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PubMed1999

Expression and characterization of recombinant mast cell tryptase.

Tryptase, a serine protease, is the major protein component in mast cells. In an animal model of asthma, tryptase has been established as an important mediator of inflammation and late airway responses induced by antigen challenge. Human tryptase is notable for its tetrameric structure, requirement of heparin for stability, and resistance to endogenous inhibitors. Human protryptase was expressed as a recombinant protein in Pichia pastoris. The recombinant protein consisted of two forms of protryptase, one containing the entire propeptide and the other containing only the Val-Gly dipeptide at its amino terminus. Isolation of active recombinant tryptase required a two column purification protocol and included a heparin- and dipeptidyl peptidase I-dependent activation step. Purified recombinant tryptase migrated as a tetramer on a gel filtration column and displayed kinetic parameters identical to those of a native tryptase obtained from HMC-1 cells, a human mast cell line. Recombinant and HMC-1 tryptase exhibited comparable sensitivities to an array of protein and low-molecular-weight inhibitors, including one that is highly specific for tryptase (APC-1167). Similarly, the recombinant enzyme cleaved both alpha- and beta-chains of fibrinogen to generate fibrinogen fragments indistinguishable from those generated by HMC-1-derived tryptase. Thus, recombinant tryptase expressed in P. pastoris displays physical and enzymatic properties essentially identical to the native enzyme. This system provides a cost-effective and easy to manipulate expression system that will enable the functional characterization of this unique enzyme.

Binding Sites↗
PubMed2004

Effect of sex and haplotype on plasma tryptase levels in healthy adults.

BACKGROUND: The total level of alpha-tryptase and ss-tryptase in serum or plasma is used as a clinical indicator of the mast cell burden. OBJECTIVE: The effect of the tryptase haplotype and of sex on the total tryptase level of healthy individuals was determined. METHODS: A novel hot-stop PCR technique was used to determine the tryptase genotype, and a standard fluoroenzyme immunoassay was used to measure total plasma tryptase levels in 106 healthy subjects. Mx modeling and the QTL association routine of Mendel 5.0 were used to analyze the data. RESULTS: Tryptase haplotypes exhibit a 1 (betaalpha/betaalpha):2 (betabeta/betaalpha):1 (betabeta/betabeta) distribution, monomorphic for ss at 1 position and allelic for ss and alpha at the other position. The betaalpha haplotype has a frequency of 0.49. The betaalpha haplotype increases total tryptase levels by 0.5 ng/mL from the overall mean, whereas female sex increases the level by 0.2 ng/mL from the mean. CONCLUSION: The tryptase haplotype and sex each have a statistically significant effect on the total plasma tryptase level of healthy subjects.

Adult↗
PubMed2004

Inhibitors of mast cell tryptase beta as therapeutics for the treatment of asthma and inflammatory disorders.

A survey of the available biological data on tryptase inhibitors suggests that there is considerable interest in tryptase as a therapeutic target particularly for the treatment of allergic asthma and inflammatory disorders. This interest was driven primarily by data from studies carried out on the cellular and in vivo actions of this serine protease over the past decade, all of which have suggested a pro-inflammatory role for tryptase. Tryptase beta is the form of interest in allergic asthma and the data from numerous studies have shown that tryptase cannot only contribute to airway bronchoconstriction and hyperresponsiveness, but may have a key role in fibrosis and ECM turnover, hallmarks of the remodeling process. Hence, inhibitors of tryptase have the potential to make an impact on fibrosis and airway wall remodelling. However, few studies, if any, have been carried out to determine the effect of tryptase inhibitors on airway remodeling and this is an area that warrants further investigation with the appropriate models because the eventual positioning of tryptase inhibitors in asthma therapy will be strengthened by data supporting an impact on airway remodeling in addition to effects on bronchial hyperresponsiveness. This review has focused on tryptase inhibitors in the pipeline and it is clear that with a few exceptions, the majority of these compounds are targeted for inhaled delivery. Finally, judging by the interest from numerous pharmaceutical companies, it appears the stage is set for tryptase inhibitors to make their mark as drugs of the future for allergic asthma and the results from clinical trials is awaited with eager anticipation.

