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PubMedDate not supplied

Identification and characterization of multiple forms of tryptase from human mast cells.

Mast cell tryptase purified from human adult skin (AS), adult lung (AL) and newborn foreskin (NS) with a monoclonal antitryptase B2 immunoaffinity Sepharose column was further fractionated by HPLC using a Mono-S cation exchange column at pH 6.5. Tryptases exhibited two clearly separated major fractions, both of which also revealed at least two overlapping peaks. Native tryptase molecules from skin consisted of two diffuse protein bands in SDS-PAGE at about 31 and 35 kDa, whereas those from lung usually exhibited a predominant diffuse band at about 29 kDa. The forms of tryptases separated by Mono-S HPLC gave a different banding pattern in SDS-PAGE. Tryptase from NS exhibited chromatographic peaks that each showed Mr values approximately 1-3 kDa higher than those of tryptase from AS. By gel filtration, the Mr values for native major fractions of tryptases derived from AS and AL were 178 kDa and 141 kDa, respectively. After carbohydrate removal by glycanase, the observed differences in Mr values in SDS-PAGE reduced to two similar sharp bands of Mr approximately 28 kDa and 30 kDa for all tryptase preparations. AS and AL tryptases and their subfractions exhibited similar enzyme kinetic values and similar immunoreactivities in a tryptase immunoassay. Inactivation rates at physiologic ionic strength were similar for both AL and AS tryptases. The results show the enzymatic and antigenic similarity between lung and skin tryptases, and suggest that tryptase is stored mainly as beta-tryptase in human mast cells. Tryptase immunoassay measures similarly both lung and skin tryptases and, thus, this assay is suitable for detection of mast cell activation, in contrast to assays for other proteinases of mast cells, e.g. chymase, cathepsin G and carboxypeptidase, that are present in MC(TC) cells mainly in skin only.

Adult↗
PubMed1995

The alpha form of human tryptase is the predominant type present in blood at baseline in normal subjects and is elevated in those with systemic mastocytosis.

Tryptase, a protease produced by all mast cells, was evaluated as a clinical marker of systemic mastocytosis. Two sandwich immunoassays were evaluated, one which used the mAb G5 for capture, the other which used B12 for capture. The B12 capture assay measured both recombinant alpha- and beta-tryptase, whereas the G5 capture assay measured primarily recombinant beta-tryptase. G5 binds with low affinity to both recombinant alpha-tryptase and tryptase in blood from normal and nonacute mastocytosis subjects, and binds with high affinity to recombinant beta-tryptase, tryptase in serum during anaphylaxis, and tryptase stored in mast cell secretory granules. B12 recognizes all of these forms of tryptase with high affinity. As reported previously, during systemic anaphylaxis in patients without known mastocytosis, the ratio of B12- to G5-measured tryptase was always < 5 and approached unity (Schwartz L.B., T.R. Bradford, C. Rouse, A.-M. Irani, G. Rasp, J.K. Van der Zwan and P.-W.G. Van der Linden, J. Clin. Immunol. 14:190-204). In this report, most mastocytosis patients with systemic disease have B12-measured tryptase levels that are elevated (> 20 ng/ml) and are at least 10-fold greater than the corresponding G5-measured tryptase level. Most of those subjects with B12-measured tryptase levels of < 20 ng/ml had only cutaneous manifestations. The B12 assay for alpha-tryptase and beta-tryptase, particularly when performed in conjunction with the G5 assay for beta-tryptase, provides a more precise measure of mast cell involvement than currently available assessments, a promising potential screening test for systemic mastocytosis and may provide an improved means to follow disease progression and response to therapy.

Acute Disease↗
PubMed2005

Structural requirements and mechanism for heparin-dependent activation and tetramerization of human betaI- and betaII-tryptase.

