined the possible contribution of plasma KKS in BK formation using airway epithelium. The HK binding proteins, urokinase plasminogen activator receptor, cytokeratin 1 and gC1qR, were expressed on transformed A549 and BEAS-2B cell lines, as well as on normal lung tissue, but Mac-1 was absent. A549 cells bound FITC-labelled HK, which was only partially inhibited by a combination of antibodies to the HK binding proteins. HK-PPK complex activation on the transformed epithelial cell lines, as well as primary epithelial cells, resulted in PK formation and liberation of BK. HK-PPK activation was inhibited by cysteine, BK and protamine, and by novobiocin, a heat shock protein 90 (HSP90) inhibitor. In summary, lung epithelial cells support the assembly and activation of the plasma KKS by a mechanism dependent on HSP90, and could contribute to KKS-mediated inflammation in lung disease.

Characterization of a tissue kallikrein inhibitor isolated from Bauhinia bauhinioides seeds: inhibition of the hydrolysis of kininogen related substrates.

Trypsin inhibitors were purified from a saline extract of Bauhinia bauhinioides seeds by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q ion-exchange chromatography or, alternatively, by affinity chromatography on trypsin-Sepharose. Both B. bauhinioides isolated inhibitors, BbTI-I and BbTI-II, inhibit trypsin being the dissociation constant 0.6 and 0.36 nM, respectively. BbTI-II only inhibits porcine pancreatic kallikrein hydrolysis of H-Pro-Phe-Arg-AMC (Ki 2.0 nM); the bradykinin-containing sequence LGMISLMKRPPGFSPFRSSRI-NH2 and the two kininogen related flanking quenched substrates Abz-MISLMKRP-EDDnp (Ki 2.0 nM) and Abz-FRSSRQ-EDDnp (Ki 2.5 nM).

[Kallikrein-kinin system in acute empyema of pleura and pyopneumothorax]. / Kallikrein-kininovaia sistema pri ostroi émpieme plevry i piopmevmotorakse.

In 56 patients with acute empyema of the pleura the level of aggregate kininogen and prekallikrein, free kallikrein and kininase activities in blood plasma and supernatant of pleural exudate were studied. It was established that epsilon-aminocapronic acid, contrykal and gordox suppress free kallikrein activity in the blood plasma and supernatant of pleural exudate. Intravenous and local application of proteolysis inhibitors resulted in improvement of the outcomes of the disease.

Identification of an endothelial cell binding site on kininogen domain D3.

High and low molecular mass kininogen, two multidomain plasma proteins, bind to endothelial cells, platelets, and neutrophils in the intravascular compartment. The specific cell attachment site on their common heavy chain is mediated by domain-3, a cystatin-like structure with inhibitory capacity for papain-like proteinases (Jiang, Y., Müller-Esterl, W., and Schmaier, A. H. (1992) J. Biol. Chem. 267, 3712-3717). In this report, the domain-3 cell binding site is determined by an antibody-directed strategy. The epitope of monoclonal antibody HKH15, which binds to domain-3 and blocks the binding of kininogens to platelets and endothelial cells, was mapped using seven synthetic peptides, which span the entire domain-3 sequence. One peptide, LDC27, specifically bound to HKH15. Fine mapping of the epitope of HKH15 revealed that a minimal 13-residue segment in LDC27, named CNA13, is the antibody binding site. LDC27 and CNA13 inhibited biotinylated high molecular mass kininogen binding to endothelial cells with apparent IC50 values of 60.3 +/- 12 and 113.3 +/- 63.7 microM, respectively. Carboxymethylated papain and affinity-purified anti-LDC27 polyclonal antibodies also inhibited the binding of biotinylated high molecular mass kininogen to endothelial cells with an apparent IC50 of 1.04 microM and 59 nM, respectively. Biotinylated LDC27 itself directly bound to endothelial cells, and domain-3 inhibited biotinylated LDC27 binding to human umbilical vein endothelial cells with an IC50 of 41 nM. Using the crystalline structure of cystatin to computer model domain-3, LDC27 and CNA13 were located in the second hairpin loop of the reactive site of cystatin-like proteins (Bode, W., Engh, R., Musil, D., Thiele, U. Huber, R., Karshikov, A., Brzin, J., Kos, J., and Turk, V. (1988) EMBO J. 7, 2593-2599). These results indicate that the major endothelial cell attachment site on kininogen domain-3 is located on its carboxyl-terminal portion and that it overlaps its cysteine protease inhibitory region.

