Tissue Kallikrein Inhibitor Research Articles

Design of kallidin-releasing tissue kallikrein inhibitors based on the specificities of the enzyme's binding subsites.

The tissue kallikrein inhibitors reported in the present work were derived by selectively replacing residues in Nalpha-substituted arginine- or phenylalanine-pNA (where pNA is p-nitroanilide), and in peptide substrates for these enzymes. Phenylacetyl-Arg-pNA was found to be an efficient inhibitor of human tissue kallikrein (Ki 0.4 microM) and was neither a substrate nor an inhibitor of plasma kallikrein. The peptide inhibitors having phenylalanine as the P1 residue behaved as specific inhibitors for kallidin-releasing tissue kallikreins, while plasma kallikrein showed high affinity for inhibitors containing (p-nitro)phenylalanine at the same position. The Ki value of the most potent inhibitor developed, Abz-Phe-Arg-Arg-Pro-Arg-EDDnp [where Abz is o-aminobenzoyl and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine], was 0.08 microM for human tissue kallikrein. Progress curve analyses of the inhibition of human tissue kallikrein by benzoyl-Arg-pNA and phenylacetyl-Phe-Ser-Arg-EDDnp indicated a single-step mechanism for reversible formation of the enzyme-inhibitor complex.

Recombinant hirustasin: production in yeast, crystallization, and interaction with serine proteases.

A synthetic gene coding for the 55-amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast alpha-factor leader sequence and expressed in Saccharomyces cerevisiae. Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease. The processed hirustasin was purified to better than 97% purity. N-terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N-terminus and the expected amino acid sequence and molecular mass. The biological activity of recombinant hirustasin was identical to that of the authentic leech protein. Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 A and 2.4 A, respectively. In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor. During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 31, the putative reactive site, to yield a modified inhibitor. In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly. Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 31. Hirustasin appears to fall into the class of protease inhibitors displaying temporary inhibition.

Mechanism of irritant-induced cough: studies with a kinin antagonist and a kallikrein inhibitor.

It has been suggested that bradykinin may play a role in stimulating cough in at least one pathological condition in humans. We have employed an animal model to investigate the possible role of this peptide in irritant-induced cough. The kinin antagonist Hoe 140 and codeine both produced dose-related inhibition of cough responses to inhalation of citric acid or bradykinin aerosols by conscious guinea pigs. The selective tissue kallikrein inhibitor CH694 inhibited cough caused by citric acid but not by bradykinin. Indomethacin pretreatment attenuated the responses to both stimuli as did phosphoramidon. It is concluded that cough produced by citric acid inhalation may be mediated, at least in part, by generation of kinins; secondary to this, a release of prostanoids also appears to participate in the response.

Kallistatin, a novel human tissue kallikrein inhibitor: Levels in body fluids, blood cells, and tissues in health and disease

Kallistatin, a human serine proteinase inhibitor, is a newly identified tissue kallikrein inhibitor. It binds strongly to tissue kallikrein but weakly to other serine proteinases such as chymotrypsin and elastase. The tissue distribution and changes in kallistatin levels in human diseases were characterized by using specific monoclonal and polyclonal antibodies against kallistatin. Kallistatin antigen levels in blood cells, fluids, and tissues measured with a specific enzyme-linked immunosorbent assay showed displacement curves that were parallel with those in purified kallistatin, indicating their immunologic identity. Expression of kallistatin mRNA in platelets, neutrophils, lymphocytes, monocytes, endothelial cells, hepatocytes, and colon and prostate carcinoma cells was identified by reverse transcription-polymerase chain reaction followed by Southern blot analysis. Plasma kallistatin concentration was 22.1 ± 3.5 μg/ml in 30 normal subjects and 21.1 ± 3.8 μg/ml in 5 patients with C1 inhibitor deficiency. A significantly reduced kallistatin level (7.2 ± 2.5 μg/ml, p < 0.001) was seen in plasma samples from 9 patients with liver disease and 10 patients with sepsis (7.7 ± 3.5 μg/ml, p < 0.001). Further, kallistatin levels in 10 women taking oral contraceptives (19.8 ± 3.8 μg/ml) and 21 pregnant women (14.9 ± 3.3 μg/ml) were significantly lower than those seen in healthy individuals. These data suggest that kallistatin is found in plasma, is produced mostly in the liver, and can be consumed during sepsis. Its consumption in sepsis may indicate a protective role to prevent blood pressure lowering.

