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Biochimica Et Biophysica Acta. General... Sep 2018Fibrin formation and dissolution are attributed to cascades of protease activation concluding with thrombin activation, and plasmin proteolysis for fibrin breakdown....
BACKGROUND
Fibrin formation and dissolution are attributed to cascades of protease activation concluding with thrombin activation, and plasmin proteolysis for fibrin breakdown. Cysteine cathepsins are powerful proteases secreted by endothelial cells and others during cardiovascular disease and diabetes. Their fibrinolytic activity and putative role in hemostasis has not been well described.
METHODS
Fibrin gels were polymerized and incubated with recombinant human cathepsins (cat) K, L, or S, or plasmin, for dose-dependent and time-dependent studies. Dissolution of fibrin gels was imaged. SDS-PAGE was used to resolve cleaved fragments released from fibrin gels and remnant insoluble fibrin gel that was solubilized prior to electrophoresis to assess fibrin α, β, and γ polypeptide hydrolysis by cathepsins. Multiplex cathepsin zymography determined active amounts of cathepsins remaining.
RESULTS
There was significant loss of α and β fibrin polypeptides after incubation with cathepsins, with catS completely dissolving fibrin gel by 24 h. Binding to fibrin stabilized catL active time; it associated with cleaved fibrin fragments of multiple sizes. This was not observed for catK or S. CatS also remained active for longer times during fibrin incubation, but its association/binding did not withstand SDS-PAGE preparation.
CONCLUSIONS
Human cathepsins K, L, and S are fibrinolytic, and specifically can degrade the α and β fibrin polypeptide chains, generating fragments unique from plasmin.
GENERAL SIGNIFICANCE
Demonstration of cathepsins K, L, and S fibrinolytic activity leads to further investigation of contributory roles in disrupting vascular hemostasis, or breakdown of fibrin-based engineered vascular constructs where non-plasmin mediated fibrinolysis must be considered.
Topics: Cathepsin K; Cathepsin L; Cathepsins; Fibrin; Fibrinolysin; Fibrinolysis; Gels; Hemostasis; Humans
PubMed: 29944896
DOI: 10.1016/j.bbagen.2018.06.015 -
Translational Vision Science &... Apr 2023We sought to evaluate the efficacy and safety of plasmin injection in the capsular bag during the cataract operation for the prevention of posterior capsule...
PURPOSE
We sought to evaluate the efficacy and safety of plasmin injection in the capsular bag during the cataract operation for the prevention of posterior capsule opacification.
METHODS
Thirty-seven anterior capsular flaps taken from phacoemulsification surgery were immersed in either 1 µg/mL plasmin (plasmin group, n = 27) or phosphate-buffered saline (control group, n = 10) for 2 minutes and photographed after fixation and nuclear staining to compare the numbers of residual lens epithelial cells. In the animal experiments, the plasmin solution was injected into the capsular bag and remained for 5 minutes during hydrodissection or after lens extraction. The degree of posterior capsular opacity of the rabbits at 2 months were photographed by slit lamp biomicroscopy. In HLE-B3 cell culture, the cell detachment rate, proliferation, and apoptosis after the plasmin digestion were analyzed.
RESULTS
The residual lens epithelial cell numbers on the capsule after plasmin treatment were 168 ± 190.7/mm2 in the 1 µg/mL plasmin group, which was significantly lower than that of the control (1012 ± 798.8/mm2; P < 0.0001). In a rabbit model, the treatment of plasmin resulted in a significantly clearer posterior capsule compared with that of the control group at 2 months postoperatively.
CONCLUSIONS
This study suggested that plasmin injection can induce effective lens epithelial cell detachment, which could be a promising adjunctive treatment to further improve the success rate in posterior capsule opacification prevention.
TRANSLATIONAL RELEVANCE
Plasmin injection for lens epithelial cell detachment could significantly decrease the number of residual lens epithelial cells. This approach could be a promising treatment incorporating the current treatment approach to further improve the success rate in posterior capsule opacification prevention.