Effect of microbial and mite proteases on low and high molecular weight kininogens. Generation of kinin and inactivation of thiol protease inhibitory activity.

Kinin release from guinea pig plasma high molecular weight kininogen (HMWK) induced by various microbial and mite proteases has been demonstrated previously (Molla, A., Yamamoto, T., Akaike, T., Miyoshi, S., and Maeda, H. (1989) J. Biol. Chem. 264, 10589-10594; Maruo, K., Akaike, T., Matsumura, Y., Kohmoto, S., Inada, Y., Ono, T., Arao, T., and Maeda, H. (1991) Biochim. Biophys. Acta 1074, 62-68). In this paper, we describe the effects of various microbial and mite proteases on low molecular weight kininogen (LMWK) and HMWK from human plasma. A protease from the house dust mite Dermatophagoides farinae (Df-protease) directly liberated kinin from both LMWK and HMWK to a significant degree. The Km, kcat, and kcat/Km values for kinin generation from LMWK were 3.24 microM, 0.61 s-1, and 1.9 x 10(5) M-1 x s-1, respectively, and those for kinin generation from HMWK were 0.56 microM, 0.12 s-1, and 2.1 x 10(5) M-1 x s-1, respectively; kcat/Km values for Df-protease were comparable with that for glandular kallikrein. In contrast, microbial proteases showed only weak kinin-releasing activity from both human plasma kininogens. Four of ten different microbial proteases liberated kinin from LMWK, and only serratial 56-kDa protease released kinin from HMWK. Furthermore, Df-protease markedly inactivated the thiol protease inhibitory activity of LMWK and HMWK, whereas all microbial proteases (as well as the endogenous protease trypsin) did not affect this inhibitory activity of both kininogens from human plasma.

[Effect of hydrochlorothiazide on various components of the kinin system and plasma renin activity in patients with arterial hypertension treated with captopril]. / Wplyw hydrochlorotiazydu na niektóre skladniki ukladu kinin oraz aktywnosc reninowa osocza u chorych na nadcisnienie tetnicze leczonych kaptoprylem.

Combined hypertension treatment with inhibitors of the converting enzyme (ICE) and diuretocs gives manifold advantages, the most important of them is a synergistic action of both drugs resulting in blood pressure decrease and prevention of hypokaliaemia. The purpose of the present study was assessment of the effect of hydrochlorothiazide in 50 mg daily doses on blood levels of kininogen and prekallikrein and on plasma renin activity (PRA) in hypertensive patients treated during captopril 150 mg daily alone during 3 weeks, without success. The study was carried out on 15 patients with essential hypertension stage III after WHO. A control group comprised 18 healthy subjects. The determinations were done three time: before treatment, after 3 weeks on captopril, and after 2 weeks of combined treatment with captopril and hydrochlorothiazide. It was found combined treatment with captopril and hydrochlorothiazide produced blood pressure normalization in patients insufficiently responding to captopril alone. During the combined treatment normalization of blood pressure was associated with a higher fall of kininogen concentration, higher rise of prekallikrein and PRA in relation to the values obtained with captopril alone. This evidenced a more pronounced reaction of the kinin system and the renin-angiotensin-aldosterone system to the combined treatment as compared with the response of both systems to captopril alone.

High molecular weight kininogen binds to platelets by its heavy and light chains and when bound has altered susceptibility to kallikrein cleavage.