Pharmacological characterization of novel tissue kallikrein inhibitors in vivo

In this study we have investigated the effect of novel tissue kallikreins on the plasma protein exudation induced by porcine pancreatic kallikrein (PPK) in the rabbit skin in vivo. The tissue kallikrein inhibitors here described were synthesized based on analogues of peptide substrates for tissue kallikreins. The intradermal injection of PPK and rabbit urinary kallikrein, but not of rabbit plasma kallikrein, significantly increased the microvascular permeability leading to local oedema formation in the rabbit skin. At the dose of 3–200 nmol/site, the intradermal co-administration of the tissue kallikrein inhibitors Bz-F-F-S-R-EDDnp (Ki = 0.1 μM; ESP5), PAC-F-S-R-EDDnp (Ki = 0.7 μM; ESP6), Bz-F-F-A-P-R-NH2 (Ki = 7.8 μM; ESP8), PAC-F-F-R-P-R-NH2 (Ki = 0.3 μM; ESP9) and Bz-F-F-S-R-NH2 (Ki = 0.3 μM; ESP11) dose-dependently inhibited the plasma protein exudation induced by PPK. The most potent compound was ESP6 (IC25 = 7.8 nmol/site) followed by ESP5 (IC25 = 14.2 nmol/site), ESP8 (IC25 = 25 nmol/site), ESP9 (IC25 = 30 nmol/site) and ESP11 (IC25 = 50.4 nmol/site). The compounds Bz-F-F-R-P-R-NH2 (Ki = 0.5 μM; ESPI), Bz-F-F-pNa (Ki = 0.4 μM; ESP3), Bz-F(NH2)-F-R-P-R-NH2 (Ki = 1.1 μM; ESP7) and Bz-F-F-S-P-R-NH2 (Ki = 4.6 μM; ESPI0) had no significant effect on the PPK-induced plasma protein exudation in doses up to 200 nmol/site. ESP6 also inhibited the PPK-induced plasma protein exudation when administered systemically. This compound may constitute a useful tool to further investigate both the physiological and pathological role of tissue kallikreins.

Tissue kallikrein inhibitors in mammals

We have discovered, purified and cloned a new kallikrein-binding protein (KBP or kallistatin) from humans and rodents. Kallistatins are members of the serine proteinase inhibitor (serpin) superfamily. They are acidic glycoproteins with molecular masses of 58–62 kDa and pI values of 4.6–5.2. Kallistatin forms a SDS-stable complex with tissue kallikrein and inhibits kallikrein's activities. Human kallistatin has a unique cleavage site with Phe-Phe-Ser at the P2-P1-P-1 positions. The protein sequence of mature human kallistatin shares 44–46% identity with other serpins such as human α1-antitrypsin, protein C inhibitor and rat kallikrein-binding protein. The kallistatin genes display the typical five exon-four intron serpin gene structure. The human kallistatin gene is localized on chromosome 14q31–32.1 and the RKBP gene is on chromosome 6. Kallistatin is evolutionarily diverse but functionally conserved in mammalian species. This overview summarizes the biochemistry, molecular biology and potential physiology and/or pathophysiology of this new tissue kallikrein inhibitor.

Synthetic inhibitors of human tissue kallikrein

We have developed a series of novel, potent low molecular weight (4-600 Da) inhibitors of TK which were stable to cleavage by the enzyme and showed a high degree of selectivity for TK over several other serine proteases. Compound 7 (CH-2856) was found to reduce eosinophilia in a model of allergic inflammation. The effects observed in vivo provide further evidence for the involvement of TK and kinins in the pathophysiology of allergic diseases such as asthma.