Topics: Animals; Rabbits; Capsule Opacification; Fibrinolysin; Lens Capsule, Crystalline; Epithelial Cells; Phacoemulsification
PubMed: 37074731
DOI: 10.1167/tvst.12.4.23 -
The British Journal of Dermatology Aug 2018Filaggrin is central to the pathogenesis of atopic dermatitis (AD). The cheeks are a common initiation site of infantile AD. Regional and temporal expression of levels... (Observational Study)
Observational Study
Early-life regional and temporal variation in filaggrin-derived natural moisturizing factor, filaggrin-processing enzyme activity, corneocyte phenotypes and plasmin activity: implications for atopic dermatitis.
BACKGROUND
Filaggrin is central to the pathogenesis of atopic dermatitis (AD). The cheeks are a common initiation site of infantile AD. Regional and temporal expression of levels of filaggrin degradation products [natural moisturizing factors (NMFs)], activities of filaggrin-processing enzymes [bleomycin hydrolase (BH) and calpain-1 (C-1)] and plasmin, and corneocyte envelope (CE) maturity in early life are largely unknown.
OBJECTIVES
We conducted a cross-sectional, observational study investigating regional and age-dependent variations in NMF levels, activity of proteases and CE maturity in stratum corneum (SC) from infants to determine whether these factors could explain the observed predilection sites for AD in early life.
METHODS
We measured NMF using a tape-stripping method at seven sites in the SC of 129 children (aged < 12 months to 72 months) and in three sites in 56 neonates and infants (< 48 h to 3 months). In 37 of these neonates and infants, corneocyte size, maturity, BH, C-1 and plasmin activities were determined.
RESULTS
NMF levels are low at birth and increase with age. Cheek SC, compared with elbow flexure and nasal tip, has the lowest NMF in the first year of life and is the slowest to reach stable levels. Cheek corneocytes remain immature. Plasmin, BH and C-1 activities are all elevated by 1 month of age in exposed cheek skin, but not in elbow skin.
CONCLUSIONS
Regional and temporal differences in NMF levels, CE maturity and protease activities may explain the predilection for AD to affect the cheeks initially and are supportive of this site as key for allergen priming in early childhood. These observations will help design early intervention and treatment strategies for AD.
Topics: Age Factors; Calpain; Cheek; Child, Preschool; Cross-Sectional Studies; Cysteine Endopeptidases; Dermatitis, Atopic; Elbow; Female; Fibrinolysin; Filaggrin Proteins; Humans; Infant; Infant, Newborn; Intermediate Filament Proteins; Male; Mutation; Skin
PubMed: 29691836
DOI: 10.1111/bjd.16691 -
Journal of Thrombosis and Haemostasis :... Jan 2021Essentials Tranexamic acid (TXA) is an antifibrinolytic drug used to reduce bleeding. Assaying plasmin generation (PG) in plasma detects clinically relevant TXA levels...
Essentials Tranexamic acid (TXA) is an antifibrinolytic drug used to reduce bleeding. Assaying plasmin generation (PG) in plasma detects clinically relevant TXA levels in vitro and ex vivo. 3.1-16.2 µg/mL TXA half-maximally inhibits PG in plasma from women undergoing cesarean delivery. PG velocity shows the strongest dose-relationship at low TXA concentrations (≤10 µg/mL). ABSTRACT: Background Tranexamic acid (TXA) is used to reduce bleeding. TXA inhibits plasmin(ogen) binding to fibrin and reduces fibrinolysis. TXA antifibrinolytic activity is typically measured by clot lysis assays; however, effects on plasmin generation (PG) are unclear due to a lack of tools to measure PG in plasma. Aims Develop an assay to measure PG kinetics in human plasma. Determine effects of TXA on PG and compare with fibrinolysis measured by rotational thromboelastometry (ROTEM). Methods We characterized effects of plasminogen, tissue plasminogen activator, fibrinogen, and α -antiplasmin on PG in vitro. We also studied effects of TXA on PG in plasma from 30 pregnant women administered intravenous TXA (5, 10, or 15 mg/kg) during cesarean delivery. PG was measured by calibrated fluorescence. PG parameters were compared with TXA measured by mass spectrometry and ROTEM of whole blood. Results The PG assay is specific for plasmin and sensitive to tissue plasminogen activator, fibrin(ogen), and α -antiplasmin. Addition of TXA to plasma in vitro dose dependently prolonged the clot lysis time and delayed and reduced PG. For all doses of TXA administered intravenously, the PG assay detected delayed time-to-peak (≤3 hours) and reduced the velocity, peak, and endogenous plasmin potential (≤24 hours) in plasma samples obtained after infusion. The PG time-to-peak, velocity, and peak correlated significantly with TXA concentration and showed less variability than the ROTEM lysis index at 30 minutes or maximum lysis. Conclusions The PG assay detects pharmacologically relevant concentrations of TXA administered in vitro and in vivo, and demonstrates TXA-mediated inhibition of PG in women undergoing cesarean delivery.
