Biomedical subjects

G Pejler

Publications and source records attributed to G Pejler.

At least 19 recordsLinked to original sources

PubMed2001

Regulation of extravascular coagulation and fibrinolysis by heparin-dependent mast cell chymase.

We recently characterized a heparin-deficient mouse strain generated by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2). The NDST-2-/- mice show severe defects in their organization of mast cell (MC) secretory granules, with an almost total absence of the various heparin-binding MC proteases. In the present report we have studied the consequences of heparin/MC protease deficiency for extravascular coagulation and fibrinolysis. Addition of prothrombin to peritoneal cells-a mixture of macrophages, lymphocytes, and MCs-resulted in formation of thrombin but the accumulation of thrombin occurred faster in the NDST-2-/-cells than in normal controls. Further, the generated thrombin was subsequently inactivated in the NDST-2+/+ cell cultures but not in the NDST-2-/- cells. Plasminogen was activated to plasmin at an apparently higher rate in peritoneal cells from NDST-2 null mice than in the normal controls. Similar to thrombin, the generated plasmin was inactivated by NDST-2+/+ but not by the NDST-2-/- cells. Subsequent experiments with normal cells showed that cell surface-associated MC chymase, in a strongly heparin-dependent manner, was responsible for both the thrombin-inactivating- and plasmin-inactivating activities. These results show that MC chymase-heparin complexes have the potential to regulate extravascular coagulation processes, as well as the plasminogen activator/plasmin system.

Amidohydrolases↗

PubMed2001

Structural requirements and mechanism for heparin-induced activation of a recombinant mouse mast cell tryptase, mouse mast cell protease-6: formation of active tryptase monomers in the presence of low molecular weight heparin.

Mast cell tryptase is stored as an active tetramer in complex with heparin in mast cell secretory granules. Previously, we demonstrated the dependence on heparin for the activation/tetramer formation of a recombinant tryptase. Here we have investigated the structural requirements for this activation process. The ability of heparin-related saccharides to activate a recombinant murine tryptase, mouse mast cell protease-6 (mMCP-6), was strongly dependent on anionic charge density and size. The dose-response curve for heparin-induced mMCP-6 activation displayed a bell-shaped appearance, indicating that heparin acts by binding to more than one tryptase monomer simultaneously. The minimal heparin oligosaccharide required for binding to mMCP-6 was 8-10 saccharide units. Gel filtration analyses showed that such short oligosaccharides were unable to generate tryptase tetramers, but instead gave rise to active mMCP-6 monomers. The active monomers were inhibited by bovine pancreatic trypsin inhibitor, whereas the tetramers were resistant. Furthermore, monomeric (but not tetrameric) mMCP-6 degraded fibronectin. Our results suggest a model for tryptase tetramer formation that involves bridging of tryptase monomers by heparin or other highly sulfated polysaccharides of sufficient chain length. Moreover, our results raise the possibility that some of the reported activities of tryptase may be related to active tryptase monomers that may be formed according to the mechanism described here.

Cell Line↗

PubMed2001

Mice lacking histidine decarboxylase exhibit abnormal mast cells.

Histidine decarboxylase (HDC) synthesizes histamine from histidine in mammals. To evaluate the role of histamine, we generated HDC-deficient mice using a gene targeting method. The mice showed a histamine deficiency and lacked histamine-synthesizing activity from histidine. These HDC-deficient mice are viable and fertile but exhibit a decrease in the numbers of mast cells while the remaining mast cells show an altered morphology and reduced granular content. The amounts of mast cell granular proteases were tremendously reduced. The HDC-deficient mice provide a unique and promising model for studying the role of histamine in a broad range of normal and disease processes.

Alleles↗

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↗

PubMed2001

Human peripheral blood mononuclear cells require factor X and platelets for expression of prothrombinase activity in response to bacterial lipopolysaccharide.

We investigated whether human peripheral blood mononuclear cells (PBMC) express prothrombinase following stimulation with bacterial lipopolysaccharide (LPS). LPS-stimulated PBMC devoid of contaminating platelets failed to activate prothrombin directly. Addition of platelets did not result in expression of prothrombinase in the absence of factor X whereas the combination of platelets and factor X resulted in strong prothrombinase activity on LPS-activated cells. The induced prothrombinase was dependent on tissue factor, as the activity was completely inhibited by an anti-tissue factor antibody. Our data suggest that platelet/monocyte cooperation is important in the generation of prothrombinase activity in response to endotoxin.