High level expression of human tissue kallikrein in the circulation induces hypotension in transgenic mice

In order to create an animal model expressing a high level of tissue kallikrein in the circulation, the human tissue kallikrein gene was placed under the control of a mouse albumin enhancer and promoter to target its expression to liver. Three lines of transgenic mice carrying the human tissue kallikrein gene were established. The major site of human tissue kallikrein synthesis was identified in the liver of transgenic mice, and a high level of human tissue kallikrein was secreted into the mouse circulation. The systolic blood pressures of these transgenic mice are about 15–20 mmHg lower than that of the control mice. Administration of aprotinin, a potent tissue kallikrein inhibitor, restored normal blood pressure in these animals. These studies show that a high level of foreign tissue kallikrein in the circulation plays a role in blood pressure regulation

Functional analysis of human tissue kallikrein in transgenic mouse models.

Clinical studies show that an inverse correlation exists between blood pressure and urinary kallikrein levels. It has been postulated that the tissue kallikrein-kinin system contributes to the maintenance of normal blood pressure. To test this hypothesis, we have established transgenic mice that overexpress human tissue kallikrein under the promoter control of the mouse metallothionein gene and a liver-targeted albumin gene. These animals secrete human tissue kallikrein in plasma at levels 10- to 40-fold higher than that found in normal human serum, and they are chronically hypotensive. This hypotensive effect can be reversed by the injection of aprotinin, a potent tissue kallikrein inhibitor, or Hoe 140, a specific bradykinin receptor antagonist. Transgenic mice overexpressing human tissue kallikrein show a sustained reduction in blood pressure throughout their life spans, indicating the lack of sufficient compensatory mechanisms to reverse the hypotensive effect of kallikrein. Somatic gene delivery of rat kallikrein-binding protein by muscle injection increases the blood pressure of the hypotensive transgenic mice to levels comparable with those in normotensive control mice. These results indicate that a direct link exists between kallikrein gene expression and alterations in blood pressure. In addition, we have developed normotensive transgenic mice that harbor the human tissue kallikrein gene containing 801 bp of its native promoter. The tissue distribution pattern of human kallikrein in these transgenic mice is similar to that in human tissues, with the highest level in the pancreas and much lower levels in the kidney and salivary gland. These transgenic mice provide new animal models for investigating the tissue-specific regulation of tissue kallikrein and its role in altering blood pressure.

High level of circulating human tissue kallikrein induces hypotension in a transgenic mouse model.

We established a unique transgenic mouse model in liver-targeted expression of human tissue kallikrein using a mouse albumin enhancer and promoter. Northern blot analysis and ELISA showed that human tissue kallikrein was predominantly expressed in the liver of transgenic mice and secreted into the circulation at a high level. The transcript was also detected in the kidney, pancreas, salivary gland and heart at a low level by reverse transcription-polymerase chain reaction followed by Southern blot analysis. Systolic blood pressures were measured by the tail-cuff method, all three independent transgenic mouse lines are hypotensive (84.6 +/- 1.0 mmHg, n = 17; 84.5 +/- 1.5 mmHg, n = 9; 83.1 +/- 0.8 mmHg, n = 13, P < 0.01) compared with the control mice (100.9 +/- 0.9 mmHg, n = 17). Administration of aprotinin, a potent tissue kallikrein inhibitor or Hoe 140, a bradykinin receptor antagonist, restored the blood pressure of transgenic mice but had no significant effect on control littermates. These studies show that over-production of tissue kallikrein in the circulation plays a role in blood pressure regulation.

Direct gene delivery of human tissue kallikrein reduces blood pressure in spontaneously hypertensive rats.

Hypertension is a multigene and multifactorial disorder affecting approximately 25% of the population. To demonstrate potential therapeutic effects of human tissue kallikrein in hypertension, spontaneously hypertensive rats were subjected to somatic gene therapy. Two human tissue kallikrein DNA constructs, one under the promoter control of the metallothionein metal response element and the other under the control of the Rous sarcoma virus 3'-LTR, were generated. We delivered naked DNA constructs into spontaneously hypertensive rats via intravenous injection. The expression of human tissue kallikrein in rats was identified in the heart, lung, and kidney by reverse transcription polymerase chain reaction followed by Southern blot analysis and an ELISA specific for human tissue kallikrein. A single injection of both human kallikrein plasmid DNA constructs caused a sustained reduction of blood pressure which began 1 wk after injection and continued for 6 wk. A maximal effect of blood pressure reduction of 46 mmHg in rats was observed 2-3 wk after injection with kallikrein DNA as compared to rats with vector DNA (n = 6, P < 0.05). The hypotensive effect caused by somatic gene delivery of human tissue kallikrein in hypertensive rats is reversed by subcutaneous injection of aprotinin, a potent tissue kallikrein inhibitor. No antibodies to either human tissue kallikrein or kallikrein DNA were detected in rat sera after injection of the human kallikrein gene. These results show that direct gene delivery of human tissue kallikrein causes a sustained reduction in systolic blood pressure in genetically hypertensive rats and indicate that the feasibility of kallikrein gene therapy for treating human hypertension should be studied.