Tryptase, a tetrameric serine protease, is a main constituent of the secretory granules in human mast cells, where it is stored in complex with heparin or chondroitin sulfate proteoglycan. Human tryptase has been implicated in a variety of clinical conditions including asthma, but the mechanisms that lead to its tetramerization/activation have not been extensively investigated. Here we addressed the activation mechanisms for human betaI and betaII-tryptase, which differ in that betaI-tryptase is N-glycosylated at Asn102 whereas betaII-tryptase has a Lys residue at position 102, and consequently lacks the corresponding N-glycosylation. We found that both tryptases were dependent on heparin for activation/tetramerization, but whereas betaI-tryptase activation preferentially occurred at acidic pH, betaII-tryptase activation was less pH-dependent. Both betaI and betaII-tryptase bound strongly to heparin-Sepharose at acidic pH but with lower affinity at neutral pH. Further, while addition of heparin to betaI-tryptase predominantly resulted in formation of active tetrameric enzyme, betaII-tryptase showed a tendency to form inactive aggregates. betaI and betaII-tryptase were similar in that the minimal heparin size to induce activation was an octasaccharide and in that the interaction with heparin and structurally related polysaccharides was dependent on high anionic charge density rather than on specific structural motifs. Addition of decasaccharides to both betaI and betaII-tryptase resulted in the formation of active monomeric enzyme, whereas intact heparin promoted assembly of tetrameric enzyme. This, together with a bell-shaped dose response curve for heparin-induced activation, suggests that the mechanism for tetramerization involves bridging of individual tryptase monomers by heparin. Taken together, this study indicates a key role for heparin in the activation of human beta-tryptase.

Animals↗
PubMed2001

Glycosylation and the activation of proteinase-activated receptor 2 (PAR(2)) by human mast cell tryptase.

1. Human mast cell tryptase appears to display considerable variation in activating proteinase-activated receptor 2 (PAR(2)). We found tryptase to be an inefficient activator of wild-type rat-PAR(2) (wt-rPAR(2)) and therefore decided to explore the factors that may influence tryptase activation of PAR(2). 2. Using a 20 mer peptide (P20) corresponding to the cleavage/activation sequence of wt-rPAR(2), tryptase was as efficient as trypsin in releasing the receptor-activating sequence (SLIGRL.). However, in the presence of either human-PAR(2) or wt-r PAR(2) expressing cells, tryptase could only activate PAR(2) by releasing SLIGRL from the P20 peptide, suggesting that PAR(2) expressed on the cells was protected from tryptase activation. 3. Three approaches were employed to test the hypothesis that PAR(2) receptor glycosylation restricts tryptase activation. (a) pretreatment of wt-rPAR(2) expressing cells or human embryonic kidney cells (HEK293) with vibrio cholerae neuraminidase to remove oligosaccharide sialic acid, unmasked tryptase-mediated PAR(2) activation. (b) Inhibiting receptor glycosylation in HEK293 cells with tunicamycin enabled tryptase-mediated PAR(2) activation. (c) Wt-rPAR(2) devoid of the N-terminal glycosylation sequon (PAR(2)T25(-)), but not rPAR(2) devoid of the glycosylation sequon located on extracellular loop-2 (PAR(2)T224A), was selectively and substantially (>30 fold) more sensitive to tryptase compared with the wt-rPAR(2). 4. Immunocytochemistry using antisera that specifically recognized the N-terminal precleavage sequence of PAR(2) demonstrated that tryptase released the precleavage domain from PAR(2)T25(-) but not from wt-rPAR(2). 5. Heparin : tryptase molar ratios of greater than 2 : 1 abrogated tryptase activation of PAR(2)T25(-). 6. Our results indicate that glycosylation of PAR(2) and heparin-inhibition of PAR(2) activation by tryptase could provide novel mechanisms for regulating receptor activation by tryptase and possibly other proteases.

Amino Acid Motifs↗
PubMed2004

Inhibition of tryptase release from human colon mast cells by protease inhibitors.

