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Sensors (Basel, Switzerland) Sep 2020This review is focused on the application of surface and volume-sensitive acoustic methods for the detection of milk proteases such as trypsin and plasmin. While trypsin... (Review)
Review
This review is focused on the application of surface and volume-sensitive acoustic methods for the detection of milk proteases such as trypsin and plasmin. While trypsin is an important protein of human milk, plasmin is a protease that plays an important role in the quality of bovine, sheep and goat milks. The increased activity of plasmin can cause an extensive cleavage of β-casein and, thus, affect the milk gelation and taste. The basic principles of surface-sensitive acoustic methods, as well as high-resolution ultrasonic spectroscopy (HR-US), are presented. The current state-of-the-art examples of the application of acoustic sensors for protease detection in real time are discussed. The application of the HR-US method for studying the kinetics of the enzyme reaction is demonstrated. The sensitivity of the acoustics biosensors and HR-US methods for protease detection are compared.
Topics: Acoustics; Animals; Caseins; Cattle; Fibrinolysin; Goats; Milk; Peptide Hydrolases; Sheep
PubMed: 33003538
DOI: 10.3390/s20195594 -
British Journal of Pharmacology Jul 2020Lung oedema in association with suppressed fibrinolysis is a hallmark of lung injury. Here, we have tested whether plasmin cleaves epithelial sodium channels (ENaC) to...
BACKGROUND AND PURPOSE
Lung oedema in association with suppressed fibrinolysis is a hallmark of lung injury. Here, we have tested whether plasmin cleaves epithelial sodium channels (ENaC) to resolve lung oedema fluid.
EXPERIMENTAL APPROACH
Human lungs and airway acid-instilled mice were used for analysing fluid resolution. In silico prediction, mutagenesis, Xenopus oocytes, immunoblotting, voltage clamp, mass spectrometry, and protein docking were combined for identifying plasmin cleavage sites.
KEY RESULTS
Plasmin improved lung fluid resolution in both human lungs ex vivo and injured mice. Plasmin activated αβγENaC channels in oocytes in a time-dependent manner. Deletion of four consensus proteolysis tracts (αΔ432-444, γΔ131-138, γΔ178-193, and γΔ410-422) eliminated plasmin-induced activation significantly. Further, immunoblotting assays identified 7 cleavage sites (K126, R135, K136, R153, K168, R178, K179) for plasmin to trim both furin-cleaved C-terminal fragments and full-length human γENaC proteins. In addition, 9 new sites (R122, R137, R138, K150, K170, R172, R180, K181, K189) in synthesized peptides were found to be cleaved by plasmin. These cleavage sites were located in the finger and the thumb, particularly the GRIP domain of human ENaC 3D model composed of two proteolytic centres for plasmin. Novel uncleaved sites beyond the GRIP domain in both α and γ subunits were identified to interrupt the plasmin cleavage-induced conformational change in ENaC channel complexes. Additionally, plasmin could regulate ENaC activity via the G protein signal.
CONCLUSION AND IMPLICATIONS
Plasmin can cleave ENaC to improve blood-gas exchange by resolving oedema fluid and could be a potent therapy for oedematous lungs.
Topics: Animals; Epithelial Sodium Channels; Fibrinolysin; Furin; Lung; Mice; Oocytes; Proteolysis; Xenopus laevis
PubMed: 32133621
DOI: 10.1111/bph.15038 -
Blood Nov 2016In this issue of , Marcos-Contreras and colleagues present convincing evidence that mechanistically links hyperfibrinolysis, typically seen as a bleeding risk, with...
In this issue of , Marcos-Contreras and colleagues present convincing evidence that mechanistically links hyperfibrinolysis, typically seen as a bleeding risk, with increased brain endothelial permeability through plasmin-mediated cleavage of high-molecular-weight kininogen (HMWK) to bradykinin (BK). This study establishes plasmin as a key effector of what might be termed “hemovascular dysfunction”: a pathological state of blood enzymatic activity resulting in vascular structural and functional disruption. Their findings point the way toward improved treatment of patients with pharmacologically (stroke and myocardial infarction) or pathologically activated fibrinolysis (trauma and surgery) through selective blockade of bradykinin activity. Combined blockade of bradykinin and plasmin activation may provide additional therapeutic benefits in hemorrhagic shock by reducing tissue edema in resuscitation while enhancing hemostasis.