Animals↗
PubMed1998

beta-Tryptase measurements post-mortem in anaphylactic deaths and in controls.

The reliability of measuring mast cell tryptase in post-mortem blood to diagnose anaphylactic deaths has been questioned because moderate elevation of tryptase can be seen also in control cases. Very high tryptase concentrations have been recorded even in a few control cases with known cause of death such as myocardial infarction or trauma. Aiming to compare findings we measured tryptase in 193 cases: 176 with known cause of death, 10 unexplained deaths and seven anaphylactic or anaphylactoid deaths (AADs). Using binary logistic regression we calculated the sensitivity and specificity of the tryptase test at different cut-off values and found 10 micrograms/l to be optimal, the sensitivity being 86% and the specificity 88%. Traumatic deaths (n = 23), sudden infant death syndrome (n = 40) or deaths after heroin-injection (n = 22) showed elevated tryptase values in 35%, 35% and 32%, respectively, and in 40% of the unexplained deaths (n = 10), which was higher than expected (12%). Heart blood tryptase level was elevated in 22% of the controls and femoral blood tryptase in 10%. No correlations were seen with age or post-mortem delay. It is concluded that tryptase measurements are useful in confirming death from AAD, and that blood should be sampled from the femoral vessels. In unexplained deaths tryptase measurement is a useful indicator, but the diagnosis is not to be based on the test alone.

Adult↗
PubMed2001

Heparin antagonists are potent inhibitors of mast cell tryptase.

Tryptase may be a key mediator in mast cell-mediated inflammatory reactions. When mast cells are activated, they release large amounts of these tetrameric trypsin-like serine proteases. Tryptase is present in a macromolecular complex with heparin proteoglycan where the interaction with heparin is known to be essential for maintaining enzymatic activity. Recent investigations have shown that tryptase has potent proinflammatory activity, and inhibitors of tryptase have been shown to modulate allergic reactions in vivo. Many of the tryptase inhibitors investigated previously are directed against the active site. In the present study we have investigated an alternative approach for tryptase regulation. We show that the heparin antagonists Polybrene and protamine are potent inhibitors of both human lung tryptase and of recombinant mouse tryptase (mouse mast cell protease 6). Protamine inhibited tryptase in a competitive manner whereas Polybrene showed noncompetitive inhibition kinetics. Treatment of tetrameric, active tryptase with Polybrene caused dissociation into monomers, accompanied by complete loss of enzymatic activity. The present report thus suggests that heparin antagonists potentially may be used in treatment of mast cell-mediated diseases such as asthma.

Animals↗
PubMed1997

Mast cell activation in arthritis: detection of alpha- and beta-tryptase, histamine and eosinophil cationic protein in synovial fluid.

1. Although mast cell hyperplasia is a feature of rheumatoid arthritis and osteoarthritis, the extent and nature of mast cell activation in joint disease have not been clearly established. 2. We have investigated the levels of mast cell tryptase and histamine and also of eosinophil cationic protein in synovial fluid collected from 31 patients with rheumatoid arthritis, 14 with seronegative spondyloarthritis and nine with osteoarthritis. Two RIAs for tryptase were employed: one with monoclonal antibody AA5, which was found to bind equally well to both alpha and beta isoforms on Western blots of the recombinant enzyme, and the other with antibody G5, which recognizes predominantly beta-tryptase. 3. alpha-Tryptase, which is likely to be released constitutively from mast cells, appeared to be the major form in synovial fluid, as the assay with antibody AA5 detected appreciably more tryptase than that with antibody G5. beta-Tryptase, which is released on anaphylactic activation of mast cells, was detected in 14 out of 45 synovial fluid samples studied, with concentrations of up to 12 micrograms/l measured by the G5 assay. The apparent levels of beta-tryptase, but not of alpha-tryptase, were closely correlated with those of histamine in the synovial fluid. Patients with osteoarthritis appeared to have a greater proportion of beta-tryptase in the synovial fluid than those with rheumatoid arthritis, as well as higher concentrations of histamine. Eosinophil cationic protein was present at high levels in the synovial fluid, although eosinophil numbers were low, and its concentrations were not correlated with the concentrations of the mast cell products. 4. These data suggest that anaphylactic degranulation of mast cells may have occurred to a greater extent in osteoarthritis than in rheumatoid arthritis, despite the relative lack of synovial inflammation in osteoarthritis. Although the eosinophil cationic protein detected may not reflect eosinophilic inflammation in the joint, the presence in synovial fluid of tryptase of both major forms, and of histamine, appears to indicate that mast cell products are secreted constitutively, as well as by processes of anaphylactic degranulation in rheumatoid arthritis, seronegative spondyloarthritis and osteoarthritis.