AIM: To investigate the ability of protease inhibitors to modulate tryptase release from human colon mast cells. METHODS: Enzymatically dispersed cells from human colon were challenged with anti-IgE or calcium ionophore A23187 in the absence or presence of tryptase and chymase inhibitors, and tryptase release was determined. RESULTS: IgE dependent tryptase release from colon mast cells was inhibited by up to approximately 37%, 40% and 36.6% by chymase inhibitors Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPFM), N-tosyl-L-phenylalanyl-chloromethyl ketone (TPCK), and alpha1-antitrypsin, respectively. Similarly, the inhibitors of tryptase leupeptin, N-tosyl-L-lysine chloromethyl ketone (TLCK) and lactoferrin were also able to inhibit anti-IgE induced tryptase release by a maximum of 39.4%, 47.6% and 36.6%, respectively. The inhibitory actions of chymase inhibitors, but not tryptase inhibitors on colon mast cells were enhanced by preincubation of them with cells for 20 min before challenged with anti-IgE. At a concentration of 10 microg/mL, protamine was able to inhibit anti-IgE and calcium ionophore induced tryptase release. However, at 100 microg/mL, protamine elevated tryptase levels in supernatants. A specific inhibitor of aminopeptidase amastatin had no effect on anti-IgE induced tryptase release. The significant inhibition of calcium ionophore induced tryptase release was also observed with the inhibitors of tryptase and chymase examined. The inhibitors tested by themselves did not stimulate tryptase release from colon mast cells. CONCLUSION: It was demonstrated for the first time that both tryptase and chymase inhibitors could inhibit IgE dependent and calcium ionophore induced tryptase release from dispersed colon mast cells in a concentration dependent of manner, which suggest that they are likely to be developed as a novel class of anti-inflammatory drugs to treat chronic of colitis in man.

Colectomy↗
PubMed2006

Mast cell tryptase in postmortem serum-reference values and confounders.

We have investigated the effects of some factors suspected of inducing spuriously increased tryptase concentrations, specifically sampling site, conjunctival petechial bleeding and prone position at the time of death as indicators of premortem asphyxia, and resuscitation efforts by external cardiac massage. Tryptase was measured in blood from the femoral vein in 60 deaths: 39 control cases who died rapidly (within minutes) from natural causes (sudden cardiac death and acute aortic dissection), 16 with death caused by prolonged asphyxia (traumatic compression of the chest and suffocation due to body position or smothering), and five anaphylactic deaths. In 44 of these cases, tryptase was measured in both heart and femoral blood. Mast cell tryptase was analyzed with a commercial FEIA method (Pharmacia Diagnostics AB, Uppsala, Sweden) measuring both alpha- and beta-tryptase. Assuming that tryptase values in the control group were gamma distributed, we calculated the upper normal limits for tryptase concentrations in femoral blood. It was found that 95% of the controls had values below 44.3 mug/l (femoral blood), SD 5.27 mug/l. All but one of the anaphylactic deaths had tryptase concentrations exceeding that limit. Tryptase was significantly elevated in femoral blood from anaphylactic deaths (p<0.007), compared with the controls. Also, in the cases where death had occurred due to asphyxia tryptase was elevated in femoral blood (p<0.04). A significant difference in tryptase concentrations was seen between blood from the heart and the femoral vessels (p<0.02) in the whole material (n=44). Tryptase concentrations in femoral blood were not influenced by prone position at death, or resuscitation efforts. It is concluded that asphyxia premortem seems to affect tryptase concentrations, that postmortem tryptase measurements should be done in serum from femoral blood, and that the normal upper limit, covering 95%, is 44.3 microg/l.

Adolescent↗
PubMed1997

Human mast cell tryptase: a stimulus of microvascular leakage and mast cell activation.