Antibodies↗

PubMed2000

Mechanism for activation of mouse mast cell tryptase: dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6.

Tryptase, a serine protease with trypsin-like substrate cleavage properties, is one of the key effector molecules during allergic inflammation. It is stored in large quantities in the mast cell secretory granules in complex with heparin proteoglycan, and these complexes are released during mast cell degranulation. In the present paper, we have studied the mechanism for tryptase activation. Recombinant mouse tryptase, mouse mast cell protease 6 (mMCP-6), was produced in a mammalian expression system. The mMCP-6 fusion protein contained an N-terminal 6 x His tag followed by an enterokinase (EK) site replacing the native activation peptide (6xHis-EK-mMCP-6). In the absence of heparin, barely detectable enzyme activity was obtained after enterokinase cleavage of 6xHis-EK-mMCP-6 over a pH range of 5.5-7.5. However, when heparin was present, 6xHis-EK-mMCP-6 yielded active enzyme when enterokinase cleavage was performed at pH 5.5-6.0 but not at neutral pH. Affinity chromatography analysis showed that mMCP-6 bound strongly to heparin-Sepharose at pH 6.0 but not at neutral pH. After enterokinase cleavage of the sample at pH 6.0, mMCP-6 occurred in inactive monomeric form as shown by FPLC analysis on a Superdex 200 column. When heparin was added at pH 6.0, enzymatically active higher molecular weight complexes were formed, e.g., a dominant approximately 200 kDa complex that may correspond to tryptase tetramers. No formation of active tetramers was observed at neutral pH. When injected intraperitoneally, mMCP-6 together with heparin caused neutrophil influx, but no signs of inflammation were seen in the absence of heparin. The present paper thus indicates a crucial role for heparin in the formation of active mast cell tryptase.

Acids↗

PubMed2000

Altered processing of fibronectin in mice lacking heparin. a role for heparin-dependent mast cell chymase in fibronectin degradation.

We have previously generated a mouse strain with a defect in its heparin biosynthesis by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2). The NDST-2(-/-) mice show reduced levels of various mast cell mediators such as histamine and various heparin-binding mast cell proteases, including chymases, tryptases, and carboxypeptidase A. In this work we have addressed the possible functional consequences of the lack of sulfated heparin. Peritoneal cells were harvested from normal and NDST-2(-/-) mice. After culturing the cells, conditioned media were collected and were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Several differences in the protein patterns were observed, including the presence of large amounts of a approximately 250-kDa protein in medium from NDST-2(-/-) mice that was absent in normal controls. Peptide microsequencing revealed identity of this protein with fibronectin. Western blot analysis showed the presence of fibronectin degradation products in cell cultures from normal mice, which were absent in cultures from NDST-2(-/-) animals. Further experiments showed that the degradation of fibronectin observed in cell cultures from NDST-2(+/+) mice was catalyzed by mast cell chymase in a strongly heparin-dependent manner. This report thus indicates a biological function for chymase/heparin proteoglycan complexes in fibronectin turnover.

Amidohydrolases↗

PubMed2000

Macrophages synthesize factor X and secrete factor X/Xa-containing prothrombinase activity into the surrounding medium.

Activation of the coagulation cascade, mediated by various monocyte/macrophage procoagulants, is an important component in the pathology of inflammatory disease. The type of procoagulant expressed may vary between different monocyte/macrophage subtypes and may differ depending on how the cells are treated. In the present study we show that both murine peritoneal macrophages and human adherent synovial cells from rheumatoid arthritis lesions express prothrombinase activity that was inhibited by anti-Factor X antibodies. Northern blot analysis showed that Factor X was transcribed by the murine peritoneal cells and Western blot analysis showed the presence of Factor X antigen. Further experiments showed that the prothrombinase activity was secreted by the cells into the medium in a detergent-sensitive form, suggesting that the prothrombinase is released on small lipid-containing vesicles.

Animals↗

PubMed1999

Mechanism by which heparin proteoglycan modulates mast cell chymase activity.