Topics: Antifibrinolytic Agents; Cesarean Section; Female; Fibrinolysin; Fibrinolysis; Humans; Pregnancy; Tissue Plasminogen Activator; Tranexamic Acid
PubMed: 33001565
DOI: 10.1111/jth.15114 -
Virulence Dec 2021Biofilm formation is a general strategy for bacterial pathogens to withstand host defense mechanisms. In this study, we found that serum proteases inhibit biofilm...
Biofilm formation is a general strategy for bacterial pathogens to withstand host defense mechanisms. In this study, we found that serum proteases inhibit biofilm formation by , and . Confocal laser-scanning microscopy analysis revealed that these proteins reduce the biomass and alter the architecture of meningococcal biofilms. To understand the underlying mechanism, the serum was fractionated through size-exclusion chromatography and anion-exchange chromatography, and the composition of the fractions that retained anti-biofilm activity against was analyzed by intensity-based absolute quantification mass spectrometry. Among the identified serum proteins, plasma kallikrein (PKLK), FXIIa, and plasmin were found to cleave neisserial heparin-binding antigen and the α-peptide of IgA protease on the meningococcal cell surface, resulting in the release of positively charged polypeptides implicated in biofilm formation by binding extracellular DNA. Further experiments also revealed that plasmin and PKLK inhibited biofilm formation of by cleaving filamentous hemagglutinin. We conclude that the proteolytic activity of serum proteases toward bacterial adhesins involved in biofilm formation could constitute a defense mechanism for the clearance of pathogens.
Topics: Adhesins, Bacterial; Biofilms; Fibrinolysin; Kallikreins; Neisseria meningitidis
PubMed: 34903146
DOI: 10.1080/21505594.2021.2003115 -
The Journal of Biological Chemistry Apr 1977The heavy polypeptide chains of human Glu-plasmin and human Lys-plasmin have been isolated in native solvents, after partial reduction and carboxymethylation of the...
The heavy polypeptide chains of human Glu-plasmin and human Lys-plasmin have been isolated in native solvents, after partial reduction and carboxymethylation of the corresponding plasmins. Two major forms of each heavy chain can be eluted, after adsorption to Sepharose/lysine, utilizing a gradient of epsilon-aminocaproic acid as the eluant. The elution profile of these heavy chains is practically identical to the elution behavior previously observed for human Glu- and Lys-plasminogen, and human Glu- and Lys-plasmin adsorbed to these columns. Sedimentation velocity analysis of the heavy chain of human Glu-plasmin, in the presence of epsilon-aminocaproic acid, demonstrated that a gross conformational alteration occurs in this peptide accompanying binding of this amino acid. A much smaller conformational alteration occurs under similar circumstances with the human Lys-plasmin heavy chain. We find that the NH2-terminal peptide released in the Glu-plasminogen to Lys-plasminogen and Glu-plasmin to Lys-plasmin conversions is also released in the Glu-plasmin heavy chain to Lys-plasmin heavy chain conversion. This reaction is catalyzed at a significant rate only by plasmin and not by urokinase. Finally, no strong interaction between streptokinase and the isolated plasmin heavy chains is observed.