We have investigated the potential of tryptase to stimulate an increase in microvascular permeability following injection into the skin of guinea pigs. Tryptase was isolated from high salt extracts of human lung tissue by octyl-agarose and heparin-agarose chromatography. Injection of purified tryptase (2.5 ng-2.5 microg/site) into the skin of guinea pigs which had been injected intravenously with Evans blue dye provoked a dose-dependent increase in microvascular permeability. The skin reactions elicited by tryptase were apparent up to 80 min following injection, while histamine-induced microvascular leakage resolved completely by 40 min. Heat-inactivation of tryptase, or preincubating the proteinase with certain proteinase inhibitors, significantly reduced the extent of microvascular leakage, suggesting dependency on an intact catalytic site. No evidence was found for a synergistic or antagonistic interaction between tryptase (2.5 ng-2.5 microg/site) and histamine (1-10 microg/site) when these mast cell products were injected together. Addition of heparin to tryptase (10:1; w/w) prior to injection was without effect on tryptase-induced microvascular leakage. Pretreatment of guinea pigs with a combination of the histamine H1 receptor antagonist pyrilamine and the histamine H2 receptor antagonist cimetidine (both 10 mg/kg), partially abolished tryptase-induced microvascular leakage as well as attenuating the reaction to histamine. Reasoning that the microvascular leakage induced by tryptase is likely to involve the release of histamine, we investigated the ability of tryptase to stimulate histamine release from dispersed guinea-pig skin and lung cells in vitro. Tryptase was found to induce concentration-dependent histamine release from both sources of tissue. Mast cell activation stimulated by tryptase in vitro was inhibited by heat treating the enzyme or by addition of proteinase inhibitors, suggesting a requirement for an intact catalytic site. Histamine release was inhibited also by preincubating cells with the metabolic inhibitors antimycin A and 2-deoxy-D-glucose indicating that the mechanism was energy-requiring and non-cytotoxic. We conclude that human mast cell tryptase may be a potent stimulus of microvascular leakage. The activation of mast cells by this proteinase may represent an amplification process in allergic inflammation.

Animals↗
PubMed1997

Interactions of mast cell tryptase with thrombin receptors and PAR-2.

Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-protein-coupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca2+. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca2+. These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.

Amino Acid Sequence↗
PubMed2002

Serum tryptase measurements in patients with myelodysplastic syndromes.

Abnormal differentiation and maturation of hemopoietic cells are characteristic features of myelodysplastic syndromes (MDS). Tryptases (alpha- and beta-type) are lineage-restricted serine proteases primarily expressed in mast cells (MC). We have analyzed expression of tryptase in 89 de novo MDS patients (refractory anemia (RA), n = 30; RA with ringed sideroblasts (RARS), n = 21; RA with excess of blasts (RAEB/RAEB-t), n = 27; chronic myelomonocytic leukemia (CMML), n = 11). Serum levels of total tryptase (alpha - protryptase + beta - tryptase) were measured by FIA. The numbers of tryptase+ cells were determined in paraffin-embedded bone marrow (bm) sections by immunohistochemistry and morphometry. In healthy individuals, serum total tryptase levels ranged between < 1 and 15 ng/ml (5.6 +/- 2.8 ng/ml). Tryptase levels of > 20 ng/ml were detected in 5/22 patients with RA (22.7%), 4/17 with RARS (23.5%), 0/16 with RAEB/RAEB-t, and 3/8 with CMML (37.5%). Thus, serum tryptase concentrations were higher in RA (16.6 +/- 14.3 ng/ml), RARS (12.9 +/- 8.2), and CMML (16.5 +/- 7.6) compared to RAEB/-t (8.7 +/- 3.8). By morphometry, elevated numbers of tryptase+ bm cells were detected in all MDS groups (RA: 139 +/- 131; RARS: 118 +/- 98; RAEB/RAEB-t: 80 +/- 79; CMML: 105 +/- 114 cells/mm2) compared to controls (54 +/- 51 cells/mm2). As assessed by Northern blotting and protein analysis, bm cells in MDS primarily produced alpha-(pro)tryptase, but little or no beta-tryptase. Together, our data show that elevated levels of tryptase are detectable in a group of patients with MDS probably because of an increase in neoplastic (mast) cells producing the enzyme(s). In addition, serum tryptase levels appear to correlate with MDS variants. Follow up studies should clarify whether an elevated tryptase concentration in MDS is of prognostic significance.

Adult↗
PubMed1995

Role of tryptase in immediate cutaneous responses in allergic sheep.