Chymases are highly basic chymotrypsin-like serine proteases expressed exclusively by mast cells. Large amounts of chymases complexed with heparin proteoglycan (PG) are released in vivo during mast cell activation. The tight binding of chymase to heparin PG results in increased activity of the protease toward certain substrates, e.g., thrombin and MeO-Suc-Arg-Pro-Tyr-pNA (S-2586). In this study, the mechanism by which heparin PG modulates chymase activity was investigated, using thrombin and various chromogenic peptide substrates as model substrates. Incubation of thrombin with oligonucleotides that block the heparin-binding site of thrombin abolished the stimulatory effect of heparin PG on thrombin inactivation. Further, thrombin mutants with defects in their heparin-binding regions were less efficiently inactivated by chymase-heparin PG than wild type thrombin. These findings suggest a model for chymase stimulation where heparin PG may promote the chymase-catalyzed cleavage of heparin-binding substrates by simultaneously binding to both chymase and substrate. Experiments in which various chromogenic peptide substrates were utilized showed that heparin PG enhanced the activity of chymase toward positively charged peptide substrates such as S-2586, whereas the cleavage of uncharged substrates was not affected by the presence of heparin PG. On the basis of the latter findings, an alternative stimulation mechanism is discussed where heparin PG may stimulate chymase activity by blocking positively charged regions in chymase, thereby reducing the level of electrostatic repulsion between chymase and positively charged substrates.

Animals↗

PubMed1999

Abnormal mast cells in mice deficient in a heparin-synthesizing enzyme.

Heparin is a sulphated polysaccharide, synthesized exclusively by connective-tissue-type mast cells and stored in the secretory granules in complex with histamine and various mast-cell proteases. Although heparin has long been used as an antithrombotic drug, endogenous heparin is not present in the blood, so it cannot have a physiological role in regulating blood coagulation. The biosynthesis of heparin involves a series of enzymatic reactions, including sulphation at various positions. The initial modification step, catalysed by the enzyme glucosaminyl N-deacetylase/N-sulphotransferase-2, NDST-2, is essential for the subsequent reactions. Here we report that mice carrying a targeted disruption of the gene encoding NDST-2 are unable to synthesize sulphated heparin. These NDST-2-deficient mice are viable and fertile but have fewer connective-tissue-type mast cells; these cells have an altered morphology and contain severely reduced amounts of histamine and mast-cell proteases. Our results indicate that one site of physiological action for heparin could be inside connective-tissue-type mast cells, where its absence results in severe defects in the secretory granules.

Amidohydrolases↗

PubMed1999

Procoagulant and anticoagulant activities of resident and inflammatory peritoneal cells.

OBJECTIVE AND DESIGN: In the present study, the involvement of both procoagulants and anticoagulants from adherent peritoneal cells in an inflammatory model was investigated. MATERIAL AND TREATMENT: Mice were injected with thioglycollate broth or lipopolysaccharide (LPS). METHODS: Cells were harvested by peritoneal lavage. Adherent peritoneal cells were cultured further +/- LPS. Subsequently, their ability to activate either Factor X or prothrombin, and their ability to inactivate thrombin through a thrombin-degrading mast cell chymase, was assayed. RESULTS: Inflammatory cells expressed reduced amounts of thrombin-inactivating activity as compared with control resident peritoneal cells. Both resident and inflammatory cells expressed potent prothrombinase activities. Further stimulation of the various cellular populations with LPS in vitro had very little effect on the prothrombinase and thrombin-inactivating activities, bud had a strong stimulatory effect on the Factor X-activating activities.

Animals↗

PubMed1998

Vitronectin expression in rheumatoid arthritic synovia--inhibition of plasmin generation by vitronectin produced in vitro.

The plasmin-generating system controls, to a great extent, the degree of connective tissue destruction as well as fibrin deposition two contributors to the pathogenesis generated in diseases such as rheumatoid arthritis. Vitronectin, an adhesive blood glycoprotein, has the potential to modulate this system by its known capacity to interact with plasminogen activator inhibitor-1, plasminogen activators, the urokinase plasminogen activator receptor, and plasminogen. The net effect of these interactions, in terms of plasmin generation, is not known as yet. In the present study, we have investigated the possible expression and role of vitronectin in rheumatoid arthritic synovia. Analysis of synovial frozen sections by immunofluorescence showed the presence of vitronectin in the 13 cases studied. In situ hybridization analysis demonstrated the presence of vitronectin mRNA in cells present in areas rich in infiltrating inflammatory cells. The adherent population of the rheumatoid arthritic synovial cells was isolated and found to synthesize and secrete vitronectin into the medium (seven of 10 isolates), as assessed by metabolic labelling and immunoprecipitation. Plasmin-generating activity was detected in the adherent synovial cells, and this activity was increased by antibodies to vitronectin. Our findings show, for the first time, that vitronectin can be endogenously produced in a pathophysiological system where it can inhibit the generation of plasmin.