Adult↗
PubMed2003

Mast cell-sperm interaction: evidence for tryptase and proteinase-activated receptors in the regulation of sperm motility.

BACKGROUND: The detection of significant levels of tryptase in human seminal plasma and follicular fluid and of tryptase-positive mast cells (MCs) in the wall of human Fallopian tubes lead us to hypothesize that tryptase may exert regulatory actions on human spermatozoa. METHODS AND RESULTS: Immunoelectronmicroscopy revealed proteinase-activated receptor 2 (PAR-2) in the membranes of the acrosomal region and midpiece of human spermatozoa. These PAR-2 were functional, as exposure of spermatozoa from healthy men (n = 12) with regular standard semen parameters to human recombinant tryptase significantly decreased motility in a dose- and time-dependent fashion. Motile spermatozoa (WHO a + b) were significantly decreased within 10 min of incubation with 1.000 ng/ml tryptase (P = 0.045). After 30 and 60 min, significant reduction of motility was also observed in the presence of lower tryptase concentrations (100 ng/ml, P = 0.037; 10 ng/ml, P = 0.046). The inhibitory effects of tryptase progressed throughout an observation period of 180 min. Furthermore, tryptase effects were reversible after washing procedures and could be inhibited by pretreatment with anti-tryptase antibody or anti-PAR-2 antiserum. CONCLUSIONS: The observations presented raise the possibility that tryptase directly interacts with human spermatozoa during their migration through the female genital tract. Genital tract MCs and their products may be as yet unrecognized factors involved in human fertility/sterility.

Acrosome↗
PubMed2005

Mast cell tryptase and chymase in chronic leg ulcers: chymase is potentially destructive to epithelium and is controlled by proteinase inhibitors.

BACKGROUND: Numerous mast cells are present in chronic leg ulcers. Tryptase and chymase are the major mediators of mast cells, but their significance is mostly dependent on their activity. In addition, the proteinases may affect ulcer epithelialization. OBJECTIVES: To study levels and activity of tryptase and chymase in wash samples and biopsies from chronic leg ulcers and the possible effect of these proteinases on keratinocyte growth and adherence. METHODS: Wash samples were taken from 16 patients and a superficial shave biopsy was taken in eight of these patients; a second biopsy series was obtained from the edge of chronic venous leg ulcers (n = 6). RESULTS: Significant levels of soluble tryptase activity and histamine, but low levels of chymase activity, were measured in wash samples from chronic ulcers. No tryptase-inhibiting activity, but clear chymase-inhibiting activity, was detected in the wash samples. In superficial wound bed biopsies, relatively marked levels of chymase activity together with histamine and tryptase activity were detected. In the second biopsy series, about 80% of the mast cells belonged to the MC(TC) type (tryptase- and chymase-immunopositive). However, about 55-61% of the chymase-immunopositive cells displayed chymase activity and 64 +/- 17% of the tryptase-positive cells revealed immunoreactivity of alpha(1)-antichymotrypsin. As the activity of chymase and tryptase was detected in the ulcer base in a ratio of 1:8, a preparation containing both chymase and tryptase was partially purified from human skin yielding a similar activity ratio of 1:11-13. Treatment of fibronectin-coated plastic surfaces with this preparation decreased the adherence of cultured human keratinocytes, this effect being attributable mainly to chymase. In 2-day cultures using growth factor/serum-deficient low- or high-calcium medium, the tryptase-chymase preparation inhibited the slow growth and at higher concentrations it even induced detachment of keratinocytes. This effect was attributed to chymase, and it was partially regulated by heparin and histamine. CONCLUSIONS: Even though chymase is partially inactivated in chronic leg ulcers, accumulated mast cells in the close proximity of the epithelium edge and their chymase may impair keratinocyte adherence and migration.