In this study, we used a specific tryptase inhibitor, APC-366 [N-(1-hydroxy-2-napthoyl)-L-arginyl-L- prolinamide hydrochloride] to investigate the effect of intradermally administered tryptase and tryptase released by antigen challenge on the immediate cutaneous reaction (ICR) in allergic sheep. The surface areas of cutaneous wheals produced by intradermal injections (0.05 ml) of 1 and 10 ng tryptase alone, tryptase combined with 3 U heparin (tryptase-heparin), or Ascaris suum antigen (10(-5) dilution) with or without pretreatment with APC-366 (1 mg/ml) were measured at 20 and 60 min after challenge. Intradermal injections of 1 and 10 ng tryptase alone (n = 7) produced an ICR of < or = 20% of that obtained after injection of histamine (5% wt/vol). Intradermal injection of tryptase-heparin (n = 7), however, resulted in 50 (1 ng) and 82% (10 ng) of the ICR to histamine (both, P < 0.05 vs. tryptase alone). APC-366 inhibited (P < 0.05) the ICR to 1 and 10 ng tryptase-heparin by > or = 70% at all times (n = 8) but had no effect on the histamine-induced ICR (n = 3). A combination of the histamine H1 antagonist chlorpheniramine (2 mg/kg iv) and the H2 antagonist metiamide (3 mg/kg iv) given 40 min before challenge (n = 8) inhibited the response to 1 and 10 ng tryptase-heparin by 42 and 62% at 20 min and by 96 and 86% at 60 min, respectively (all, P < 0.05). APC-366 also blocked the ICR to A. suum antigen by 68% (P < 0.05) in nine sheep. These results indicate that intradermal injection of tryptase-heparin can induce an ICR. This ICR can be inhibited by APC-366 or a combination of the histamine H1 and H2 antagonists, suggesting that the tryptase response is mediated by histamine. APC-366 also blocks the mast cell-mediated ICR to intradermally injected A. suum antigen. Collectively, these results suggest that tryptase may modulate mast cell histamine release.

Animals↗
PubMed1996

Inhaled tryptase causes bronchoconstriction in sheep via histamine release.

Allergen-induced bronchoconstriction involves mast cell activation. Tryptase is a mast cell serine protease that is released during this process, but little is known about the action of tryptase in the airway. The purpose of this study was to determine: (1) if aerosolized tryptase causes bronchoconstriction, and (2) the mechanism by which this occurs. We measured mean pulmonary flow resistance (RL) in five allergic sheep before and after consecutive inhalations of 100 and 500 ng tryptase (in 2 ml total volume). Inhaled tryptase at 100 and 500 ng increased RL (mean +/- SE) by 33 +/- 12 and 122 +/- 8% (p < 0.05) over baseline. The response was reproducible upon repeat challenges. These studies were repeated in the same animals after pretreatment with aerosolized APC 366 (9 mg/3 ml), a specific tryptase inhibitor. In APC-366-treated sheep, tryptase increased RL by 10 +/- 3 and 6 +/- 2% (p < 0.05 versus control values) at 100 and 500 ng, respectively. The response to tryptase was also blocked by pretreating the sheep intravenously with the histamine H1-antagonist chlorpheniramine (2 mg/kg), in which RL increased only 5 +/- 4 and 7 +/- 6% after 100 and 500 ng tryptase. APC 366, however, did not block histamine-induced bronchoconstriction. Consistent with these findings was the observation that segmental bronchial challenge with tryptase (1 microgram) resulted in a significant increase in histamine levels in bronchoalveolar lavage. Inhaled tryptase (500 ng) also caused airway hyperresponsiveness to aerosolized carbachol 2 h after tryptase challenge. This tryptase-induced airway hyperresponsiveness could be blocked either by pretreating the sheep with APC 366 (30 min before challenge) or by treating the sheep 30 min after challenge. These results indicate that inhaled tryptase causes bronchoconstriction and airway hyperresponsiveness in allergic sheep by an event that may involve mast cell activation.

Administration, Inhalation↗
PubMed1998

Inhibitors of tryptase for the treatment of mast cell-mediated diseases.