Arthritis, Rheumatoid↗

PubMed1997

Secretory granule proteases in rat mast cells. Cloning of 10 different serine proteases and a carboxypeptidase A from various rat mast cell populations.

Two of the major rat mast cell proteases, rat mast cell protease 1 (RMCP-1) and RMCP-2, have for many years served as important phenotypic markers for studies of various aspects of mast cell (MC) biology. However, except for these proteases only fragmentary information has been available on the structure and complexity of proteases expressed by different subpopulations of rat MCs. To address these questions, cDNA libraries were constructed from freshly isolated rat peritoneal MCs and from the rat mucosal MC line RBL-1. cDNA clones for 10 different serine proteases (RMCP-1-10), and the MC carboxypeptidase A were isolated and characterized. Six of these proteases have not been isolated previously. Based on their protease content, three separate subpopulations of MCs were identified. Connective tissue MCs (CTMCs) from the ear and peritoneum express the chymases RMCP-1 and -5, the tryptases RMCP-6, and -7 and the carboxypeptidase A. However, based on a large difference in the level of expression of RMCP-7, CTMCs of these two organs may be regarded as two separate subpopulations. RMCP-2 and the three closely related proteases of the RMCP-8 subfamily were identified as the major mucosal MC proteases in rat. In contrast to what has been reported for human MCs, no expression of cathepsin G or cathepsin G-like proteases was detected in any of the rat MC populations. To determine mRNA frequencies for the various proteases expressed by normal tissue MCs, an unamplified peritoneal MC cDNA library was screened with a panel of mono-specific cDNA probes. These results showed that peritoneal MCs are highly specialized effector cells with mRNA frequencies for the major proteases in the range of several percent of the total mRNA pool.

Amino Acid Sequence↗

PubMed1997

Cloning, structural analysis, and expression of the pig IgE epsilon chain.

As a step in the evolutionary studies of immunoglobulin E (IgE) and for the purpose of developing new reagents that will facilitate a more detailed analysis of IgE-mediated inflammatory reactions in a large animal model, we here present the cloning of the epsilon chain of IgE in the domestic pig (Sus scrufa). A partial cDNA clone for the epsilon chain of pig IgE was isolated by polymerase chain reaction (PCR) amplification using degenerate primers directed against conserved regions in the second (CH2) and the fourth (CH4) constant domains of IgE. cDNA derived from mRNA isolated from the spleen and lymph nodes of a pig actively sensitized with a protein extract from the nematode Ascaris suum was used as template. Screening of a spleen cDNA library with the partial cDNA clone as probe resulted in isolation of a clone that contained the entire coding region. The nucleotide sequence was determined and was found to conform with the previously identified mammalian epsilon-chain sequences. The highest degree of similarity was found to sheep IgE. A DNA construct encoding a baculovirus signal sequence, a histidine hexapeptide, and the CH2-CH3-CH4 domains of the pig IgE epsilon chain was obtained by PCR amplification. The construct was ligated into the baculovirus expression vector pVL1392. Infection of High Five insect cells with recombinant baculovirus resulted in expression and secretion of a soluble 6 x His-CH2-CH3-CH4 protein product.

Amino Acid Sequence↗

PubMed1996

Lactoferrin regulates the activity of heparin proteoglycan-bound mast cell chymase: characterization of the binding of heparin to lactoferrin.

Rat mast cell protease 1 (RMCP-1) is a secretory granule serine protease (chymase) that is recovered in vivo in a macromolecular complex with heparin proteoglycan (PG). We have previously shown that heparin activates RMCP-1 and that RMCP-1, when bound to heparin PG, is largely resistant to inhibition by a variety of macromolecular protease inhibitors. In the search for alternative mechanisms in the regulation of RMCP-1 activity, we hypothesized that heparin antagonists, by interfering with the RMCP-1/heparin PG interaction, might influence the activity of heparin-bound mast cell chymase. In the present study, lactoferrin (LF), a heparin-binding protein, was assessed for RMCP-1 inhibiting activity. LF proved to decrease the activity of heparin PG-associated RMCP-1, although a portion of the enzyme activity was resistant to regulation. The mechanism of regulation was shown to involve the displacement of RMCP-1 from heparin PG, and LF caused an approx. 6-fold increase in the apparent Km of the RMCP-1-heparin PG complex for the chromogenic substrate S-2586. The interaction of LF with heparin was characterized. Pig mucosal heparin and endogenous heparin PG were equally effective in binding LF, whereas heparan sulphate bound with lower affinity. None of dermatan sulphate, chondroitin sulphate or hyaluronan were effective in binding LF. Further, the 6-O-, 2-O- and N-sulphate groups in heparin were of approximately equal importance for binding. Octasaccharides were the smallest heparin oligosaccharides showing significant binding to LF.

