Inactivation of thrombin by a complex between rat mast-cell protease 1 and heparin proteoglycan.
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ABSTRACT:
SUBMITTER: Pejler G
PROVIDER: S-EPMC1138300 | biostudies-other | 1994 Apr
REPOSITORIES: biostudies-other
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Project description:The susceptibility of rat mast-cell heparin to oxidative degradation was examined. Heparin as a component of intact mast-cell granules (MCG) was degraded following ingestion by normal human neutrophils. In contrast, neutrophils from patients with chronic granulomatous disease (CGD), which do not respond to stimulation with respiratory-burst activity, exhibited a greatly diminished ability to degrade phagocytosed MCG heparin. MCG-associated heparin also was cleaved by H2O2 plus Fe2+ (Fenton's reagent). Isolated heparin proteoglycan (average Mr approx. 750,000) was rapidly cleaved to smaller molecules similar in size to commercial pig heparin upon exposure to Fenton's reagent. This cleavage was inhibited by catalase and by the hydroxyl-radical (OH.)-scavenger mannitol, but not by superoxide dismutase (SOD). The cleavage products retained approx. 26% of the anticoagulant activity of the native molecule. The heparin proteoglycan was also cleaved by acetaldehyde/xanthine oxidase/FeSO4, a system that generates superoxide (O2.-), H2O2 and OH.. Whereas the cleavage at relatively high iron ion concentrations was inhibited by catalase and mannitol but not by SOD, at lower iron ion concentrations the cleavage was inhibited by catalase, mannitol and SOD. These findings suggest the involvement of OH., which at high Fe2+ concentrations is generated by Fenton's reagent (H2O2 plus Fe2+), and at low iron ion concentrations is generated by the iron-ion-catalysed interaction between O2.- and H2O2 (Haber-Weiss reaction). These studies suggest that oxygen radicals generated by activated phagocytes may contribute to the degradation in vivo of both solubilized and granule-associated proteoglycan heparin.
| S-EPMC1149655 | biostudies-other
Project description:Background:Thrombin could elicit degranulation of mast cells involved in numerous physiologic and pathologic processes; however, the detailed scrutiny of this procedure and further research of possible cell signaling pathways are lacking. Methods:P815 mouse mast cells were exposed to various concentrations of thrombin for 16?h. Expression of protease-activated receptor (PAR)1, PAR2, PAR3, and PAR4 mRNA in P815 was analyzed by quantitative real-time PCR (qRT-PCR) and the fittest concentration of thrombin was decided. Then, secretions of mediators from P815 stimulated by thrombin 0.2?U/ml were determined using enzyme-linked immunosorbent assay (ELISA) and Luminex liquichip; the possible cell signaling pathways were measured by immunoblotting. Furthermore, inhibition of thrombin inhibitor (hirudin), PAR1 inhibitor (SCH79797), and MAPK inhibitors (SB203580, PD98059, and SP600125) on the mediator section was evaluated by ELISA and Luminex liquichip. Results:Thrombin 0.2?U/ml induced the elevated expression of PAR1, PAR2, PAR3, and PAR4, as well as the increasing level of phospho-I?B?, phospho-SAPK/JNK MAPK, phospho-P38 MAPK (Thr180/Tyr182), and phospho-ERK1/2 MAPK (p44/42) in P815. Secretion of vascular endothelial growth factor (VEGF), tumor necrosis factor-? (TNF-?), interleukin- (IL-) 2, IL-6, chemokine ligand- (CCL-) 2, chemokine (C-X-C motif) ligand- (CXCL-) 1, and CXCL-5 from P815 increased apparently; this effect could be diminished by hirudin, whereas SCH79797 and MAPK inhibitors (SB203580, PD98059, and SP600125) diminish the secretions with weaker effect. Conclusion:We found the expression of PAR mRNA in P815, activation of signaling pathways of nuclear factor-kappaB (NF-?B), and mitogen-activated protein kinases (MAPKs) including C-Jun NH2-terminal kinase (JNK), P38, and extracellular signal-regulated kinase 1/2 (ERK1/2), and the release of multiple inflammatory mediators stimulated by thrombin, as well as the inhibition of the inflammatory releases by hirudin, SCH79797, and MAPK inhibitors including SB203580, PD98059, and SP600125.
| S-EPMC6878808 | biostudies-literature
Project description: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.