Adult↗
PubMed2004

Can serum mast cell tryptase help diagnose anaphylaxis?

OBJECTIVE: We aimed to determine the utility of serum mast cell tryptase to diagnose anaphylaxis. METHODS: As part of a venom immunotherapy trial, we performed 64 sting challenges. Blood samples were taken before the sting (baseline), and 15 min and 60 min after the sting. Tryptase was measured in baseline, 15 minute and 60 minute serum samples. Histamine was measured in baseline and 15 minute plasma samples. Eleven people had undisputed severe anaphylactic reactions; tryptase and histamine levels were assessed against this clinical gold standard diagnosis. RESULTS: Excluding mild reactions from the analysis, peak tryptase readings had sensitivity of 0.36 and specificity of 0.93 using the recommended cut-off range (< 12.0 microg/L). Receiver-operator curve analysis found a cut-off of 9.0 microg/L would improve diagnostic performance (sensitivity 0.55, specificity 0.93). Serial tryptase measurement was significantly more discriminatory; an increase in tryptase of 2.0 microg/L or greater had a sensitivity of 0.73 and specificity of 0.98. The addition of histamine measurements, defining a positive result by either a rise in tryptase or a rise in histamine, appeared to further increase sensitivity (0.90). CONCLUSIONS: Clinicians should use caution when using serum tryptase to refute or support a diagnosis of anaphylaxis. Serial tryptase measurement increases sensitivity and specificity. Further studies using serial tryptase determinations in general emergency department practice, perhaps supplemented by histamine determinations, are warranted.

Anaphylaxis↗
PubMed1994

Modulation of thrombin and thrombin receptor peptide mitogenicity by human lung mast cell tryptase.

In previous studies, mast cell tryptase acted as a potent mitogen for fibroblasts from human lung and rodent embryonic tissue but failed to stimulate growth of cultured rat aortic vascular smooth muscle cells (VSMC). The current study shows that tryptase inhibits DNA synthesis in VSMC stimulated by thrombin. However, it does not affect the stimulation of DNA synthesis by the synthetic thrombin receptor peptide Ser-Phe-Phe-Leu-Arg-Asn-Pro (SFFLRNP), which mimics the amino-terminus of thrombin receptor proteolytically activated by thrombin. Nor does tryptase alter the mitogenic response of VSMC to purified growth factors, such as platelet-derived growth factor (PDGF). These data suggest that tryptase inhibits thrombin-induced DNA synthesis without interfering with intracellular mitogenic signaling pathways activated by thrombin or other growth factors. This study further suggests that tryptase neither cleaves nor inactivates thrombin. Therefore, inhibition of thrombin's mitogenic effects by tryptase is not mediated by destruction of thrombin itself. The inhibition by tryptase of thrombin-induced DNA synthesis in VSMC contrasts with the stimulatory effect of tryptase on fibroblasts, in which synergy is observed with thrombin, with thrombin receptor peptide and with other growth factors. These data provide in vitro evidence that mast cell tryptase interferes with thrombin-stimulated vascular smooth muscle growth and suggest that tryptase is a multifunctional growth factor whose actions are cell specific.

Animals↗
PubMed2005

Quantitation of minimal residual disease in acute myeloid leukemia by tryptase monitoring identifies a group of patients with a high risk of relapse.