Topics: Fibrinolysin; Humans
PubMed: 27856467
DOI: 10.1182/blood-2016-09-735720 -
Arteriosclerosis, Thrombosis, and... Jan 2021Coronavirus disease 2019 (COVID-19) is associated with derangement in biomarkers of coagulation and endothelial function and has been likened to the coagulopathy of...
OBJECTIVE
Coronavirus disease 2019 (COVID-19) is associated with derangement in biomarkers of coagulation and endothelial function and has been likened to the coagulopathy of sepsis. However, clinical laboratory metrics suggest key differences in these pathologies. We sought to determine whether plasma coagulation and fibrinolytic potential in patients with COVID-19 differ compared with healthy donors and critically ill patients with sepsis. Approach and Results: We performed comparative studies on plasmas from a single-center, cross-sectional observational study of 99 hospitalized patients (46 with COVID-19 and 53 with sepsis) and 18 healthy donors. We measured biomarkers of endogenous coagulation and fibrinolytic activity by immunoassays, thrombin, and plasmin generation potential by fluorescence and fibrin formation and lysis by turbidity. Compared with healthy donors, patients with COVID-19 or sepsis both had elevated fibrinogen, d-dimer, soluble TM (thrombomodulin), and plasmin-antiplasmin complexes. Patients with COVID-19 had increased thrombin generation potential despite prophylactic anticoagulation, whereas patients with sepsis did not. Plasma from patients with COVID-19 also had increased endogenous plasmin potential, whereas patients with sepsis showed delayed plasmin generation. The collective perturbations in plasma thrombin and plasmin generation permitted enhanced fibrin formation in both COVID-19 and sepsis. Unexpectedly, the lag times to thrombin, plasmin, and fibrin formation were prolonged with increased disease severity in COVID-19, suggesting a loss of coagulation-initiating mechanisms accompanies severe COVID-19.
CONCLUSIONS
Both COVID-19 and sepsis are associated with endogenous activation of coagulation and fibrinolysis, but these diseases differently impact plasma procoagulant and fibrinolytic potential. Dysregulation of procoagulant and fibrinolytic pathways may uniquely contribute to the pathophysiology of COVID-19 and sepsis.
Topics: Biomarkers; Blood Coagulation; Blood Coagulation Disorders; COVID-19; Cross-Sectional Studies; Female; Fibrinolysin; Humans; Male; Middle Aged; Pandemics; SARS-CoV-2; Sepsis
PubMed: 33196292
DOI: 10.1161/ATVBAHA.120.315338 -
Tuberculosis (Edinburgh, Scotland) Jul 2019Plasminogen and plasmin are fundamental components of the fibrinolytic system that interact with microorganisms generating different immunopathological effects. The...
Plasminogen and plasmin are fundamental components of the fibrinolytic system that interact with microorganisms generating different immunopathological effects. The molecules of Mycobacterium tuberculosis interplaying with plasminogen have already been identified and characterized. In this work, we studied the effects of plasmin(ogen) bound toMycobacterium bovisCalmette-Guérin (BCG) on phagocytosis in THP1 macrophages as well as in granuloma formation and development on in vitrohuman granuloma model. For this purpose, BCG was coated with plasminogen and plasmin, obtained after activation of zymogen by tissue plasminogen activator. The results showed a significant reduction in the number of bacteria phagocytosed by macrophages in presence of plasminogen or plasmin on BCG surface. On the other hand, at 3 days BCG/plasminogen/plasmin induced an increase granuloma numbers with respect to those induced by uncoated bacteria. BCG/plasminogen/environments also showed a significant increase of IL-6 secretion. At 7 days, a reduced number of granulomas and an increased number of bacteria was observed with respect to uncoated BCG environment. Altogether, these results showed that plasmin(ogen) on the mycobacterial surface affects phagocytosis, granuloma development and the cytokine context, thus resulting in an increased number of bacteria in granulomas.
Topics: Cells, Cultured; Cytokines; Fibrinolysin; Granuloma; Humans; Macrophages; Mycobacterium bovis; Phagocytosis; Plasminogen; Tuberculin; Tuberculosis
PubMed: 31378266
DOI: 10.1016/j.tube.2019.05.008 -
Arthritis Research & Therapy Jul 2019This study evaluates the utility of urinary pro-thrombotic molecules such as tissue factor (TF), anti-thrombotic molecules such as tissue factor pathway inhibitor...
OBJECTIVE
This study evaluates the utility of urinary pro-thrombotic molecules such as tissue factor (TF), anti-thrombotic molecules such as tissue factor pathway inhibitor (TFPI), and fibrinolytic molecules such as plasmin and d-dimer as biomarkers of lupus nephritis (LN).
