Browse
Submit Data
Databases
API
Help

Dataset Information

7 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1.

ABSTRACT: Protease nexin-1 (PN-1) is a proteinase inhibitor that is secreted by human fibroblasts in culture. PN-1 inhibits certain regulatory serine proteinases by forming a covalent complex with the catalytic-site serine residue; the complex then binds to the cell surface and is internalized and degraded. The fibroblast surface was recently shown to accelerate the rate of complex-formation between PN-1 and thrombin. The present paper demonstrates that the accelerative activity is primarily due to cell-surface heparan sulphate, with a much smaller contribution from chondroitin sulphate. This conclusion is supported by the effects of purified glycosaminoglycans on the second-order rate constant for the inhibition of thrombin by PN-1. Also, treatment of 35SO4(2-)-labelled cells with heparitin sulphate lyase or chondroitin sulphate ABC lyase demonstrated two discrete pools of 35S-labelled glycosaminoglycans; subsequent treatment of plasma membranes with these glycosidases showed that heparitin sulphate lyase treatment abolished about 80% of the accelerative activity and chondroitin sulphate ABC lyase removed the remaining 20%. These results show that two components are responsible for the acceleration of PN-1-thrombin complex-formation by human fibroblasts. Although dermatan sulphate is also present on fibroblasts, it did not accelerate the inhibition of thrombin by PN-1.

SUBMITTER: Farrell DH

PROVIDER: S-EPMC1148156 | biostudies-other | 1987 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Hematopoietic protease nexin-1 protects against lung injury by preventing thrombin signaling in mice.

Project description:Coagulation and fibrinolytic system deregulation has been implicated in the development of idiopathic pulmonary fibrosis, a devastating form of interstitial lung disease. We used intratracheal instillation of bleomycin to induce pulmonary fibrosis in mice and analyzed the role of serine protease inhibitor E2 (serpinE2)/protease nexin-1 (PN-1), a tissue serpin that exhibits anticoagulant and antifibrinolytic properties. PN-1 deficiency was associated, after bleomycin challenge, with a significant increase in mortality, as well as a marked increase in active thrombin in bronchoalveolar lavage fluids, an overexpression of extracellular matrix proteins, and an accumulation of inflammatory cells in the lungs. Bone marrow transplantation experiments showed that protective PN-1 was derived from hematopoietic cell compartment. A pharmacological strategy using the direct thrombin inhibitor argatroban reversed the deleterious effects of PN-1 deficiency. Concomitant deficiency of the thrombin receptor protease-activated receptor 4 (PAR4) abolished the deleterious effects of PN-1 deficiency in hematopoietic cells. These data demonstrate that prevention of thrombin signaling by PN-1 constitutes an important endogenous mechanism of protection against lung fibrosis and associated mortality. Our findings suggest that appropriate doses of thrombin inhibitors or PAR4 antagonists may provide benefit against progressive lung fibrosis with evidence of deregulated thrombin activity.

| S-EPMC6156894 | biostudies-other

Sulfated glycosaminoglycans accelerate transthyretin amyloidogenesis by quaternary structural conversion.

Project description:Glycosaminoglycans (GAGs), which are found in association with all extracellular amyloid deposits in humans, are known to accelerate the aggregation of various amyloidogenic proteins in vitro. However, the precise molecular mechanism(s) by which GAGs accelerate amyloidogenesis remains elusive. Herein, we show that sulfated GAGs, especially heparin, accelerate transthyretin (TTR) amyloidogenesis by quaternary structural conversion. The clustering of sulfate groups on heparin and its polymeric nature are essential features for accelerating TTR amyloidogenesis. Heparin does not influence TTR tetramer stability or TTR dissociation kinetics, nor does it alter the folded monomer-misfolded monomer equilibrium directly. Instead, heparin accelerates the conversion of preformed TTR oligomers into larger aggregates. The more rapid disappearance of monomeric TTR in the presence of heparin likely reflects the fact that the monomer-misfolded amyloidogenic monomer-oligomer-TTR fibril equilibria are all linked, a hypothesis that is strongly supported by the light scattering data. TTR aggregates prepared in the presence of heparin exhibit a higher resistance to trypsin and proteinase K proteolysis and a lower exposure of hydrophobic side chains comprising hydrophobic clusters, suggesting an active role for heparin in amyloidogenesis. Our data suggest that heparin accelerates TTR aggregation by a scaffold-based mechanism, in which the sulfate groups comprising GAGs interact primarily with TTR oligomers through electrostatic interactions, concentrating and orienting the oligomers, facilitating the formation of higher molecular weight aggregates. This model raises the possibility that GAGs may play a protective role in human amyloid diseases by interacting with proteotoxic oligomers and promoting their association into less toxic amyloid fibrils.

| S-EPMC3035766 | biostudies-literature

Molecular organization of the rat glia-derived nexin/protease nexin-1 promoter.