PURPOSE: Recent data suggest that tryptase is produced by blast cells in a group of patients with acute myeloid leukemia (AML). In these patients, serum tryptase levels are elevated at diagnosis and decrease to normal (<15 ng/mL) or near normal values in those achieving complete hematologic remission (CR) after chemotherapy. PATIENTS: In this study, we examined the value of tryptase as a marker of minimal residual AML. In 61 patients with de novo AML exhibiting elevated serum tryptase (>15 ng/mL) at diagnosis, tryptase levels were measured serially during and after chemotherapy by a fluoroenzyme immunoassay. RESULTS: Of the 61 patients, 42 (68.9%) entered hematologic CR in response to induction chemotherapy. Twenty-nine of these 42 patients also entered biochemical remission (BR) defined by a decrease of tryptase levels to normal (<15 ng/mL). The remaining 13 patients exhibited elevated enzyme levels despite of hematologic CR. As assessed by multivariate analysis, the elevated tryptase in CR was found to be an independent prognostic variable concerning disease-free survival. Thus, AML relapses occurred in 15 of 29 patients with CR + BR (52%) and in 12 of 13 patients with CR without BR (92%), resulting in a significantly reduced probability of continuous CR for patients with CR without BR (P < 0.05). In all patients with continuous hematologic CR, tryptase levels remained constantly normal, whereas a recurrent elevation of tryptase in CR was invariably followed by a hematologic relapse. CONCLUSION: A persistently elevated tryptase level in AML in CR is indicative of minimal residual AML and associated with a high risk of relapse.

Acute Disease↗
PubMed2000

Characteristics of monkey tryptase purified from cheek pouch vascular tissues.

Tryptase purified from rat and dog tissues has been reported, although the characteristics of these enzymes are different from human tryptase. For pathophysiological studies of human tryptase, studies on species that have a similar tryptase to humans is needed. In this study, we purified monkey tryptase from cheek pouch vascular tissues using heparin affinity and gel filtration columns. The monkey tryptase, which had a molecular weight of 130 kDa by gel filtration, consisted of a tetramer of 33 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal sequence showed high homology with tryptases from other species. The optimum pH and temperature were 7.5-9.0 and 25-40 degrees C, respectively. The enzyme was labile in high-KCl buffer, and the optimum KCl concentration was 0.1 M. The enzyme activity was completely inhibited by diisopropyl phosphorofluoridate and leupeptin but not by soybean trypsin inhibitor and alpha-antitrypsin. The enzyme hydrolyzed vasoactive intestinal peptide but did not affect angiotensin I, somatostatin and bradykinin. In the present study, we first isolated monkey tryptase from cheek pouch vascular tissues and showed that the characteristics of monkey tryptase are very similar to those of human tryptase.

Animals↗
PubMed2005

Mast cell tryptase stimulates DLD-1 carcinoma through prostaglandin- and MAP kinase-dependent manners.

We found that striptease-positive mast cells were abundant in the invasive front of human colon adenocarcinoma by examining 30 cases. Because tryptase has been suggested to be the agonist proteinase for protease-activated receptor-2 (PAR-2), we investigated the effects of stimulation of PAR-2 by tryptase on the cell signaling and proliferation of DLD-1, a human colon carcinoma cell line. PAR-2 stimulation by tryptase induced the increase in [Ca(2+)](i), which was desensitized by the prior application of PAR-2 activating peptide (AP). The proliferative responses of DLD-1 to tryptase and PAR-2 AP were associated with the phosphorylation of MEK and MAP kinase. Inhibition of MEK by PD98059 completely inhibited the proliferation-enhancing effects of tryptase and PAR-2 AP as well as phosphorylation of MAP kinase. Moreover, tryptase and PAR-2 AP stimulated the production of prostaglandin E2 and the inhibition of prostaglandin synthesis by indomethacin or NS398 resulted in the complete inhibition of the proliferative responses to tryptase and PAR-2 AP. Furthermore, the tryptase-stimulated proliferation of DLD-1 was concentration-dependently inhibited by nafamostat mesilate, a specific inhibitor of tryptase. These results as a whole indicated that tryptase has proliferative effects on DLD-1 through cyclooxygenase- and MAP kinase-dependent manners acting on PAR-2 by its proteolytic activity.

Benzamidines↗
PubMed2004

Histidines are critical for heparin-dependent activation of mast cell tryptase.