Topics: Fibrinolysin; Glutamates; Humans; Immunodiffusion; Lysine; Macromolecular Substances; Molecular Weight
PubMed: 139407
DOI: No ID Found -
Nature Communications Dec 2021Cardiac ATTR amyloidosis, a serious but much under-diagnosed form of cardiomyopathy, is caused by deposition of amyloid fibrils derived from the plasma protein...
Cardiac ATTR amyloidosis, a serious but much under-diagnosed form of cardiomyopathy, is caused by deposition of amyloid fibrils derived from the plasma protein transthyretin (TTR), but its pathogenesis is poorly understood and informative in vivo models have proved elusive. Here we report the generation of a mouse model of cardiac ATTR amyloidosis with transgenic expression of human TTR. The model is characterised by substantial ATTR amyloid deposits in the heart and tongue. The amyloid fibrils contain both full-length human TTR protomers and the residue 49-127 cleavage fragment which are present in ATTR amyloidosis patients. Urokinase-type plasminogen activator (uPA) and plasmin are abundant within the cardiac and lingual amyloid deposits, which contain marked serine protease activity; knockout of α-antiplasmin, the physiological inhibitor of plasmin, enhances amyloid formation. Together, these findings indicate that cardiac ATTR amyloid deposition involves local uPA-mediated generation of plasmin and cleavage of TTR, consistent with the previously described mechano-enzymatic hypothesis for cardiac ATTR amyloid formation. This experimental model of ATTR cardiomyopathy has potential to allow further investigations of the factors that influence human ATTR amyloid deposition and the development of new treatments.
Topics: Amyloid; Amyloid Neuropathies, Familial; Animals; Cardiomyopathies; Fibrinolysin; Humans; Mice, Transgenic; Plaque, Amyloid; Prealbumin; Protein Folding; Proteolysis
PubMed: 34876572
DOI: 10.1038/s41467-021-27416-z -
Memorias Do Instituto Oswaldo Cruz Aug 2011Enolase is the eighth enzyme in the glycolytic pathway, a reaction that generates ATP from phosphoenol pyruvate in cytosolic compartments. Enolase is essential,... (Review)
Review
Enolase is the eighth enzyme in the glycolytic pathway, a reaction that generates ATP from phosphoenol pyruvate in cytosolic compartments. Enolase is essential, especially for organisms devoid of the Krebs cycle that depend solely on glycolysis for energy. Interestingly, enolase appears to serve a separate function in some organisms, in that it is also exported to the cell surface via a poorly understood mechanism. In these organisms, surface enolase assists in the invasion of their host cells by binding plasminogen, an abundant plasma protease precursor. Binding is mediated by the interaction between a lysine motif of enolase with Kringle domains of plasminogen. The bound plasminogen is then cleaved by specific proteases to generate active plasmin. Plasmin is a potent serine protease that is thought to function in the degradation of the extracellular matrix surrounding the targeted host cell, thereby facilitating pathogen invasion. Recent work revealed that the malaria parasite Plasmodium also expresses surface enolase, and that this feature may be essential for completion of its life cycle. The therapeutic potential of targeting surface enolases of pathogens is discussed.
Topics: Animals; Cell Membrane; Fibrinolysin; Life Cycle Stages; Phosphopyruvate Hydratase; Plasminogen; Plasmodium
PubMed: 21881761
DOI: 10.1590/s0074-02762011000900011 -
PloS One 2021Ischemic stroke is a common and debilitating disease with limited treatment options. Protease activated receptor 1 (PAR1) is a fundamental cell signaling mediator in the...
BACKGROUND
Ischemic stroke is a common and debilitating disease with limited treatment options. Protease activated receptor 1 (PAR1) is a fundamental cell signaling mediator in the central nervous system (CNS). It can be activated by many proteases including thrombin and plasmin, with various down-stream effects, following brain ischemia.
