Figure - available from: Frontiers in Cardiovascular Medicine
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Serpin-dependent mechanism of protease inhibition. (A) Natively folded serpins present a reactive center loop (RCL) which acts as a bait for interaction with active serine proteases. Upon association, initiation of proteolytic activity by the protease triggers the formation of a Michaelis complex and covalent bonding of the serpin with the protease. (B) The primary outcome of Michaelis complex formation is reconfiguration of the serpin, with the RCL inserting as the third of five strands in the core beta sheet and permanent denaturation of the target serine protease. (C) The secondary and less frequent outcome is completion of proteolytic activity and dissociation of the active serine protease, while the RCL continues to insert as the third of five strands in the serpin core beta sheet.
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The making and breaking of clots orchestrated by the thrombotic and thrombolytic serine protease cascades are critical determinants of morbidity and mortality during infection and with vascular or tissue injury. Both the clot forming (thrombotic) and the clot dissolving (thrombolytic or fibrinolytic) cascades are composed of a highly sensitive and...
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The balance between proteases and protease inhibitors plays a critical role in tissue remodeling during cardiovascular diseases. Different serine protease inhibitors termed serpins, which are expressed in the cardiovascular system, can exert a fine-tuned regulation of protease activities. Among them, protease nexin-1 (PN-1, encoded by SERPINE2 ) is...
Tick saliva has been extensively studied in the context of tick-host interactions because it is involved in host homeostasis modulation and microbial pathogen transmission to the host. Accumulated knowledge about the tick saliva composition at the molecular level has revealed that serine protease inhibitors play a key role in the tick-host interact...
Plasminogen activator inhibitor 1 (PAI-1) is a member of the serine protease inhibitor (serpin) superfamily. PAI-1 is the principal inhibitor of the plasminogen activators, tissue plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). Turbulence in the levels of PAI-1 tilts the balance of the hemostatic system resulting in ble...
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... The primary mechanism of haemostasis regulates bleeding which involves the formation of clots and their solubilization [5]. Active serine proteases and their regulation by inhibitors in the circulating blood form the basis for both clot-formation and clot-solubilization cascades [6]. ...
... The brinolysis starts with the conversion of plasminogen to its active form plasmin which solubilizes the blood clot [7]. Dysregulation of these proteases involved in coagulation pathway leads to blood related disorders, chronic lung disease and neurodegenerative diseases [5]. The aetiology of vascular disorders such as atherosclerosis, thrombosis, and aneurysms are also linked to protease activity [8]. ...
Thrombosis is the formation of abnormal blood clots in the blood vessels that obstruct blood flow and lead to cause thrombosis. Current treatments for thrombosis are associated with serious side effects. Therefore there is a need for alternative natural therapy. To isolate and characterize fibrinolytic protease from M.oleifera and evaluation of its fibrinolytic efficiency.
Fresh leaves of Moringa oleifera Lam were taken, fibrinolytic protease was isolated and characterized for its potential to solubilize fibrin under in-vitro conditions and its blood clot solubilization efficiency under ex-vivo experiments. The isolated protease showed a single protein band on native-PAGE. It showed optimum fibrinolytic activity at pH 8.0, 37 oC at 50µg concentration. Its fibrinolytic activity was also confirmed by fibrin zymography. Km and Vmax of isolated protease was determined by the Lineweaver Burk plot. The isolated protease could solubilize 96.41% of blood clot by 96 hrs under ex-vivo conditions. In-vitro fibrin hydrolysis and ex-vivo blood clot solubilization activities shown by isolated protease from leaves of Moringa oleifera Lam suggest its fibrinolytic potential to dissolve blood clots. Being a natural molecule and from a dietary plant it can be explored as an alternative natural therapy against thrombosis.
... The immune system response to SARS-CoV-2 reveals a different point of view on COVID-19 and fibrinolysis. Numerous inflammatory conditions are linked to the elevated circulating suPAR level [11]. ...
... Our finding was consistent with Zhang et al., who examined SERPINE treatment of severe immune-mediated lung damage in SARS-CoV-2 viral infections in mice models and found its effectiveness in preventing disease-mediated damage [29]. It is worth noting that the role of serpins and anti-PLAUR in the fibrinolysis process in COVID-19 is complex, and investigations on the potential therapeutic modulation of these processes with natural, virus-derived, or engineered serpins, expanding the consideration of these proteins beyond only regulation of the fibrinolytic system, may be a valuable pursuit as many of these modulators are already found to be safe and effective, and in some cases, FDA-approved [11]. Designing inhibitors that cross species barriers according to the species-specific residues of uPA and block uPAR-uPA interactions simultaneously would be helpful. ...