The carboxyl terminus of bradykinin and amino terminus of the light chain of kininogens comprise an endothelial cell binding domain.

Bradykinin (BK), a potent vasoactive nonapeptide formed by proteolytic cleavage of kininogen, mediates its activity by binding to specific cell surface receptors. Delivery of BK to these receptors is limited by cell-bound and plasma kininases that degrade BK to inactive fragments. Binding of its parent compound, kininogen, to specific endothelial cell receptors may provide an environment in which the degradation of BK by kininases is restricted. The determinants that mediate kininogen binding to endothelial cells have not been fully elucidated. The present studies suggest that part of BK and the amino-terminal amino acids of kininogens' common light chain constitute part of this recognition sequence. Human umbilical vein endothelial cells (HUVEC) in culture at 37 degrees C express 2-3-fold more binding sites for biotinylated high molecular weight kininogen (biotin-HK) containing BK than for biotin-HK from which BK has been liberated by plasma kallikrein. Binding of BK-free biotin-HK was not restored by preincubating HUVEC with BK, arguing against the possibility that BK released from biotin-HK stimulated expression of additional HK receptors. The extent of biotin-HK binding to HUVEC at 37 degrees C directly correlated with the amount of BK retained within the protein. Four lines of evidence suggest that part of BK and the amino terminus of the light chain of kininogens are part of the sequence recognized by the endothelial cell kininogen receptor. First, monoclonal antibodies to the carboxyl terminus of BK (MBK3) and the common light chains of the kininogens (HKL6, HKL8) inhibited biotin-HK binding to HUVEC. Second, a synthetic, dimeric bradykinin receptor antagonist blocked biotin-HK (IC50 = 9 microM) binding to HUVEC as did two synthetic tissue kallikrein inhibitors modeled after the carboxyl-terminal sequence of BK. Third, synthetic peptides containing the carboxyl-terminal portion of BK and the amino terminus of kininogens' common light chain, MKRPPGFSPFRSSRIG and GFSPFRSSRIG, blocked binding of biotin-HK (IC50 = 2-3 mM), whereas an overlapping peptide, SPFRSSRIGEIKEETT, at 5 mM and a scrambled peptide, FSGPKRSPIMGRPSFR, did not. Fourth, biotinylated GMKRPPGFSPFRSSRIG specifically bound to HUVEC, and its binding was blocked by HK. Since the presence of the nonapeptide BK in HK contributes to maximal binding of HK to HUVEC, there is a novel type of BK receptor in which part of the nonapeptide is recognized within the context of its parent molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacological characterization of rabbit corpus cavernosum relaxation mediated by the tissue kallikrein-kinin system.