Human tryptase is a structurally unique and mast cell specific trypsin-like serine protease. Recent biological and immunological investigations have implicated tryptase as a mediator in the pathology of numerous allergic and inflammatory conditions including rhinitis, conjunctivitis, and most notably asthma. A growing body of data further implicates tryptase in certain gastrointestinal, dermatological, and cardiovascular disorders as well. The recent availability of potent, and selective tryptase inhibitors, though, has facilitated the validation of this protease as an important therapeutic target as well. Herein, we describe the design and potency of four classes of selective tryptase inhibitors, of which the first three types are synthetic and the fourth is natural in origin: 1) peptidic inhibitors (e.g., APC-366), 2) dibasic inhibitors (i.e., pentamidine-like), 3) Zn(2+)-mediated inhibitors (i.e., BABIM-like), and 4) heparin antagonists (e.g., lactoferrin). These inhibitors have been tested in the airways and skin of allergic sheep. Aerosol administration of tryptase inhibitors from each structural class 30 minutes before, and 4 hours and 24 hours after allergen challenge, abolishes late phase bronchoconstriction and airway hyperresponsiveness in a dose-dependent manner. Moreover, intradermal injection of APC-366 blocks the cutaneous response to antigen. These studies provide the essential proof-of-concept for the further pursuit of tryptase inhibitors for the treatment of asthma, and perhaps other allergic diseases. Results from clinical studies with the first generation tryptase inhibitor APC-366, currently in phase II trials for the treatment of asthma, provide additional support for a pathological role for tryptase in this disease. Notable advances in the area of tryptase inhibitor design at Axys Pharmaceuticals, Inc. include a novel, zinc-mediated, serine protease inhibitor technology (described herein), and the discovery of a unique class of extremely potent and selective dibasic tryptase inhibitors. Independently, an X-ray crystal structure of active tryptase tetramer complexed with 4-amidinophenyl pyruvic acid has been reported. It is anticipated that these discoveries will further accelerate the design of structurally novel tryptase inhibitors as well as the development of new drugs for the treatment of mast cell tryptase-mediated disorders.

Animals↗
PubMed1985

The fibrinogenolytic activity of purified tryptase from human lung mast cells.

The capacity of purified tryptase from human lung mast cells to metabolize human fibrinogen, fibrin, and plasminogen was evaluated. Tryptase (5 micrograms/ml) inactivated the thrombin-induced clotting activity of fibrinogen (100 micrograms/ml) with essentially similar t 1/2 values of 4.6 min in the absence of heparin and 5.8 min in the presence of heparin (20 micrograms/ml) that were not appreciably different than with lysine-Sepharose-purified plasmin (5 micrograms/ml). Fibrinogen treated with tryptase together with heparin lost all detectable clotting activity by 4 hr at 37 degrees C, whereas fibrinogen treated with tryptase alone resulted in destruction of only 80% of fibrinogen clotting equivalents after 16 hr. Tryptase alone was observed to cleave only the alpha-chains of fibrinogen by electrophoresis of tryptase-treated, denatured, and reduced fibrinogen in polyacrylamide gradient gels. Tryptase together with heparin cleaved first the alpha-chain and then the beta-chain, the latter cleavage corresponding to complete loss of fibrinogen clotting activity by 4 hr. No fibrinogen fragments with anticoagulant activity were generated by tryptase. In contrast, plasmin left no residual clotting activity after 4 hr of incubation and generated fibrinogen fragments with anticoagulant activity. Plasmin sequentially cleaved the alpha, beta, and gamma subunits of fibrinogen. Tryptase alone (6 micrograms/ml) or together with heparin (20 micrograms/ml) failed to activate plasminogen (0.6 mg/ml) after a 60-min incubation at 37 degrees C. Addition of urokinase to tryptase-treated or untreated plasminogen resulted in essentially identical plasmin activities (0.32 and 0.34 U/ml, respectively), indicating that tryptase neither activates nor destroys plasminogen. Tryptase (700 ng) also failed to substantially solubilize cross-linked fibrin (2.6 micrograms) or the corresponding amount of fibrinogen bound to plastic microtiter plates with or without heparin. The failure to solubilize fibrinogen and, possibly, fibrin is consistent with the observation that the apparent m.w. by SDS polyacrylamide gel electrophoresis of unreduced fibrinogen is not appreciably altered by prior treatment with tryptase, even though cleavage of alpha-and beta-chains is revealed after reduction. Fibrinogenolysis by tryptase complements other mast cell mediators with anticoagulant properties such as heparin and suggests a significant prevention of coagulation by activated mast cells.

Blood Coagulation Tests↗
PubMed2006

Expression and characterization of recombinant gamma-tryptase.