Urokinase receptors regulate the interplay between inflammation, immunity, and blood clotting. The soluble urokinase plasminogen activator system is an immunologic regulator affecting endothelial function and its related receptor; the soluble urokinase plasminogen activator receptor (suPAR) has been reported to impact kidney injury. This work aims to measure serum levels of suPAR in COVID-19 patients and correlate the measurements with variable clinicolaboratory parameters and patient outcomes. In this prospective cohort study, 150 COVID-19 patients and 50 controls were included. The circulating suPAR levels were quantified by Enzyme-linked immunosorbent assay (ELISA). Routine COVID-19 laboratory assessments, including CBC, CRP, LDH, serum creatinine, and estimated glomerular filtration rates, were performed. The need for oxygen therapy, CO-RAD score, and survival rates was assessed. Bioinformatic analysis and molecular docking were run to explore the urokinase receptor structure/function and to characterize molecules as potential anti-suPAR therapeutic targets, respectively. We found higher circulating suPAR levels in COVID-19 patients vs. controls (p < 0.001). Circulating suPAR levels positively correlated with COVID-19 severity, the need for O2 therapy, the total leukocytes count, and the neutrophils to lymphocyte ratio, while they were negatively correlated with the O2 saturation level, albumin, blood calcium, lymphocytic count, and GFR. In addition, the suPAR levels were associated with poor prognostic outcomes such as a high incidence of acute kidney injury (AKI) and mortality rate. Kaplan–Meier curves showed a lower survival rate with higher suPAR levels. The logistic regression analysis confirmed the significant association of suPAR levels with the occurrence of AKI related to COVID-19 and with increased mortality probability within three months of COVID-19 follow-up. Some compounds that can act similarly to uPAR were discovered and tested by molecular docking to identify the possible ligand–protein interactions. In conclusion, higher circulating suPAR levels were associated with COVID-19 severity and could be considered a putative predictor of AKI development and mortality.
... Specificity of fibrinolytic protease(s) of selected fungal isolates for different substrates: Coagulation cascade and fibrinolysis are complex processes and are tightly regulated by sequence of events affecting the series of receptors, inhibitors and cofactors 9,39 . There are plentiful proteins present in the blood plasma and all are vital or important for normal functioning of the living system. ...
The diseases related to the cardiovascular system significantly contribute towards global mortality. Dysregulation/malfunctioning of coagulation and fibrinolytic pathways especially under pathophysiological conditions disturbs the homeostasis and causes cardiovascular complications. Thrombosis is one such disorder wherein normal blood flow is obstructed due to an abnormal fibrin clot. Serious issues with the available therapeutics (thrombolytics) viz. fatal side effects, low specificity and efficacy and high cost, motivate the research for discovery of potentially novel, safe, efficacious and cost-effective thrombolytics. In the present study, an effectual fibrin specific fibrinolytic protease (thrombolytic) was produced from a newly isolated fungus Aspergillus flavus SH71 by using low-cost agricultural residues as substrates. Molasses as a sole carbon source and linseed cake as an exclusive nitrogen source supported the protease production of 188.99 U/mL and 215.67 U/mL respectively which were higher than that obtained in control (133.91 U/mL). Also, some other agro-based substrates (orange peel, rice bran, malt, mixed fruit brunch and soymeal) supported higher enzyme titre than control. Furthermore, a bioprocess was developed for fibrinolytic protease production by optimization of process variables i.e. concentration of molasses and linseed cake, medium pH and incubation time based on design of experiments and a substantial yield enhancement was achieved (1.97 folds).
... Under inflammatory conditions, these modulate hyaluronic acid molecules, and these further create serum-derived hyaluronan-associated proteins complex and build tunnels for the leukocytes migrating within the extracellular matrix. 29 Similar to α-2macroglobulin and antithrombin III, ITIH is assigned to proteases inhibitors. However, apart from their inflammatory contribution, ITIH 1 and 2 are also recognized for regulating implantation and placentation. ...
Kidney transplantation remains the therapeutic option for patients with end‐stage kidney disease. Current immunosuppressive regimens are efficient in combating acute kidney rejection. However, insights into chronic kidney allograft injury remains limited. Simultaneously, pregnancy is more common after kidney transplantation than during dialysis treatment. Due to ethical issues, comprehensive studies on the impact of immunosuppressive regimens on pregnancy are challenging. The study aimed to investigate the proteomic status of lymphocytes obtained from pregnant female rats under immunosuppressive treatment. The experiment involved a group of 10 female, pregnant Wistar rats, five of which were treated with tacrolimus, mofetil mycophenolate, and glucocorticosteroids; five were used as control. The lymphocytes were obtained and analyzed with mass spectrometry. Measurements were processed by a database search in the ProteinPilot software with a cutoff of 1% false discovery rate. The outcomes were verified statistically by a t‐test (p value < 0.05) regarding proteins up‐ and downregulation. A total of 2082 proteins were identified in all experiments. Eight hundred five proteins were quantified in an absolute manner in a data‐independent acquisition‐total protein approach analysis. Ninety‐five proteins were recognized as present at different concentrations in analyzed groups and were annotated to intracellular pathways. The proteins involved in nonsense‐mediated decay and L13a‐mediated translational silencing of ceruloplasmin expression were recognized as downregulated. The set of proteins clinically identified as acute phase proteins was upregulated. Despite the blockade of adaptive cellular immunity, the lymphocytes in the analyzed group reveal sustained proinflammatory status with decreased ability to regulate translation. This potentially affects pregnancy and immunity.