The unstimulated platelet surface contains a specific and saturable binding site for high molecular weight kininogen (HK) and low molecular weight kininogen (LK). Investigations were performed with purified heavy and light chains of HK to determine which portion(s) of the HK molecule binds to the platelet surface. Purified 64-Kd heavy chain of HK and 56-Kd light chain of HK, independently, inhibited 125I-HK binding to unstimulated platelets with a 50% inhibitory concentration (IC50) of 84 nmol/L (apparent Ki, 30 nmol/L) and 30 nmol/L (apparent Ki, 11 nM), respectively. The ability of each of the purified chains of HK to independently inhibit 125I-HK binding was not due to cleavage, reduction, and alkylation of the protein, because two-chain HK, produced by treating HK the same way as purifying the separate chains, inhibited binding similarly to intact HK. Further, purified LK alone inhibited 125I-HK binding to platelets (Ki, 17 +/- 1 nmol/L, n = 7). The 64-Kd heavy chain of HK was a competitive inhibitor on a reciprocal plot of 125I-HK-platelet binding with an apparent Ki of 28 +/- 6 nmol/L (n = 4). Independently, purified 56-Kd light chain of HK was also found to be a competitive inhibitor of 125I-HK-platelet binding, with an apparent Ki of 11 +/- 3 nmol/L (mean +/- SEM, n = 4). These indirect studies indicated that HK binds to platelets by two portions of the molecule, one on the heavy chain and another on the light chain. Studies with 125I-light chain of HK showed that it specifically bound directly to platelets in the presence of zinc, since it was blocked by HK, light chain of HK, or EDTA, but not by LK, C1s, C1 inhibitor, plasmin, factor XIII, or fibrinogen. Purified light chain of HK did not inhibit direct 125I-LK binding to platelets. HK was found to bind to platelets in an unmodified form. HK bound to platelets was cleaved by plasma or urinary kallikrein at a slower rate than the same concentration of soluble HK or HK bound and subsequently eluted from the platelet surface. Cleavage of platelet-bound HK correlated with bradykinin liberation. These studies indicate that HK has two domains on its molecule that bind to platelets. Further, platelet-bound HK is protected from kallikreins' proteolysis. This latter finding suggests that cell binding may modify the rate of bradykinin liberation from HK.

Discrimination between rat thiostatin (T-kininogen) and one of its cystatin-like inhibitory fragments by a monoclonal antibody, and localization of the epitope.

A monoclonal antibody (mAb D3) raised against rat thiostatin (T-kininogen) strongly interacted with a fragment, identified as cystatin-like domain 3, which inhibits cysteine proteinases but did not recognize intact, native thiostatin. The antigen-antibody reaction requires cleavage of the single peptide chain of thiostatin in its inter-domain 2-3 region. This mAb can also differentiate between the two molecular varieties of thiostatin, reacting only with immobilized domain 3 from T1 thiostatin, which differs from the T2 variety by only 10 out of 125 residues. mAb D3 did not react with an N-terminally truncated domain 3 of T1 thiostatin prepared by submaxillary gland kallikrein k10 proteolysis. This suggests that the epitope, or an essential part of it, is located on a stretch of 12 residues at the N-terminal of the T1 thiostatin domain 3. This sequence in T1 thiostatin differs from that in T2 thiostatin by four amino acids, two of which are arginyl residues in T1. Chemical modification of these residues located at positions 246 and 250 decreased the reactivity of T1 domain 3 towards the antibody, suggesting that at least one of them is a critical residue of the epitope. Arginine 246 is part of a small disulfide loop between cysteines 245 and 248 which is also necessary for antibody recognition. This antibody does not change the inhibitory properties of purified domain 3 towards papain or rat liver cathepsin L, indicating that the N-terminal part of domain 3 is not involved in inhibition. mAb D3 was used to demonstrate the presence of inhibitory thiostatin fragments in ascites fluid but not in plasma from normal or turpentine-injected rats.

Neutralization of kinin-releasing enzymes of crotalid venoms by monospecific and polyspecific antivenoms.