Tryptases are trypsin-like serine proteases whose expression is restricted to cells of hematopoietic origin, notably mast cells. gamma-Tryptase, a recently described member of the family also known as transmembrane tryptase (TMT), is a membrane-bound serine protease found in the secretory granules or on the surface of degranulated mast cells. The 321 amino acid protein contains an 18 amino acid propeptide linked to the catalytic domain (cd), followed by a single-span transmembrane domain. gamma-Tryptase is distinguished from other human mast cell tryptases by the presence of two unique cysteine residues, Cys(26) and Cys(145), that are predicted to form an intra-molecular disulfide bond linking the propeptide to the catalytic domain to form the mature, membrane-anchored two-chain enzyme. We expressed gamma-tryptase as either a soluble, single-chain enzyme with a C-terminal His tag (cd gamma-tryptase) or as a soluble pseudozymogen activated by enterokinase cleavage to form a two-chain protein with an N-terminal His tag (tc gamma-tryptase). Both recombinant proteins were expressed at high levels in Pichia pastoris and purified by affinity chromatography. The two forms of gamma-tryptase exhibit comparable kinetic parameters, indicating the propeptide does not contribute significantly to the substrate affinity or activity of the protease. Substrate and inhibitor library screening indicate that gamma-tryptase possesses a substrate preference and inhibitor profile distinct from that of beta-tryptase. Although the role of gamma-tryptase in mast cell function is unknown, our results suggest that it is likely to be distinct from that of beta-tryptase.

Enteropeptidase↗
PubMed1993

Bis(5-amidino-2-benzimidazolyl)methane and related amidines are potent, reversible inhibitors of mast cell tryptases.

Tryptase is the major secretory protease of human mast cells and is proposed to be involved in neuropeptide processing and tissue inflammation. Exploration of the biology of tryptase has been hindered by the lack of potent, selective inhibitors. The current study explores the properties of aromatic diamidines as inhibitors of dog and human tryptase. The strongest inhibitors of tryptase in this series are bis(5-amidino-2-benzimidazolyl)methane (BABIM) and (5-amidino-2-benzimidazolyl)-(5-(N,N'-dimethylamidino)-2- benzimidazolyl)methane, which exhibit K(i) values of 1.8 and 1.4 nM, respectively, in blocking the hydrolysis of tosyl-L-Gly-Pro-Lys-4-nitroanilide by human tryptase. These compounds are approximately 10,000-fold more potent than benzamidine, and are the strongest reversible inhibitors of tryptase described to date. Other aromatic mono- and diamidines, including amiloride and pentamidine, are less potent. Nonetheless, they abolish tryptase activity at high inhibitor concentrations. The rank order of tryptase inhibitor potency parallels that of inhibitors tested against trypsin. BABIM, the only highly active member of this series whose potency against other targets has been examined previously, is a far stronger inhibitor of tryptase than of other trypsin-like serine proteases, including those involved with hemostasis, fibrinolysis and the complement system. Therefore, BABIM appears to have selective affinity for tryptase. In addition to inhibiting tryptase-induced hydrolysis of peptide-based chromogenic substrates, BABIM blocks completely the reversal of vasoactive intestinal peptide-induced relaxation of isolated trachea by dog tryptase. Thus, BABIM and related amidines are potent inhibitors of mast cell tryptases that may be useful in exploring mast cell protease biology.

Amiloride↗
PubMed1989

Time course of appearance and disappearance of human mast cell tryptase in the circulation after anaphylaxis.

Tryptase, a neutral protease of human mast cells, is a potentially important indicator of mast cell involvement in various clinical conditions. The current study examined the time course of appearance and disappearance of tryptase in the circulation after an anaphylactic event and the stability of both endogenous and exogenous tryptase in terms of freeze-thawing and temperature. The peak level of tryptase after an experimentally induced systemic anaphylactic reaction occurred 1-2 h after the initiating bee sting in each of three subjects, in contrast to histamine levels which peaked at 5-10 min. In some cases elevated levels of tryptase may not be detected during the initial 15-30 min. Tryptase levels then declined under apparent first order kinetics with a t1/2 of approximately 2h. Similar disappearance kinetics were observed for two subjects presenting in the emergency room with immediate type reactions, one with severe asthma after indomethacin ingestion, the other with systemic anaphylaxis after a bee sting. Histamine levels declined rapidly and were back to baseline by 15-60 min. Measured levels of tryptase in serum or plasma were not diminished by up to four freeze-thaw cycles. Incubation of serum samples taken from subjects with elevated levels of tryptase at 22 and 37 degrees C indicated that greater than 50% of endogenous tryptase was still detected after 4 d. Purified tryptase added to serum or plasma and incubated as above was less stable: approximately 50% of exogenous tryptase in serum and approximately 15% in plasma was detected after 2d of incubation. Therefore, optimally samples should be stored frozen, but even those stored at room temperature for up to 4 d should be satisfactory. The best time to obtain samples for tryptase determinations is 1-2 h after the precipitating event, but depending on the magnitude of the initial response elevated levels of tryptase may be present in the circulation for several hours.