... We recently reported that three of these same genes were also amongst the most highly up-regulated in the same earlier study of cows with a low energy balance status, many of which were experiencing either mastitis or endometritis [43]. The serpin superfamily are serine/cysteine protease inhibitors which are strongly associated with a variety of inflammatory conditions, particularly through targeting areas with active thrombosis and/or thrombolysis [46,47]. The term GO:0002020~protease binding was also identified as up-regulated in E. coli(+) cows in the present study. ...
The risk and severity of clinical infection with Escherichia coli as a causative pathogen for bovine mastitis is influenced by the hosts’ phenotypic and genotypic variables. We used RNA-Seq analysis of circulating leukocytes to investigate global transcriptomic profiles and genetic variants from Holstein cows with naturally occurring cases of clinical mastitis, diagnosed using clinical symptoms and milk microbiology. Healthy lactation-matched cows served as controls (CONT, n = 6). Blood samples were collected at two time periods during the recovery phase post diagnosis: EARLY (10.3 ± 1.8 days, n = 6) and LATE (46.7 ± 11 days, n = 3). Differentially expressed genes (DEGs) between the groups were identified using CLC Genomics Workbench V21 and subjected to enrichment analysis. Variant calling was performed following GATKv3.8 best practice. The comparison of E. coli(+) EARLY and CONT cows found the up-regulation of 1090 DEGs, mainly with immune and inflammatory functions. The key signalling pathways involved NOD-like and interleukin-1 receptors and chemokines. Many up-regulated DEGs encoded antimicrobial peptides including cathelicidins, beta-defensins, S100 calcium binding proteins, haptoglobin and lactoferrin. Inflammation had largely resolved in the E. coli(+) LATE group, with only 29 up-regulated DEGs. Both EARLY and LATE cows had up-regulated DEGs encoding ATP binding cassette (ABC) transporters and haemoglobin subunits were also up-regulated in LATE cows. Twelve candidate genetic variants were identified in DEGs between the infected and CONT cows. Three were in contiguous genes WIPI1, ARSG and SLC16A6 on BTA19. Two others (RAC2 and ARHGAP26) encode a Rho-family GTPase and Rho GTPase-activating protein 26. These results show that the initial inflammatory response to E. coli continued for at least 10 days despite prompt treatment and provide preliminary evidence for genetic differences between cows that may predispose them to infection.
... This observation could be consistent with the molecular action of www.nature.com/scientificreports/ BB94, which is a broad spectrum inhibitor of zinc MMPs, whereas the enzymatic breakdown of fibrin by HT1080s is associated with expression of serine proteases and not directly with MMP activity 61,62 . Rat tail T1C and bovine skin T1C were prepared using the same protocol and differed only with the tissue source of telocollagen. ...
Cells are known to continuously remodel their local extracellular matrix (ECM) and in a reciprocal way, they can also respond to mechanical and biochemical properties of their fibrous environment. In this study, we measured how stiffness around dermal fibroblasts (DFs) and human fibrosarcoma HT1080 cells differs with concentration of rat tail type 1 collagen (T1C) and type of ECM. Peri-cellular stiffness was probed in four directions using multi-axes optical tweezers active microrheology (AMR). First, we found that neither cell type significantly altered local stiffness landscape at different concentrations of T1C. Next, rat tail T1C, bovine skin T1C and fibrin cell-free hydrogels were polymerized at concentrations formulated to match median stiffness value. Each of these hydrogels exhibited distinct fiber architecture. Stiffness landscape and fibronectin secretion, but not nuclear/cytoplasmic YAP ratio differed with ECM type. Further, cell response to Y27632 or BB94 treatments, inhibiting cell contractility and activity of matrix metalloproteinases, respectively, was also dependent on ECM type. Given differential effect of tested ECMs on peri-cellular stiffness landscape, treatment effect and cell properties, this study underscores the need for peri-cellular and not bulk stiffness measurements in studies on cellular mechanotransduction.