METHODS
A permanent middle cerebral artery occlusion (PMCAo) model was used in PAR1 KO and WT C57BL/6J male mice. Mice were evaluated for neurological deficits (neurological severity score, NSS), infarct volume (Tetrazolium Chloride, TTC), and for plasmin and thrombin activity in brain slices.
RESULTS
Significantly low levels of plasmin and thrombin activities were found in PAR1 KO compared to WT (1.6±0.4 vs. 3.2±0.6 ng/μl, p<0.05 and 17.2±1.0 vs. 21.2±1.0 mu/ml, p<0.01, respectively) along with a decreased infarct volume (178.9±14.3, 134.4±13.3 mm3, p<0.05).
CONCLUSIONS
PAR1 KO mice have smaller infarcts, with lower thrombin and plasmin activity levels. These findings may suggest that modulation of PAR1 is a potential target for future pharmacological treatment of ischemic stroke.
Topics: Animals; Brain; Fibrinolysin; Ischemic Stroke; Male; Mice; Mice, Knockout; Receptor, PAR-1; Thrombin
PubMed: 33720950
DOI: 10.1371/journal.pone.0248431 -
The Biochemical Journal Sep 1981Active-site-inhibited plasmin was prepared by inhibition with d-valyl-l-phenylalanyl-l-lysylchloromethane or by bovine pancreatic trypsin inhibitor (Kunitz inhibitor)....
Active-site-inhibited plasmin was prepared by inhibition with d-valyl-l-phenylalanyl-l-lysylchloromethane or by bovine pancreatic trypsin inhibitor (Kunitz inhibitor). Active-site-inhibited Glu-plasmin binds far more strongly to fibrin than Glu-plasminogen [native human plasminogen with N-terminal glutamic acid (residues 1-790)]. This binding is decreased by alpha(2)-plasmin inhibitor and tranexamic acid, and is, in the latter case, related to saturation of a strong lysine-binding site. In contrast, alpha(2)-plasmin inhibitor and tranexamic acid have only weak effects on the binding of Glu-plasminogen to fibrin. This demonstrates that its strong lysine-binding site is of minor importance to its binding to fibrin. Active-site-inhibited Lys-plasmin and Lys-plasminogen (Glu-plasminogen lacking the N-terminal residues Glu(1)-Lys(76), Glu(1)-Arg(67) or Glu(1)-Lys(77))display binding to fibrin similar to that of active-site inhibited Glu-plasmin. In addition, alpha(2)-plasmin inhibitor or tranexamic acid similarly decrease their binding to fibrin. Glu-plasminogen and active-site-inhibited Glu-plasmin have the same gross conformation, and conversion into their respective Lys- forms produces a similar marked change in conformation [Violand, Sodetz & Castellino (1975) Arch. Biochem. Biophys.170, 300-305]. Our results indicate that this change is not essential to the degree of binding to fibrin or to the effect of alpha(2)-plasmin inhibitor and tranexamic acid on this binding. The conversion of miniplasminogen (Glu-plasminogen lacking the N-terminal residues Glu(1)-Val(441)) into active-site-inhibited miniplasmin makes no difference to the degree of binding to fibrin, which is similarly decreased by the addition of tranexamic acid and unaffected by alpha(2)-plasmin inhibitor. Active-site-inhibited Glu-plasmin, Lys-plasmin and miniplasmin have lower fibrin-binding values in a plasma system than in a purified system. Results with miniplasmin(ogen) indicate that plasma proteins other than alpha(2)-plasmin inhibitor and histidine-rich glycoprotein decrease the binding of plasmin(ogen) to fibrin.
Topics: Binding Sites; Electrophoresis, Polyacrylamide Gel; Fibrin; Fibrinolysin; Glutamates; Humans; Lysine; Peptide Fragments; Plasminogen; Protein Binding; Tranexamic Acid; alpha-2-Antiplasmin
PubMed: 6459779
DOI: 10.1042/bj1970619