METHODS
Urine samples from 113 biopsy-proven LN patients (89 active LN and 24 inactive LN), 45 chronic kidney disease patients, and 41 healthy controls were examined for d-dimer, plasmin, TF, and TFPI levels by ELISA. The area under the receiver operating characteristic curve (AUC) analysis, multivariate regression analysis, and Bayesian network analysis were performed to assess the diagnostic value of the assayed molecules in LN.
RESULTS
Although urinary d-dimer, plasmin, TF, and TFPI were all elevated in active LN compared to all control groups, and correlated with rSLEDAI and SLICC RAS disease activity indices, urine plasmin emerged as the strongest independent predictor of eGFR and renal disease status, by multivariate regression analysis and Bayesian network analysis. Whereas urine plasmin discriminated active LN from inactive disease with an AUC of 0.84, the combination of urine plasmin and TFPI discriminated ALN from ILN with an AUC of 0.86, with both surpassing the specificity and positive predictive value of traditional markers such as anti-dsDNA and complement C3.
CONCLUSION
Both thrombogenic and thrombolytic cascades appear to be upregulated in lupus nephritis, with proteins from both cascades appearing in the urine. Of the coagulation cascade proteins surveyed, urine plasmin emerges as the strongest predictor of eGFR and clinical renal disease in patients with LN.
Topics: Adult; Bayes Theorem; Biomarkers; Female; Fibrin Fibrinogen Degradation Products; Fibrinolysin; Humans; Lipoproteins; Lupus Nephritis; Male; Middle Aged; Multivariate Analysis; Regression Analysis; Sensitivity and Specificity; Thromboplastin; Young Adult
PubMed: 31319876
DOI: 10.1186/s13075-019-1959-y -
Journal of Thrombosis and Haemostasis :... Apr 2023Fibrinolysis is a series of enzymatic reactions that degrade insoluble fibrin. Plasminogen activators convert the zymogen plasminogen to the active serine protease... (Review)
Review
Assays to quantify fibrinolysis: strengths and limitations. Communication from the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee on fibrinolysis.
Fibrinolysis is a series of enzymatic reactions that degrade insoluble fibrin. Plasminogen activators convert the zymogen plasminogen to the active serine protease plasmin, which cleaves and solubilizes crosslinked fibrin clots into fibrin degradation products. The quantity and quality of fibrinolytic enzymes, their respective inhibitors, and clot structure determine overall fibrinolysis. The quantity of protein can be measured by antigen-based assays, and both quantity and quality can be assessed using functional assays. Furthermore, variations of commonly used assays have been reported, which are tailored to address the role(s) of specific fibrinolytic factors and cellular elements (eg, platelets, neutrophils, and red blood cells). Although the concentration and/or activity of a protein can be quantified, how these individual components contribute to the overall fibrinolysis outcome can be challenging to determine. This difficulty is due to temporal changes within and around the thrombi during the clot breakdown, particularly the fibrin matrix structure, and composition. Furthermore, terms such as "fibrinolytic activity/potential," "plasminogen activation," and "plasmin activity" are often used interchangeably despite having different definitions. The purpose of this review is to 1) summarize the assays measuring fibrinolysis activity and potential, 2) facilitate the interpretation of data generated by these assays, and 3) summarize the strengths and limitations of these assays.
Topics: Humans; Fibrinolysis; Fibrinolysin; Thrombosis; Plasminogen; Fibrin; Serine Proteases; Communication
PubMed: 36759279
DOI: 10.1016/j.jtha.2023.01.008 -
Developments in Ophthalmology 2016The field of vitreoretinal surgery has evolved substantially over the last several decades. Scientific advances have improved our understanding of disease... (Review)
Review
The field of vitreoretinal surgery has evolved substantially over the last several decades. Scientific advances have improved our understanding of disease pathophysiology, and new surgical adjuncts and techniques have decreased surgical time and improved patient outcomes. Pharmacologic agents have recently been developed for intraocular use in order to enhance vitreous removal and even as a nonsurgical treatment for pathology due to an abnormal vitreoretinal interface. Plasmin can successfully cause vitreous liquefaction and induce a posterior vitreous detachment. Additionally, ocriplasmin has been approved for symptomatic vitreomacular adhesion and others appear to be promising for pharmacologic manipulation of the vitreous. The ability to induce vitreous liquefaction and a complete posterior vitreous detachment (PVD) with a single intravitreal injection has potential implications for the management of multiple vitreoretinopathies. Enzymatic vitrectomy may help to reduce vitreous viscosity, thereby facilitating removal during vitrectomy and reducing surgical time, especially when using smaller-gauge vitrectomy instruments. The induction of a PVD also has the potential to reduce intraoperative complications. As we improve our understanding of the molecular flux in the vitreous cavity, pharmacologic vitreodynamics will likely become more important as it may allow for improved manipulation of intravitreal molecules.