| S-EPMC1218013 | biostudies-other
Project description:Heparin proteoglycans (HEP-PGs) carry standard heparin-mediated anticoagulant properties as well as novel antiplatelet functions, a combination that may be significant for targeting multiple pathways in a single therapy. Recent work developing semisynthetic HEP-PG mimetics has shown promising results also in vivo, however flow conditions in vitro that replicate in vivo hemodynamics have not been reported. In this work, we present several assays (platelet calcium mobilization, aggregometry, microfluidic tests at venous and arterial hemodynamics) to characterize specific mechanistic effects of dual antiplatelet and anticoagulant (APAC) constructs as mimetics of HEP-PGs. Three APACs with different conjugation levels of heparin chains (CL10, CL18, HICL) were shown to decrease platelet deposition to collagen surfaces in PPACK-treated whole blood at venous shear rate (200 s-1). FXIIa-inhibited whole blood (CTI: corn trypsin inhibitor, 40 μg/mL) perfused over collagen/tissue factor showed reduced both platelet and fibrin deposition when treated with APACs. IC50 values for platelet and fibrin inhibition were calculated for each molecule at venous shear rate. Increasing the shear rate to arterial flows (1000 s-1) and using APAC as the sole anticoagulant, resulted in a more potent antiplatelet effect of APAC, suggesting an added effect on von Willebrand Factor (vWF) function. Additionally, APAC caused an inhibition of calcium mobilization specific to thrombin and collagen stimulation and a dose-dependent reduction in collagen-mediated platelet aggregation. Understanding the sensitivity of APAC activity to shear rate, platelet signaling and procoagulant pathways is important for applications in which APAC administration may have beneficial therapeutic effects.
| S-EPMC6179373 | biostudies-literature
Project description:The inactivation of thrombin by heat and by its physiological inhibitor, antithrombin-III, shows quite different dependence on heparin concentration. Heparin at 250 microgram/ml protects thrombin against heat inactivation, and thrombin behaves heterogeneously in this reaction. In the absence of heparin, the thermodynamic activation parameters change with temperature (deltaH+ = 733 kJ/mol and 210 kJ/mol at 50 and 58 degrees C respectively). When heparin is present, heat inactivation of the protected thrombin species proceeds with deltaH+ = 88 kJ/mol and is independent of temperature in the same range. On the other hand, heparin at 0.125-2.5 microgram/ml accelerates the thrombin-antithrombin-III reaction. Thrombin does not show heterogeneity in this reaction and the time courses at any heparin concentration and any temperature between 0 and 37 degrees C appear to follow first-order kinetics. Activation enthalpy is independent of heparin concentration or temperature, deltaH+ = 82-101 kJ/mol, varying slightly with antithrombin-III concentration and thrombin specific activity. Heparin seems to exert its effect by increasing activation entropy. On the basis of these data we suggest a mechanism of action of heparin in the thrombin-antithrombin-III reaction which accounts for all the important features of the latter and seems to unify the different hypotheses that have been advanced.
| S-EPMC1185853 | biostudies-other
Project description:Mast cells (MCs) are characterized by an abundance of lysosome-like secretory granules filled with immunomodulatory compounds including histamine, cytokines, lysosomal hydrolases, MC-restricted proteases, and serglycin proteoglycans. The latter are essential for promoting the storage of other granule compounds and are built up of the serglycin core protein to which highly sulfated and thereby negatively charged glycosaminoglycan (GAG) side chains of heparin or chondroitin sulfate type are attached. In the search for mechanisms operating in regulating MC granule homeostasis, we here investigated the role of mitogen-activated protein kinase (MAPK) signaling. We show that inhibition of MEK1/2 (a MAPK kinase) leads to increased metachromatic staining of MC granules, indicative of increased proteoglycan content. Indeed, MEK1/2 inhibition caused a profound increase in the expression of the gene coding for the serglycin core protein and of genes coding for various enzymes involved in the biosynthesis/sulfation of the GAGs attached to the serglycin core protein. This was accompanied by corresponding increases in the levels of the respective GAGs. Deletion of the serglycin core protein abrogated the induction of enzymes operative in proteoglycan synthesis, indicating that availability of the serglycin proteoglycan core protein has a regulatory function impacting on the expression of the various serglycin-modifying enzymes. MEK1/2 inhibition also caused a substantial increase in the expression of granule-localized, proteoglycan-binding proteases. Altogether, this study identifies a novel role for MAPK signaling in regulating the content of secretory granules in MCs.
| S-EPMC6060404 | biostudies-literature
Project description:Serine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-β-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced μs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased μs-ms dynamics in a β-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two β-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.