... Based on the secreted protease profiling of MRC-5containing degraded scaffolds we next evaluated whether we could slow down GAF degradation. Urokinase acts on plasmin activation by cleaving its precursor, plasminogen, and a well-known contaminant of FBG extracted from bovine and human plasmas (Markus and Ambrus, 1960;Yaron et al., 2021) (Figure 5). We compared the stability of MRC-5 cellcontaining GAFs prepared with bovine, human and plasminogen-depleted human FBGs. ...
In tissue engineering, cell origin is important to ensure outcome quality. However, the impact of the cell type chosen for seeding in a biocompatible matrix has been less investigated. Here, we investigated the capacity of primary and immortalized fibroblasts of distinct origins to degrade a gelatin/alginate/fibrin (GAF)-based biomaterial. We further established that fibrin was targeted by degradative fibroblasts through the secretion of fibrinolytic matrix-metalloproteinases (MMPs) and urokinase, two types of serine protease. Finally, we demonstrated that besides aprotinin, specific targeting of fibrinolytic MMPs and urokinase led to cell-laden GAF stability for at least forty-eight hours. These results support the use of specific strategies to tune fibrin-based biomaterials degradation over time. It emphasizes the need to choose the right cell type and further bring targeted solutions to avoid the degradation of fibrin-containing hydrogels or bioinks.
... A PEGylated version of the Myxomavirus derived SERPIN Serp-1 has also been developed demonstrating improved efficacy in a mouse model of diffuse alveolar hemorrhage (259). Serp-1 is a broad acting SERPIN with anti-inflammatory and antifibrinolytic activities (260). In addition, however, Serp-1 also functions as a heparin dependent inhibitor of thrombin (261,262). ...
Appropriate activation of coagulation requires a balance between procoagulant and anticoagulant proteins in blood. Loss in this balance leads to hemorrhage and thrombosis. A number of endogenous anticoagulant proteins, such as antithrombin and heparin cofactor II, are members of the serine protease inhibitor (SERPIN) family. These SERPIN anticoagulants function by forming irreversible inhibitory complexes with target coagulation proteases. Mutations in SERPIN family members, such as antithrombin, can cause hereditary thrombophilias. In addition, low plasma levels of SERPINs have been associated with an increased risk of thrombosis. Here, we review the biological activities of the different anticoagulant SERPINs. We further consider the clinical consequences of SERPIN deficiencies and insights gained from preclinical disease models. Finally, we discuss the potential utility of engineered SERPINs as novel therapies for the treatment of thrombotic pathologies.
... The role of serpins in the regulation of inflammation is well known because the most abundant serpin in human serum is alpha-1-antitrypsin, which is a major protective factor against the damaging effects of neutrophil elastase (Mangan et al., 2008;Yaron et al., 2021). Other human serpins, such as antichymotrypsin, also have an anti-inflammatory function. ...
Tick saliva has been extensively studied in the context of tick-host interactions because it is involved in host homeostasis modulation and microbial pathogen transmission to the host. Accumulated knowledge about the tick saliva composition at the molecular level has revealed that serine protease inhibitors play a key role in the tick-host interaction. Serpins are one highly expressed group of protease inhibitors in tick salivary glands, their expression can be induced during tick blood-feeding, and they have many biological functions at the tick-host interface. Indeed, tick serpins have an important role in inhibiting host hemostatic processes and in the modulation of the innate and adaptive immune responses of their vertebrate hosts. Tick serpins have also been studied as potential candidates for therapeutic use and vaccine development. In this review, we critically summarize the current state of knowledge about the biological role of tick serpins in shaping tick-host interactions with emphasis on the mechanisms by which they modulate host immunity. Their potential use in drug and vaccine development is also discussed.
... Interest in these hydrolytic enzymes is due to their well-characterized, widespread, and diverse roles, in a host of physiological and pathological processes. For example, it has long been established, that in addition to their fibrinolytic role in clot dissolution (Yaron et al., 2021), the trypsin-like serine proteinases urokinase (uPA), tissue-type plasminogen activator (tPA) and plasmin play critical roles in a number of processes including extracellular matrix remodelling (Lu et al., 2011), wound healing and carcinogenesis (Aimes et al., 2003;Nyberg et al., 2006;Pawar et al., 2019). ...
The trypsin-like proteases (TLPs) play widespread and diverse roles, in a host of physiological and pathological processes including clot dissolution, extracellular matrix remodelling, infection, angiogenesis, wound healing and tumour invasion/metastasis. Moreover, these enzymes are involved in the disruption of normal lung function in a range of respiratory diseases including allergic asthma where several allergenic proteases have been identified. Here, we report the synthesis of a series of peptide derivatives containing an N-alkyl glycine analogue of arginine, bearing differing electrophilic leaving groups (carbamate and triazole urea), and demonstrate their function as potent, irreversible inhibitors of trypsin and TLPs, to include activities from cockroach extract. As such, these inhibitors are suitable for use as activity probes (APs) in activity-based profiling (ABP) applications.