1. The roles of the tissue kallikrein-kinin system and nitric oxide (NO) release in Phoneutria nigriventer venom-induced relaxations of rabbit corpus cavernosum (RbCC) smooth muscle have been investigated by use of a bioassay cascade. 2. Phoneutria nigriventer venom (10-30 micrograms), porcine pancreatic kallikrein (100 mu), rabbit urinary kallikrein (10 mu), bradykinin (BK, 0.3-3 nmol), acetylcholine (ACh, 0.3-30 nmol) and glyceryl trinitrate (GTN, 0.5-10 nmol) caused relaxations of the RbCC strips. Captopril (1 microM) substantially potentiated Phoneutria nigriventer venom- and BK-induced RbCC relaxations without affecting those elicited by GTN. 3. The bradykinin B2 receptor antagonist, Hoe 140 (D-Arg-[Hyp3,Thi5,D- Tic7,Oic8]-BK, 50 nM), aprotinin (10 micrograms ml-1) and the tissue kallikrein inhibitor, Pro-Phe-Aph-Ser-Val- Gln-NH2 (KIZD-06, 1.3 microM) significantly inhibited Phoneutria nigriventer venom-induced RbCC relaxations, without affecting those provoked by GTN and ACh. The B1 receptor antagonist, [Leu9]des Arg10BK (0.5 microM) and soybean trypsin inhibitor (SBTI, 10 micrograms ml-1) had no effect on Phoneutria nigriventer venom-induced RbCC relaxations. 4. The relaxations induced by Phoneutria nigriventer venom, porcine pancreas kallikrein, BK and ACh were significantly inhibited by N omega-nitro-L-arginine methyl ester (L-NAME, 10 microM) but not by D-NAME (10 microM). L-NAME did not affect GTN-induced relaxations. L-Arginine (300 microM), but not D-arginine (300 microM), significantly reversed the inhibitory effect of L-NAME. 5. Our results indicate that Phoneutria nigriventer venom activates the tissue kallikrein-kininogen-kinin system in RbCC strips leading to NO release and suggest a functional role for this system in penile erection.

Activation of serpins and their cognate proteases in muscle after crush injury

Direct muscle injury was induced in rats in order to evaluate alterations in the balance of serine proteases and inhibitors (serpins) as a response to tissue damage. It was previously found that certain proteases, specifically urokinase-like plasminogen activator (uPA) and others, required activation in order to effect regeneration. We hypothesized that the magnitude and temporal sequence of serpin activation would follow, pari passu, activation of their cognate proteases. In addition to uPA, tissue PA (tPA) and tissue kallikrein were the proteases studied. The serpins we analyzed were protease nexin I (PNI), PA inhibitor 1 (PAI-1, and the kallikrein-binding protein (KBP). uPA nearly doubled 48 h after injury, while there was no change in amidolytic activity after addition of fibrin monomer as an estimation of tPA activity. Tissue kallikrein activity, barely detectable in normal muscle, slowly increased, nearly tripling at 7 days after injury. Greater magnitude and more rapid changes in muscle serpins occurred over the same post-injury time course. By 24 h PNI increased threefold, while PAI-1 increased more slowly, reaching double the control values by 5 days after injury. Surprisingly, KBP, the serpin-class inhibitor of tissue kallikrein, had the most robust response, increasing tenfold over control 48 h after crush injury of muscle. These results further implicate the serpin:protease balance in tissue injury. Participation of complex receptors, such as the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP), various growth factors, cytokines, and other molecules, in regulating this balance is implicated by these data.

Human tissue kallikrein induces hypotension in transgenic mice.

We investigated the role of the kallikrein-kinin system in blood pressure control by developing transgenic mice overexpressing human tissue kallikrein. Two lines of transgenic mice carrying the human tissue kallikrein gene under the control of the mouse metallothionein metal-responsive promoter were established. Human tissue kallikrein was identified in pancreas, salivary gland, kidney, liver, and spleen of the transgenic mice by a specific radioimmunoassay for human tissue kallikrein. The immunoreactive human tissue kallikrein reached high levels in the circulation. The linear displacement curves for the transgenic product were parallel with the human tissue kallikrein standard curve, indicating their immunologic identity. The expression of human tissue kallikrein transcript in the transgenic mice was further confirmed by Northern blot analysis and by reverse transcription-polymerase chain reaction followed by Southern blot. Both lines of transgenic mice had significantly lowered blood pressure (86.4 +/- 13.5 mm Hg [mean +/- SD], n = 8 and 78.9 +/- 12.4 mm Hg, n = 8) compared with control mice (100.9 +/- 5.0 mm Hg, n = 8). Induction with zinc did not lower the blood pressure further despite elevated expression of the transgene. Administration of aprotinin, a potent tissue kallikrein inhibitor, restored the blood pressure of the transgenic mice but had no significant effect on control littermates. Our findings raise the possibility of tissue kallikrein being a powerful modulator of blood pressure and provide a new animal model for the study of blood pressure regulation.