Project description:The first three exons and the promoter of rat glia-derived nexin, also called protease nexin-1 (GDN/PN-1), have been identified through analysis of rat genomic clones. A 1.6 kilobase (kb) fragment containing 105 base pairs of the first exon and 5'-flanking sequences was sequenced. The 5'-flanking sequence and the first exon were found to be GC-rich, indicating that the 5' region of the rat GDN/PN-1 gene resides within a CpG island. A TATA box-like sequence, but no CAAT box, was found. The rat GDN/PN-1 promoter contains five SP1 consensus sites, four consensus sites for the MyoD1 transcription factor, and one binding site for the transcription factors NGFI-A, NGFI-C, Krox-20, and Wilms tumor factor. The presence of these consensus sequences is consistent with the known expression pattern of GDN/PN-1. Primer extension and RNase protection assays identified one transcriptional start site. The 1.6 kb promoter fragment cloned in a reporter plasmid was found to induce firefly luciferase expression in a cell-specific manner. A positive regulatory element is localized in the region -1545 to -389. In vitro CpG methylation blocked transcription from the GDN/PN-1 promoter in rat hepatoma cells but not in C6 rat glioma cells.

| S-EPMC6081634 | biostudies-literature

Data regarding the effects of thrombin and dabigatran-inhibited thrombin on protease-activated receptor 1 and activation of human atrial fibroblasts.

Project description:The data presented here are related to the research paper entitled "Thrombin induces protease-activated receptor 1 signaling and activation of human atrial fibroblasts and dabigatran prevents these effects" (Altieri et al., 2018) [1]. Data show that silencing of protease-activated receptor 1 (PAR1) prevents the activation of Fib isolated from atrial appendages of patients without atrial fibrillation (AF), as assessed by immunofluorescence for α-smooth muscle actin (αSMA) and Picro-Sirius red staining. Moreover, it is reported that primary atrial Fib obtained from two subjects with permanent AF express PAR1 and PAR2 and display enhanced αSMA immunoreactivity and collagen synthesis in response to thrombin, but not to dabigatran-bound thrombin, alike Fib from non-fibrillating atria.

| S-EPMC5997940 | biostudies-literature

A simple two-step purification of protease nexin.

Project description:This paper describes a simple purification procedure for protease nexin, a serine proteinase inhibitor secreted by cultured human fibroblasts that regulates proteinase activity at and near the cell surface. The first step in the procedure takes advantage of the high-affinity binding of protease nexin to dextran sulphate-Sepharose. This step eliminates the need for prior concentration of the serum-free fibroblast-conditioned medium, since protease nexin binds to the resin in the presence of physiological saline. The use of dextran sulphate also provides an affinity resin with considerably less variability than the heparin-based resins previously used. Final purification to homogeneity involves a combination of DEAE-Sepharose in-line with dextran sulphate-Sepharose to simultaneously purify and concentrate the protein. Purified protease nexin is shown by Ouchterlony analysis and peptide mapping to be immunologically and structurally distinct from antithrombin III and heparin cofactor II, two plasma proteinase inhibitors with similar properties.

| S-EPMC1147074 | biostudies-other

Glycosaminoglycans accelerate biomimetic collagen mineralization in a tissue-based in vitro model.

Project description:Mammalian teeth are attached to the jawbone through an exquisitely controlled mineralization process: unmineralized collagen fibers of the periodontal ligament anchor directly into the outer layer of adjoining mineralized tissues (cementum and bone). The sharp interface between mineralized and nonmineralized collagenous tissues makes this an excellent model to study the mechanisms by which extracellular matrix macromolecules control collagen mineralization. While acidic phosphoproteins, localized in the mineralized tissues, play key roles in control of mineralization, the role of glycosaminoglycans (GAGs) is less clear. As several proteoglycans are found only in the periodontal ligament, it has been hypothesized that these inhibit mineralization of collagen in this tissue. Here we used an in vitro model based on remineralization of mouse dental tissues to determine the role of matrix GAGs in control of mineralization. GAGs were selectively removed from demineralized mouse periodontal sections via enzymatic digestion. Proteomic analysis confirmed that enzymatic GAG removal does not significantly alter protein content. Analysis of remineralized tissue sections by transmission electron microscopy (TEM) shows that GAG removal reduced the rate of remineralization in mineralized tissues compared to the untreated control, while the ligament remained unmineralized. Protein removal with trypsin also reduced the rate of mineralization, but to a lesser extent than GAG removal, despite a much larger effect on protein content. These results indicate that GAGs promote mineralization in mineralized dental tissues rather than inhibiting mineral formation in the ligament, which may have broader implications for understanding control of collagen mineralization in connective tissues.

| S-EPMC7293698 | biostudies-literature

Mechanisms and specificity of factor XIa and trypsin inhibition by protease nexin 2 and basic pancreatic trypsin inhibitor.