The amounts of kinin-releasing enzymes in the venoms of Crotalus atrox, Crotalus adamanteus, Crotalus scutulatus scutulatus and Agkistrodon piscivorus piscivorus were measured by determining the amounts of kinin released from a sheep kininogen substrate by means of a specific radioimmunoassay. Four monospecific and two commercial polyspecific antivenom IgG samples were tested for their ability to reduce the kinin-releasing activities of the four crotalid venoms measured in vitro. All of the antivenom IgG samples were able to neutralize venom kininogenase activity to varying extents. On of the commercial polyspecific antivenoms was of equal or higher potency than the corresponding monospecific antivenoms for three of the venoms tested, indicating a high degree of cross-neutralization. F(ab')2 and especially Fab fragments of that polyspecific antivenom IgG were also effective in reducing the kinin-releasing activities of the four crotalid venoms.

[Effect of captopril on various components of the kinin system and plasma renin activity in patients with idiopathic arterial hypertension]. / Wplyw kaptoprylu na niektóre skladniki ukladu kinin oraz aktywnosc reninowa osocza u chorych na nadcisnienie tetnicze samoistne.

Authors studied effect of captopril on serum kininogen and prekallikrein concentrations, as well as on plasma renin activity (PRA) in patients with primary hypertension. The control group consisted of 18 healthy persons, 5 patients were in I, 12 in II and 8 in III WHO class. Monotherapy with 150 mg per day of captopril had been performed for 3 weeks. Patients were 3 times examined: 1--before therapy, 2--after 24 hours of treatment, 3--after 3 weeks of captopril therapy. It was proved, that captopril lowers arterial pressure with coexisting PRA increase and induces changes in kinins system such as: decrease of kininogen+ concentration and increase of serum prekallikrein level in comparison with their pretreatment values. Maximum PRA increase and blood pressure decrease were observed after 24 hours of captopril administration, whereas changes in kinins system were taking place during the whole observation period. Presented studies indicate that antihypertensive action of captopril is related to Renin-Angiotensin-Aldosterone System as well as to plasma kinins one.

Significant effects of Z-Gln-Val-Val-OME, common sequences of thiol proteinase inhibitors on thiol proteinases.

The first studies on a series of the small synthetic thiol proteinase inhibitors, conservative common sequences in several thiol proteinase inhibitors, are described. Among the many interesting findings with synthetic thiol proteinase inhibitors was the observation that the most effective analogue, Z-Gln-Val-Val-Ala-Gly-OMe, whose amino and carboxyl groups were protected with Z and OMe, respectively, showed inhibitory activity on papain and cathepsin B and protected papain from egg cystatin, human low-molecular-weight kininogen and T-kininogen-induced inhibition but not from leupeptin-induced inhibition. Moreover, it was revealed that Z-Gln-Val-Val-OMe was the smallest peptide to exhibit a protective effect on papain.

The relationship between rat major acute phase protein and the kininogens.

The rat major acute phase protein (alpha 1-MAP) is a cysteine protease inhibitor. The stoichiometry of the interaction between the inhibitor and enzyme was shown to be 1:2. A cDNA clone specific for rat alpha 1-MAP was isolated from a cDNA library prepared from an inflamed rat liver RNA template. The 1458-base pair insert was sequenced and positively identified by alignment with a partial amino acid sequence obtained by radiosequence analysis of the primary translation product for alpha 1-MAP. Complete sequence analysis determined the alpha 1-MAP cDNA coded for the entire protein with the exception of the first four amino acids of the signal peptide, all of which were identified by radiosequencing. The coding sequence spans 1282 nucleotides, followed by 115 base pairs of a 3' untranslated region. Two putative active sites, suggested by the enzyme-inhibitor ratio, have been identified by analysis of internal duplications of the alpha 1-MAP sequence and the alignment of these regions with the sequences of several low molecular weight cysteine protease inhibitors. A computer homology analysis of the protein sequence revealed a 59.3% overall identity between rat alpha 1-MAP and bovine low molecular weight (LMW) kininogen. The homology included the signal peptide regions. LMW kininogen is a precursor of bradykinin. alpha 1-MAP does contain a bradykinin sequence; the flanking amino acids are different, however. Evidence for the expression of the LMW and a high molecular weight kininogen from the same gene, and the high degree of homology between these proteins and the rat acute phase protein suggest that all three proteins belong to a precisely regulated gene family.

Reduced cofactor function of human high molecular weight kininogen induced by human plasma kallikrein.