Animals↗

PubMed1996

Mast cell chymase in complex with heparin proteoglycan is regulated by protamine.

Protamines are polycationic proteins that are widely used for neutralisation of the anticoagulant action of heparin. However, several reports have shown adverse, mast cell-dependent reactions to protamine. The exact mechanism by which protamine causes these adverse effects is not clear. In the present study, the possibility that protamine may influence mast cell chymase function was investigated. Mast cell chymase is in vivo recovered in a macromolecular complex with heparin proteoglycan, and this interaction is essential for expression of optimal enzymatic activity. Protamine was shown to strongly reduce the activity of mast cell chymase by a mechanism that involved displacement of the chymase from heparin proteoglycan.

Animals↗

PubMed1995

Regulation of rat mast cell protease 1 activity. Protease inhibition is prevented by heparin proteoglycan.

Rat mast cell protease 1 (RMCP-1) is a chymotrypsin-like serine protease (chymase) that is specifically expressed by connective-tissue-type mast cells. It is stored in the secretory granules of the cells in a complex with heparin proteoglycan, and the chymase/heparin proteoglycan complexes are released following mast cell activation. The present study was undertaken to examine if the association with heparin proteoglycan influenced the regulation of RMCP-1 by various macromolecular protease inhibitors. Endogenous mast cell heparin proteoglycan was shown to significantly block the inhibition of RMCP-1 by the serpins alpha 1-protease inhibitor and alpha 1-antichymotrypsin, as well as the inhibition by alpha 2-macroglobulin, soybean trypsin inhibitor and plasma. The blocking of protease inhibition showed an optimum at a RMCP-1/proteoglycan ratio of 5:1 (by mass), corresponding to approximately 80 RMCP-1 molecules bound/proteoglycan molecule. Chymase activity present on intact peritoneal mast cells, i.e. present in its native complex with heparin proteoglycan, was also shown to be largely resistant to inhibition by alpha 1-antichymotrypsin and alpha 1-protease inhibitor. Heparin 10-saccharides and 20-saccharides were inefficient in preventing the interaction of RMCP-1 with alpha 1-antichymotrypsin, whereas pig mucosal heparin (approximately 50 monosaccharide units) blocked protease inhibition. We have previously shown that heparin potentiates the catalytic activity of RMCP-1 and, in the present study, we show that the mechanism for chymase activation involves a sixfold reduction of the Km,app value of RMCP-1 for the chromogenic substrate S-2586. Thus, the association of mast cell chymase with heparin proteoglycan may serve both to potentiate the catalytic activity of the enzyme and to increase the life-span of the chymases by preventing their inhibition after exocytosis.

Animals↗

PubMed1995

Identification of the proteolytic thrombin fragments formed after cleavage with rat mast cell protease 1.

We have previously identified rat mast cell protease 1 (RMCP-1), a chymotrypsin-like secretory granule serine protease, as a potent inactivator of thrombin. The present study outlines the cleavage pattern obtained after degradation of thrombin by RMCP-1. The cleavage sites in thrombin were identified by N-terminal amino acid sequence analysis of recovered thrombin fragments. Incubation of thrombin with RMCP-1 resulted in the rapid formation of a 37-kDa fragment, due to cleavage of the Phe1G-Gly1F bond in the thrombin A chain (numbering of amino acid residues according to topological equivalencies with chymotrypsinogen). Further incubation resulted in cleavage of the Trp148-Thr149 bond in the B chain, along with the formation of fragments of 27 kDa and 15 kDa. When the RMCP-1/thrombin mixtures were incubated further, successive degradation of the 37-kDa, 27-kDa and 15-kDa fragments was observed, along with cleavage of the Tyr117-Ile118 bond in the B chain and the formation of fragments of 12, 9 and 6 kDa. No residual thrombin activity was detected after the degradation process had proceeded to this stage. Heparin was shown to markedly enhance the rate of thrombin degradation by RMCP-1.

Amino Acid Sequence↗