Tissue kallikrein activity and kinin release in human endothelial cells.

The kininogenase, tissue kallikrein (EC 3.4.21.8), has been identified in different blood vessels. The enzyme was mainly found in vascular smooth muscle cells. It is not known whether it is present and functionally active in vascular endothelial cells. The following study investigates the presence of tissue kallikrein in endothelial cells from human umbilical veins and pulmonary arteries. Tissue kallikrein was demonstrated in three ways: 1) by immunostaining in endothelial cells; 2) by measurement of tissue kallikrein activity using a colorimetric assay; 3) by the measurement of kinin release in intact and homogenised endothelial cells with a radioimmunoassay. Immunostaining demonstrated the presence of tissue kallikrein in endothelial cells from human umbilical veins and endothelial cells from human pulmonary arteries. Tissue kallikrein-like activity, measured by the degradation of D-Val-cyclohexyl-Ala-Arg-4-nitraniline, was 3.57 +/- 0.5 mU/10(6) endothelial cells from human umbilical veins and 7.52 +/- 0.84 mU/10(6) endothelial cells from human pulmonary arteries. Intracellular kinin concentrations were 424 +/- 83 pg/10(6) cells in endothelial cells from human umbilical veins and 576 +/- 146 pg/10(6) cells in endothelial cells from human pulmonary arteries, and they increased in a time-dependent manner after homogenisation. The increase was abolished by aprotinin (1000 kIU), an inhibitor of tissue kallikrein in both cell types. Addition of exogenous kallikrein (5 mU) to homogenised cells led to a five fold increase of kinin concentrations after five minutes, indicating a sufficient resource of cellular kininogen. Removal of extracellularly bound kininogen by washing with dextran sulphate (100 mg/l) resulted in an approximately 75% reduction of the cellular kinin release.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions in vitro and in vivo between porcine tissue kallikrein and porcine plasma proteinase inhibitors.

The elimination of radio-iodinated porcine tissue kallikrein, after intravenous injection in the pig, showed a rapid initial clearance from plasma (T1/2 approximately 10 min), followed by a phase of slower elimination (T1/2 approximately 100 min). Gel filtration of plasma samples showed complexes with alpha 1-alpha 2-macroglobulin (A1a2-M) and alpha 1-proteinase inhibitor (A1-PI), which decreased with time. The urinary excretion of undegraded tissue kallikrein was about 1.8%. Gel filtration of urine showed a minor peak representing free tissue kallikrein and a major peak representing degradation products. On average, 8.3% was found in the liver and 1.3% in the kidneys post mortem, indicating that these are the primary organs for the elimination of tissue kallikrein. The in vivo findings were supported by in vitro experiments. A1a2-M were found to be the major inhibitors of tissue kallikrein, when a mixture of the enzyme and porcine plasma was analysed by gel filtration, immunoelectrophoresis, crossed immunoelectrophoresis and autoradiography. A1-PI was only a minor inhibitor of tissue kallikrein. Both the A1a2-M and A1-PI complex formation was found to be time-dependent and slow; unbound glandular kallikrein was still detected after 12 h, even when there was a molar surplus of A1a2-M and A1-PI. The complexes with A1a2-M and the unbound tissue kallikrein were found to be enzymatically active against low-molecular-weight chromogenic substrate. The total tissue kallikrein-inhibiting capacity of plasma seemed to be exceeded at a concentration of 800 K U/l when analysed using the rat uterus bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinins and kallikrein in nasal secretion after antigen challenge in patients with allergic rhinitis

This study showed that nasal allergy patients have increased nasal airway resistance and tissue kallikrein activity in nasal lavage in accordance with the severity of symptoms. Nasal airway resistance and tissue kallikrein activity were significantly increased in the nasal lavage collected 15 min after antigen challenge, placement of a disk containing house dust antigen. The free kinin level in the nasal lavage collected 15 min after the antigen challenge was also markedly increased. Pretreatment of patients with d-chlor-pheniramine maleate (histamine H1 receptor antagonist) blunted this increase in nasal airway resistance and tissue kallikrein activity 15 min after the antigen challenge, suggesting that histamine may accelerate excretion of tissue kallikrein in nasal secretions. Camostat mesilate inhibited the in vitro tissue kallikrein activity in nasal lavage collected from allergy patients. Administration of camostat mesilate significantly inhibited nasal airway resistance and tissue kallikrein activity in nasal lavage after the antigen challenge, indicating that kinin generated in the nasal cavity increased nasal airway resistance. These results suggest that tissue kallikrein inhibitors may serve as anti-allergic drugs by suppressing the nasal symptoms of nasal allergy patients.