Mast cell tryptase is a tetrameric serine protease that is stored in complex with negatively charged heparin proteoglycans in the secretory granule. Tryptase has potent proinflammatory properties and has been implicated in diverse pathological conditions such as asthma and fibrosis. Previous studies have shown that tryptase binds tightly to heparin, and that heparin is required in the assembly of the tryptase tetramer as well as for stabilization of the active tetramer. Because the interaction of tryptase with heparin is optimal at acidic pH, we investigated in this study whether His residues are of importance for the heparin binding, tetramerization, and activation of the tryptase mouse mast cell protease 6. Molecular modeling of mouse mast cell protease 6 identified four His residues, H35, H106, H108, and H238, that are conserved among pH-dependent tryptases and are exposed on the molecular surface, and these four His residues were mutated to Ala. In addition, combinations of different mutations were prepared. Generally, the single His-Ala mutations did not cause any major defects in heparin binding, activation, or tetramerization, although some effect of the H106A mutation was observed. However, when several mutations were combined, large defects in all of these parameters were observed. Of the mutants, the triple mutant H106A/H108A/H238A was the most affected with an almost complete inability to bind to heparin and to form active tryptase tetramers. Taken together, this study shows that surface-exposed histidines mediate the interaction of mast cell tryptase with heparin and are of critical importance in the formation of active tryptase tetramers.

Amino Acid Sequence↗
PubMed2004

Inhibition of tryptase and chymase induced nucleated cell infiltration by proteinase inhibitors.

AIM: To investigate the ability of proteinase inhibitors to modulate nucleated cell infiltration into the peritoneum of mice induced by tryptase and chymase. METHODS: Human lung tryptase and skin chymase were purified by a similar procedure involving high salt extraction, heparin agarose affinity chromatography followed by S-200 Sephacryl gel filtration chromatography. The actions of proteinase inhibitors on tryptase and chymase induced nucleated cell accumulation were examined with a mouse peritoneum model. RESULTS: A selective chymase inhibitor Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPPF) was able to inhibit approximately 90 % neutrophil, 73 % eosinophil, 87 % lymphocyte and 60 % macrophage accumulation induced by chymase at 16 h following injection. Soy bean trypsin inhibitor (SBTI), chymostatin, and alpha1-antitrypsin showed slightly less potency than ZIGPPF in inhibition of the actions of chymase. While all tryptase inhibitors tested were able to inhibit neutrophil, eosinophil, and macrophage accumulation provoked by tryptase at 16 h following injection, only leupeptin, APC366, and aprotinin were capable of inhibiting tryptase induced lymphocyte accumulation. The inhibitors of tryptase tested were also able to inhibit tryptase induced neutrophil and eosinophil accumulation at 6 h following injection. When being injected alone, all inhibitors of chymase and tryptase at the concentrations tested by themselves had no significant effect on the accumulation of nucleated cells in the peritoneum of mice at both 6 h and 16 h. CONCLUSION: Proteinase inhibitors significantly inhibited tryptase and chymase-induced nucleated cell accumulation in vivo, and therefore they are likely to be developed as a novel class of anti-inflammatory drugs.

Animals↗
PubMed2004

Modulation of tryptase and histamine release from human lung mast cells by protease inhibitors.

Inhibition of IgE dependent histamine release from human mast cells by protease inhibitors has been observed in skin, tonsil and synovial tissues. However, little is known about the actions of protease inhibitors on tryptase release from human lung mast cells. We therefore examined the ability of protease inhibitors to modulate tryptase and histamine release from human lung mast cells. IgE dependent tryptase release from dispersed lung mast cells was inhibited to a maximum of approximately 53.8% and 44.5% by N-a-tosyl-L-lysine chloromethyl ketone (TLCK) and N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), respectively. A similar degree of inhibition of calcium ionophore A23187 (CI) induced tryptase release was also observed with these two inhibitors. Preincubation of TLCK or TPCK with the mast cells at 37 degrees C for 20 minutes before addition of anti-IgE or CI did not improve their ability to inhibit anti-IgE and CI induced tryptase release. At a concentration of 10 microg/ml, protamine inhibited anti-IgE or CI induced tryptase release; but at 100 microg/ml, it increased anti-IgE and CI induced release of tryptase from lung mast cells. A concentration dependent inhibition of anti-IgE and CI induced release of histamine from lung mast cells was also observed with TLCK, TPCK and protamine. The maximum inhibition of anti-IgE induced histamine release was approximately 40.7%, 40.2% and 33.4% with TLCK, TPCK and protamine, respectively. At the concentrations tested, TLCK and TPCK by themselves did not stimulate tryptase and histamine release from lung mast cells. A specific inhibitor of aminopeptidase, amastatin, had no effect on anti-IgE induced tryptase and histamine release and was used as control. In conclusion, it was demonstrated that protease inhibitors are able to inhibit IgE dependent tryptase and histamine release from human lung mast cells, which suggested that they could be developed to a novel class of anti-inflammatory drugs to treat allergic conditions in man.