Most reports in the literature state that human plasma kallikrein does not destroy the capacity of human high molecular weight kininogen (HMrK) to function as a cofactor in the contact phase activation of factor XII. In the present work preparations of highly purified human plasma kallikrein that showed high plasminogen activator (PGA) activities rapidly reduced the cofactor function of human HMrK. Gel electrophoresis with SDS without reduction showed that all kallikrein preparations tested contained two protein bands, one major band with a Mr of about 83,000, and one weak band with a Mr of 80,000. The main band is probably identical with kallikrein I, which Levison & Tomalin (1982b), using Ac-Pro-Phe-Arg-OMe-HCl as substrate, found to be ten times more active (in terms of kcat/Km) than kallikrein II with Mr 3000 daltons lower. The rate of HMrK destruction in our experiments varied with the kallikrein preparation used, but assays of their hydrolytic activities against benzoyl arginine ethylester (BAEe) or the plasma kallikrein selective tripeptide substrate H-D-Pro-Phe-Arg-pNA (S-2302) did not discriminate between enzyme preparations with different HMrK-destroying capacities. Assay of PGA activities demonstrated a correlation between the level of PGA measured, and the HMrK-destroying capacity.

Inactivation of human high molecular weight kininogen by human mast cell tryptase.

Tryptase, the major neutral protease of human pulmonary mast cell secretory granules, rapidly inactivates human high m.w. kininogen (HMWK) in vitro. HMWK (5600 nM) lost 50% of its capacity to release kinin in response to kallikrein after a 5-min incubation with tryptase (31 nM), even though kinin activity was neither generated nor, when bradykinin was incubated with tryptase, destroyed by tryptase. The procoagulant activity of HMWK (51 nM) and the purified procoagulant chain (40 nM) that is derived from HMWK were each 72% inactivated after 7 min of incubation with tryptase (0.04 nM and 0.02 nM, respectively). Human urinary and pancreatic kallikrein did not inactivate this procoagulant activity under conditions in which kinin generation occurs. Complete cleavage of native single-chain HMWK by tryptase occurred in less than 10 min as analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide slab gels. The major products formed during the initial 2 min were proteins of 100,000 and 95,000 apparent m.w., and by 10 to 30 min were fragments of 74,000 and 67,000 apparent m.w. Reduction of these cleavage products yielded two major fragments of 67,000 and 66,000 apparent m.w. that were both present by 0.17 min. The presence of lower m.w. products, thought to be primarily from the carboxy-terminal procoagulant region of HMWK, were also detected with and without reduction. The capacity of tryptase to inactivate HMWK is consistent with the ability of other mast cell-derived mediators, such as heparin proteoglycan and prostaglandin D2, to suppress blood coagulation and thrombosis, and may play an important role in the biology of mast cell-dependent events in vivo.

Activation of factor XII in plasma from rats pretreated with tranexamic acid. Inhibition of a plasmin-induced loss of the functional activity of high molecular weight kininogen.

Incubation of plasma from rats pretreated with tranexamic acid (40 mg/100 g) with acetone (23% V/V) yielded enzyme preparations in which all the plasminogen present was recovered as plasmin and a plasmin-like substance without affinity for lysine-Sepharose. This substance, designated "plasmin", was separated from plasmin and kallikrein in a three-step procedure using columns of lysine-Sepharose, DEAE-Sephadex A-50, and arginine-Sepharose. The ratios of fibrinolytic, caseinolytic, LEe esterase, BAEe esterase and kininogenase activities of "plasmin" corresponded well with those of rat plasmin and human plasmin. Both rat plasmin and "plasmin" destroyed the capacity of high molecular weight kininogen (HMWK) to function as a cofactor in the activation of factor XII in rat plasma, without causing a corresponding release of the kinin part of the molecule. Rat plasma kallikrein induced full release of kinin from HMWK, but the functional capacity was retained. It is suggested that the reduced extent of activation of factor XII observed in plasma from rats injected intravenously with dextran, or rat plasma that has been passed through a column with lysine-Sepharose, is due to the loss of functional HMWK caused by plasmin activated in vivo or on the column.