Project description:Factor XIa (FXIa) inhibition by protease nexin-2 (PN2KPI) was compared with trypsin inhibition by basic pancreatic trypsin inhibitor (BPTI). PN2KPI was a potent inhibitor of FXIa (K(i) ? 0.81 nM) and trypsin (K(i) ? 0.03 nM), but not of other coagulation proteases (thrombin, FVIIa, FIXa, FXa, FXIIa, plasmin, kallikrein, K(i) > 185 nM). PN2KPI was ?775-fold more potent than BPTI in FXIa inhibition, but both exhibited similar potencies against trypsin. Studies of FXIa and trypsin inhibition by PN2KPI and BPTI and P1 site swap mutants (PN2KPI-R15 K, BPTI-K15 R) demonstrated that FXIa inhibition by PN2KPI and P1 site swap mutants and trypsin inhibition by PN2KPI and BPTI conform to a single-step, slow equilibration inhibitory mechanism, whereas FXIa-inhibition by BPTI follows a classical, competitive inhibitory mechanism. Mutation of P1 impaired FXIa inhibition by PN2KPI-R15 K ?14-fold, enhanced FXIa inhibition by BPTI-K15 R ?150-fold, and had no effect on trypsin inhibition. Arginine at the P1 site of either PN2KPI or BPTI confers high affinity and specificity for FXIa, whereas either arginine or lysine suffices for trypsin inhibition. Thus, PN2KPI is a highly specific inhibitor of FXIa among coagulation enzymes, but the flexibility of trypsin renders it susceptible to inhibition by both wild-type and mutant forms of PN2KPI and BPTI.

| S-EPMC3031312 | biostudies-literature

Protease nexin 1 inhibits hedgehog signaling in prostate adenocarcinoma.

Project description:Prostate adenocarcinoma (CaP) patients are classified into low-, intermediate-, and high-risk groups that reflect relative survival categories. While there are accepted treatment regimens for low- and high-risk patients, intermediate-risk patients pose a clinical dilemma, as treatment outcomes are highly variable for these individuals. A better understanding of the factors that regulate the progression of CaP is required to delineate risk. For example, aberrant activation of the Hedgehog (Hh) pathway is implicated in CaP progression. Here, we identify the serine protease inhibitor protease nexin 1 (PN1) as a negative regulator of Hh signaling in prostate. Using human CaP cell lines and a mouse xenograft model of CaP, we demonstrate that PN1 regulates Hh signaling by decreasing protein levels of the Hh ligand Sonic (SHH) and its downstream effectors. Furthermore, we show that SHH expression enhanced tumor growth while overexpression of PN1 inhibited tumor growth and angiogenesis in mice. Finally, using comparative genome hybridization, we found that genetic alterations in Hh pathway genes correlated with worse clinical outcomes in intermediate-risk CaP patients, indicating the importance of this pathway in CaP.

| S-EPMC3484431 | biostudies-literature

Effect of heparin on the glia-derived-nexin-thrombin interaction.

Project description:In order to determine the specificity of the interaction between thrombin and glia-derived nexin (GdN), the inactivation of proteolytically modified human thrombin species by GdN has been studied. The second-order rate constants for the inactivation of alpha-, beta T-, gamma T- and epsilon-thrombin by GdN were 1.41, 0.63, 0.33 and 1.91 microM-1.s-1 respectively. The kinetic properties of gdN were also investigated in the presence of different types of heparin, fractionated according to antithrombin III-binding affinity. Association rate constants of both gdN and antithrombin III with alpha-thrombin were obtained using unfractionated, low- and high-affinity heparin types. The different heparin types gave optimal rates of inhibition at similar heparin concentrations for both inhibitors. At optimal heparin concentrations, the rate of inactivation of alpha-thrombin by GdN was 0.5-1.2 nM-1.s-1, which suggests that, under these conditions, the interaction is diffusion-controlled.

| S-EPMC1135555 | biostudies-other

Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein.

Project description:Protease nexin 1 (PN1) is an endogenous serine protease inhibitor (SERPIN), expressed at high levels in the prostate, and capable of inhibiting the proliferation of prostate cancer cells. We previously showed that PN1-uPA complexes inhibited Sonic Hedgehog (SHH) signalling through engagement of the LRP receptor. Here, we describe an alternative anti-proliferative mechanism through which PN1 expression leads to apoptosis. In prostate cancer cells, increased expression of PN1 led to substantial reduction of XIAP levels and apoptosis mediated through the uPAR, but not the LRP receptor. The alterations in XIAP were effected in two ways 1) via alteration in the NF-?B pathway, a pathway known to signal XIAP transcription and 2) by promoting XIAP instability. The AKT pathway is known to phosphorylate XIAP at serine 87 leading to protein stability and PN1 expression is shown to interfere with this process. As a result of both mechanisms, programmed cell death is substantially increased. Consistent with these observations, reduced PN1 protein correlated with elevated p65/XIAP expression and with higher Gleason scores in human prostate tissue arrays. Thus, PN1 expression appears to differentially down-regulate distinct oncogenic pathways depending upon the cell surface receptor engaged by its complexes and demonstrates a novel molecular mechanism by which the protein can promote tumor cell apoptosis.

| S-EPMC4414153 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data