| S-EPMC8087772 | biostudies-literature
Project description:INTRODUCTION:Thrombin is a primary target of most anticoagulants. Yet, thrombin's dual and opposing role in pro- as well as anti- coagulant processes imposes considerable challenges in discovering finely tuned regulators that maintain homeostasis, rather than disproportionately changing the equilibrium to one side. In this connection, we have been studying exosite 2-mediated allosteric modulation of thrombin activity using synthetic agents called low molecular weight lignins (LMWLs). Although the aromatic scaffold of LMWLs is completely different from the polysaccharidic scaffold of heparin, the presence of multiple negatively charged groups on both ligands induces binding to exosite 2 of thrombin. This work characterizes the nature of interactions between LMWLs and thrombin to understand the energetic cooperativity between exosite 2 and active site of thrombin. MATERIALS AND METHODS:The thermodynamics of thrombin-LMWL complexes was studied using spectrofluorimetric titrations as a function of ionic strength and temperature of the buffer. The contributions of enthalpy and entropy to binding were evaluated using classic thermodynamic equations. Label-free surface plasmon resonance was used to assess the role of sodium ion in LMWL binding to thrombin at a fixed ionic strength. RESULTS AND CONCLUSIONS:Exosite 2-induced conformational change in thrombin's active site is strongly dependent on the structure of the ligand, which has consequences with respect to regulation of thrombin. The ionic and non-ionic contributions to binding affinity and the thermodynamic signature were highly ligand specific. Interestingly, LMWLs display preference for the sodium-bound form of thrombin, which supports the existence of an energetic coupling between exosite 2 and sodium-binding site of thrombin.
| S-EPMC5943173 | biostudies-literature
Project description:TSG-6 (TNF-?-stimulated gene/protein 6), a hyaluronan (HA)-binding protein, has been implicated in the negative regulation of inflammatory tissue destruction. However, little is known about the tissue/cell-specific expression of TSG-6 in inflammatory processes, due to the lack of appropriate reagents for the detection of this protein in vivo. Here, we report on the development of a highly sensitive detection system and its use in cartilage proteoglycan (aggrecan)-induced arthritis, an autoimmune murine model of rheumatoid arthritis. We found significant correlation between serum concentrations of TSG-6 and arthritis severity throughout the disease process, making TSG-6 a better biomarker of inflammation than any of the other arthritis-related cytokines measured in this study. TSG-6 was present in arthritic joint tissue extracts together with the heavy chains of inter-?-inhibitor (I?I). Whereas TSG-6 was broadly detectable in arthritic synovial tissue, the highest level of TSG-6 was co-localized with tryptases in the heparin-containing secretory granules of mast cells. In vitro, TSG-6 formed complexes with the tryptases murine mast cell protease-6 and -7 via either heparin or HA. In vivo TSG-6-tryptase association could also be detected in arthritic joint extracts by co-immunoprecipitation. TSG-6 has been reported to suppress inflammatory tissue destruction by enhancing the serine protease-inhibitory activity of I?I against plasmin. TSG-6 achieves this by transferring heavy chains from I?I to HA, thus liberating the active bikunin subunit of I?I. Because bikunin is also present in mast cell granules, we propose that TSG-6 can promote inhibition of tryptase activity via a mechanism similar to inhibition of plasmin.
| S-EPMC3123119 | biostudies-literature
Project description:To study the interaction between low-density lipoprotein (LDL) and granules from rat serosal mast cells in vitro, mast cells were stimulated with the degranulating agent 48/80 to induce exocytosis of the secretory granules. Subsequent incubation of the exocytosed granules with 125I-LDL resulted in binding of the labelled LDL to the granules. When increasing amounts of agent 48/80 were added to mast-cell suspensions, a dose-dependent release of granules was observed and a parallel increase in the amount of 125I-LDL bound to granules resulted. 125I-LDL bound to a single class of high-affinity binding sites on the granules. At saturation, 105 ng of LDL were bound per microgram of granule protein. The lipoprotein binding to mast-cell granules was apolipoprotein(apo)-B + E-specific. Thus 125I-LDL binding to the granules was effectively compared for by LDL (apo-B) or by dimyristoyl phosphatidylcholine vesicles containing apo-E, but not by high-density lipoprotein (HDL3) containing apo-AI as their major protein component. Neutralization by acetylation of the positively charged amino groups of apo-B of LDL or presence of a high ionic strength in the incubation medium prevented LDL from binding to the granules, indicating the presence of ionic interactions between the positively charged amino acids of LDL and negatively charged groups of the granules. It could be demonstrated that LDL bound to the negatively charged heparin proteoglycan of the granules. Thus treatment of granules with heparinase resulted in loss of their ability to bind LDL, and substances known to bind to heparin, such as Toluidine Blue, avidin, lipoprotein lipase, fibronectin and protamine, all effectively competed with LDL for binding to the granules. The results show that LDL is efficiently bound to the heparin proteoglycan component of mast-cell granules once the mast cells are stimulated to release their granules into the extracellular space.
| S-EPMC1147600 | biostudies-other