Topics: Fibrinolysin; Fibrinolytic Agents; Humans; Intravitreal Injections; Peptide Fragments; Tissue Adhesions; Vitrectomy; Vitreoretinal Surgery; Vitreous Body
PubMed: 26501959
DOI: 10.1159/000438962 -
Blood Jul 2021Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor-dependent thrombin generation and...
Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor-dependent thrombin generation and plasmin activity have been shown to promote liver injury after APAP overdose. However, the contribution of the contact and intrinsic coagulation pathways has not been investigated in this model. Mice deficient in individual factors of the contact (factor XII [FXII] and prekallikrein) or intrinsic coagulation (FXI) pathway were administered a hepatotoxic dose of 400 mg/kg of APAP. Neither FXII, FXI, nor prekallikrein deficiency mitigated coagulation activation or hepatocellular injury. Interestingly, despite the lack of significant changes to APAP-induced coagulation activation, markers of liver injury and inflammation were significantly reduced in APAP-challenged high-molecular-weight kininogen-deficient (HK-/-) mice. Protective effects of HK deficiency were not reproduced by inhibition of bradykinin-mediated signaling, whereas reconstitution of circulating levels of HK in HK-/- mice restored hepatotoxicity. Fibrinolysis activation was observed in mice after APAP administration. Western blotting, enzyme-linked immunosorbent assay, and mass spectrometry analysis showed that plasmin efficiently cleaves HK into multiple fragments in buffer or plasma. Importantly, plasminogen deficiency attenuated APAP-induced liver injury and prevented HK cleavage in the injured liver. Finally, enhanced plasmin generation and HK cleavage, in the absence of contact pathway activation, were observed in plasma of patients with acute liver failure due to APAP overdose. In summary, extrinsic but not intrinsic pathway activation drives the thromboinflammatory pathology associated with APAP-induced liver injury in mice. Furthermore, plasmin-mediated cleavage of HK contributes to hepatotoxicity in APAP-challenged mice independently of thrombin generation or bradykinin signaling.
Topics: Acetaminophen; Animals; Chemical and Drug Induced Liver Injury; Factor XII; Female; Fibrinolysin; Fibrinolysis; Humans; Kininogens; Male; Mice; Mice, Knockout; Prekallikrein; Proteolysis
PubMed: 33827130
DOI: 10.1182/blood.2020006198 -
Biomolecules Mar 2023Dyspnea and progressive hypoxemia are the main clinical features of patients with coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory... (Review)
Review
Dyspnea and progressive hypoxemia are the main clinical features of patients with coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pulmonary pathology shows diffuse alveolar damage with edema, hemorrhage, and the deposition of fibrinogens in the alveolar space, which are consistent with the Berlin Acute Respiratory Distress Syndrome Criteria. The epithelial sodium channel (ENaC) is a key channel protein in alveolar ion transport and the rate-limiting step for pulmonary edema fluid clearance, the dysregulation of which is associated with acute lung injury/acute respiratory distress syndrome. The main protein of the fibrinolysis system, plasmin, can bind to the furin site of γ-ENaC and induce it to an activation state, facilitating pulmonary fluid reabsorption. Intriguingly, the unique feature of SARS-CoV-2 from other β-coronaviruses is that the spike protein of the former has the same furin site (RRAR) with ENaC, suggesting that a potential competition exists between SARS-CoV-2 and ENaC for the cleavage by plasmin. Extensive pulmonary microthrombosis caused by disorders of the coagulation and fibrinolysis system has also been seen in COVID-19 patients. To some extent, high plasmin (ogen) is a common risk factor for SARS-CoV-2 infection since an increased cleavage by plasmin accelerates virus invasion. This review elaborates on the closely related relationship between SARS-CoV-2 and ENaC for fibrinolysis system-related proteins, aiming to clarify the regulation of ENaC under SARS-CoV-2 infection and provide a novel reference for the treatment of COVID-19 from the view of sodium transport regulation in the lung epithelium.
Topics: Humans; SARS-CoV-2; COVID-19; Furin; Fibrinolysin; Respiratory Distress Syndrome; Ion Transport; Sodium
PubMed: 37189326
DOI: 10.3390/biom13040578