Adult↗
PubMed1999

Increased mast cell tryptase in sudden infant death - anaphylaxis, hypoxia or artefact?

BACKGROUND: Increased concentrations of mast cell tryptase in post mortem blood have frequently been observed in sudden infant deaths but the cause of this has not yet been clarified. OBJECTIVE: The aim was to evaluate factors (immunological, morphological and anamnestic data) behind the observed increase in mast cell tryptase in sudden infant deaths with elevated tryptase. METHODS: Mast cell tryptase and total immunoglobulin (Ig) E were measured in post mortem sera from 44 infants younger than 1.5 years. Radioallergosorbent tests were performed for possible allergens (mixture for relevant food allergens, Phadiatop and latex). IgG subclasses, IgM, and complement factors (C3, C4 and factor B) were measured with radial immunodiffusion. Mast cells, labelled with antibodies against mast cell tryptase, were counted in the lungs and heart. The circumstances of death and medical history of the deceased infant and family were obtained through police and hospital records. RESULTS: In 40% of the SIDS cases tryptase was elevated (>10 microg/L). Total IgE in serum was increased in 33% compared with clinical reference values but showed no association with mast cell tryptase. RAST tests were positive in three cases. In one of these cases both tryptase and total IgE were elevated. The only variable that was associated with high tryptase values was prone position at death (P < or = 0.05 ). Allergy or asthma in the family were alleged in 50% of the cases, but was not associated with elevated tryptase or IgE. Children with elevated total IgE also displayed high concentrations of IgG1 and IgG2. Infants who died in the spring had significantly higher IgE than the others (P < or = 0.05). CONCLUSION: The results do not support the hypothesis that the elevated tryptase concentrations in sudden infant death are caused by allergy. The association between prone position at death and elevated tryptase could hypothetically be explained by mast cell degranulation due to, for example, a hypoxic stimulus in these infants.

Anaphylaxis↗
PubMed2002

Characterization of mast-cell tryptase-expressing peripheral blood cells as basophils.

BACKGROUND: Mast-cell tryptase is a protease with proinflammatory activity, the expression of which by peripheral blood leukocytes (PBLs) has not been fully characterized. OBJECTIVE: We examined tryptase expression in human PBLs to further characterize this tryptase-expressing cell population for lineage and disease association. METHODS: PBLs were fixed, permeabilized, stained with antibodies to tryptase and a panel of mast cell- and basophil-specific markers, and analyzed by means of flow cytometry. RESULTS: Tryptase expression was restricted to a population of cells that stained positive for IgE and negative for the panel of lineage markers (IgE(+), lin(-)). This IgE(+), lin(-) population did not stain for the mast-cell markers Kit or chymase but did stain for the basophil-specific granule proteins recognized by the 2D7 and BB1 mAbs. Per-cell tryptase expression demonstrated a greater than 100-fold range of expression among donors but did not correlate with disease status (asthma or mastocytosis), FEV(1), or serum tryptase concentration. Tryptase was released by purified basophils after anti-IgE activation. CONCLUSIONS: The phenotype of tryptase-expressing PBLs and their lack of increase in patients with mastocytosis demonstrates that these cells are basophils. Per-cell basophil tryptase expression is highly variable between donors, with some donors expressing levels approaching those of mast cells. As such, anti-tryptase antibodies cannot be used to distinguish these 2 cell types from one another by means of flow cytometry. These results demonstrate that tryptase represents an additional mediator through which basophils may contribute to allergic inflammation.

Asthma↗