Kallistatin: a novel human tissue kallikrein inhibitor. Purification, characterization, and reactive center sequence.

A novel human tissue kallikrein inhibitor designated as kallistatin has been purified from plasma to apparent homogeneity by polyethylene glycol fractionation and successive chromatography on heparin-Agarose, DEAE-Sepharose, hydroxylapatite, and phenyl-Superose columns. A purification factor of 4350 was achieved with a yield of approximately 1.35 mg per liter of plasma. The purified inhibitor migrates as a single band with an apparent molecular mass of 58 kDa when analyzed on SDS-polyacrylamide gel electrophoresis under reducing conditions. It is an acidic protein with pI values ranging from 4.6 to 5.2. No immunological cross-reactivity was found by Western blot analyses between kallistatin and other serpins. Kallistatin inhibits human tissue kallikrein's activity toward kininogen and tripeptide substrates. The second-order reaction rate constant (ka) was determined to be 2.6 x 10(4) M-1 s-1 using Pro-Phe-Arg-MCA. The inhibition is accompanied by formation of an equimolar, heat- and SDS-stable complex between tissue kallikrein and kallistatin, and by generation of a small carboxyl-terminal fragment from the inhibitor due to cleavage at the reactive site by tissue kallikrein. Heparin blocks kallistatin's complex formation with tissue kallikrein and abolishes its inhibitory effect on tissue kallikrein's activity. The amino-terminal residue of kallistatin is blocked. Sequence analysis of the carboxyl-terminal fragment generated from kallistatin reveals the reactive center sequence from P1' to P15', which shares sequence similarity with, but is different from known serpins including protein C inhibitor, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. The results show that kallistatin is a new member of the serpin superfamily that inhibits human tissue kallikrein.

Mapping the binding site of tissue kallikrein: preparation and testing of all possible substrate analog inhibitors homologous with the sequence of kininogen between Ser386 and Gln392.

Programs aimed at converting peptide inhibitors of proteolytic enzymes into more traditional drug structures require an understanding of the role played by the individual amino acid residues in the inhibitor. To this end, all possible substrate analogues occurring within the sequence Ser386-Pro-Phe-Arg-Ser-Val-Gln392 from bovine kininogen were synthesized and tested as inhibitors of tissue kallikrein (EC 3.4.21.35, beta-PPK). Of the 21 sequences which can be formed from the heptapeptide, 11 have inhibitory constants which could be measured in the chromogenic assay employed in these studies. No dipeptide and only one tripeptide, Ac-Phe-Arg-Ser-NH2 (Ki = 718 microM), measurably inhibits the enzyme. All longer peptides inhibit beta-PPK. The heptapeptide Ac-Ser-Pro-Phe-Arg-Ser-Val-Gln-NH2 is the most effective inhibitor in this series (Ki = 101 microM). Each amino acid residue in the sequence appears to alter binding in a relatively independent manner. The N-terminal seryl residue (P4) and the prolyl residue (P3) slightly improve the Ki of the various inhibitors. The phenylalanyl residue at P2 appears to have a more pronounced effect on Ki. The arginyl residue at P1 and the seryl residue at P1' appear to be the most important residues in the inhibitory sequence. They contribute approximately one-third and one-fourth of the binding energy to the interaction between the substrate analogues and beta-PPK, respectively. The valyl residue at P2', and the C-terminal glutaminyl residue improve Ki of each of the peptides tested. Almost 80% of the binding energy of the substrate analogue inhibitors comes from the core sequence Phe-Arg-Ser which occurs between P2 and P1'. Molecular models developed from the Chen-Bode coordinates of the aprotinin-beta-PPK complex have been used to interpret the results of these studies.