Female↗
PubMed1996

Induction of vascular permeability enhancement by human tryptase: dependence on activation of prekallikrein and direct release of bradykinin from kininogens.

Tryptase is a trypsin-type serine protease that is released from mast cells. Bradykinin (BK) is released directly from kininogens or through activation of either Hageman factor or subsequent plasma prekallikrein. Its nasal administration or inhalation induces allergy-like symptoms. Although elevated levels of tryptase and BK in allergic fluids have been detected, the role of this proteinase and the mechanism of BK production at allergic reaction sites are still unknown. To investigate the pathologic functions of tryptase, the enzyme, purified from human lung, was incubated with normal human plasma, deficient plasmas, kininogens, or prekallikrein. High molecular weight kininogen was then added, and the mixtures were examined for vascular permeability enhancement (VPE) activity, a representative function of bradykinin, using guinea pig skin. Tryptase-treated plasma induced VPE in a dose-dependent manner; activity was lost in the absence of a kininase inhibitor but not an antihistamine drug. Tryptase produced VPE activity from normal or Hageman factor-deficient plasma, but only 30% of this activity was produced from prekallikrein-deficient plasma. Significantly, no activity was obtained from kininogen-deficient plasma. Deficient plasma that were reconstituted with each missing factor resulted in VPE-inducing capacity by tryptase, equivalent to that found with normal plasma. Incubation of tryptase with high or low molecular weight kininogen induced VPE activity in a dose- and incubation time-dependent manner. Prekallikrein incubated with tryptase also generated a soybean trypsin inhibitor-sensitive VPE-inducing activity from high molecular weight kininogen. The loss of tryptase VPE-producing activity as a function of incubation time was found to be a result of spontaneous inactivation of the enzyme and not of the degradation of high molecular weight kininogen by the enzyme. We conclude that tryptase induces VPE by releasing BK, primarily through prekallikrein activation, but also through direct release from kininogens. This indicates that this mast cell-derived proteinase contributes to kinin production in allergic diseases.

Animals↗
PubMed1990

Interactions of human mast cell tryptase with biological protease inhibitors.

Tryptase from human mast cells has been shown (in vitro) to catalyze the destruction of fibrinogen and high-molecular-weight kininogen as well as the activation of C3a and collagenase. Although large amounts of tryptase are released in tissues by degranulating mast cells and levels as high as 1000 ng/ml have been measured in the circulation following systemic anaphylaxis, no specific physiologic inhibitor has yet been found for the protease. The current work tests several more inhibitors for their effects on tryptase and examines any effect of tryptase on these inhibitors. First, antileukoprotease and low-molecular-weight elastase inhibitor from human lung and hirudin and antithrombin III had no effect on tryptase activity in vitro. Second, the possibility that tryptase, being insensitive to the effects of inhibitors, might instead destroy them was also considered. Tryptase failed to cleave and inactivate antileukoprotease, low-molecular-weight elastase inhibitor, alpha 1 protease inhibitor, alpha 2 macroglobulin, and antithrombin III. Third, based on the knowledge that tryptase stability is regulated by its interaction with heparin, antithrombin III was used as a model heparin-binding protein to demonstrate that a protein competitor for heparin-binding sites, presumably by displacement of tryptase, destabilizes this enzyme. Conversely, tryptase, in excess, blocked the binding of antithrombin III to heparin, thereby attenuating the heparin-mediated inhibition of thrombin by antithrombin III.

Antithrombin III↗