Plasmin Formation Research Articles

Lack of Interactions Between Alteplase/Tenecteplase and the Adenosine A1R/A3R Agonist AST-004.

AST-004, a small molecule agonist of the adenosine A1 and A3 receptors, is a potential cerebroprotectant for patients with acute stroke and is currently in clinical trials. Drug-drug interactions are critically important to assess in the context of acute stroke care. Lytic therapy with tPA (tissue-type plasminogen activator)-induced plasmin formation (alteplase) is the only available pharmacotherapy for acute stroke. Consequently, it is imperative to evaluate potential interactions between AST-004 and tPAs such as alteplase and tenecteplase. The interactions between AST-004 and tPAs were evaluated in 3 ways in preparation for AST-004 phase II trials. First, the metabolic stability of AST-004 was determined in the presence of alteplase and plasmin. Second, the potential for AST-004 to influence the thrombolytic efficacy of alteplase and tenecteplase was evaluated with an in vitro assay system utilizing a fluorogenic substrate of plasmin. Finally, the potential for AST-004 to influence the thrombolytic efficacy of alteplase was also determined with an in vitro thrombolysis assay of human blood thrombi. Neither alteplase nor plasmin affected the stability of AST-004 in vitro. In 2 different in vitro systems, AST-004 had no effect on the ability of alteplase or tenecteplase to generate plasmin, and AST-004 had no effect on the thrombolytic efficacy of alteplase to lyse blood clots in human blood. These studies indicate that there will be no interactions between AST-004 and tPAs such as alteplase or tenecteplase in patients with stroke undergoing thrombolytic therapy.

Plasminogen influence on the PAI-1 release by human platelets

РАІ-1 (plasminogen activator inhibitor type 1), as a major physiological inhibitor of tissue plasminogen activator and urokinase, plays a key role in the regulation of fibrinolysis in vivo. Besides, PAI-1 suppresses plasmin formation and affects cell migration through interaction with vitronectin. РАІ-1 is secreted from α-granules of platelets upon stimulation of cells by agonists. The aim of our study was to explore the effects of Glu- and Lys-forms of plasminogen on PAI-1 secretion by platelets and to evaluate the possible role of plasminogen in modulation of agonist-induced PAI-1 release. The secretion of PAI-1 by platelets was investigated by the Western blot analysis. It has been established that depending on the agonist, PAI-1 can be released from platelets in a free form, in a complex with a tissue plasminogen activator, as well as in the form of high-molecular complexes that contain a tissue activator and vitronectin molecules. The revealed induction of PAI-1 secretion under the action of Gly- and Lys-forms of plasminogen indicates their ability to activate intracellular signaling pathways that regulate the release of platelet α-granules. Our findings may be of importance for elucidating the pathogenetic mechanisms of many diseases associated with abnormally enhanced platelet function and PAI-1-related disorders. Keywords: Glu- and Lys-plasminogen, PAI-1, platelets, vitronectin, α-granules

Phenotypes of Disseminated Intravascular Coagulation.

Two phenotypes of disseminated intravascular coagulation (DIC) are systematically reviewed. DIC is classified into thrombotic and fibrinolytic phenotypes characterized by thrombosis and hemorrhage, respectively. Major pathology of DIC with thrombotic phenotype is the activation of coagulation, insufficient anticoagulation with endothelial injury, and plasminogen activator inhibitor-1-mediated inhibition of fibrinolysis, leading to microvascular fibrin thrombosis and organ dysfunction. DIC with fibrinolytic phenotype is defined as massive thrombin generation commonly observed in any type of DIC, combined with systemic pathologic hyperfibrinogenolysis caused by underlying disorder that results in severe bleeding due to excessive plasmin formation. Three major pathomechanisms of systemic hyperfibrinogenolysis have been considered: (1) acceleration of tissue-type plasminogen activator (t-PA) release from hypoxic endothelial cells and t-PA-rich storage pools, (2) enhancement of the conversion of plasminogen to plasmin due to specific proteins and receptors that are expressed on cancer cells and endothelial cells, and (3) alternative pathways of fibrinolysis. DIC with fibrinolytic phenotype can be diagnosed by DIC diagnosis followed by the recognition of systemic pathologic hyperfibrin(ogen)olysis. Low fibrinogen levels, high fibrinogen and fibrin degradation products (FDPs), and the FDP/D-dimer ratio are important for the diagnosis of systemic pathologic hyperfibrin(ogen)olysis. Currently, evidence-based treatment strategies for DIC with fibrinolytic phenotypes are lacking. Tranexamic acid appears to be one of the few methods to be effective in the treatment of systemic pathologic hyperfibrin(ogen)olysis. International cooperation for the elucidation of pathomechanisms, establishment of diagnostic criteria, and treatment strategies for DIC with fibrinolytic phenotype are urgent issues in the field of thrombosis and hemostasis.

A High-Throughput Small-Angle X-ray Scattering Assay to Determine the Conformational Change of Plasminogen.

Plasminogen (Plg) is the inactive form of plasmin (Plm) that exists in two major glycoforms, referred to as glycoforms I and II (GI and GII). In the circulation, Plg assumes an activation-resistant "closed" conformation via interdomain interactions and is mediated by the lysine binding site (LBS) on the kringle (KR) domains. These inter-domain interactions can be readily disrupted when Plg binds to lysine/arginine residues on protein targets or free L-lysine and analogues. This causes Plg to convert into an "open" form, which is crucial for activation by host activators. In this study, we investigated how various ligands affect the kinetics of Plg conformational change using small-angle X-ray scattering (SAXS). We began by examining the open and closed conformations of Plg using size-exclusion chromatography (SEC) coupled with SAXS. Next, we developed a high-throughput (HTP) 96-well SAXS assay to study the conformational change of Plg. This method enables us to determine the Kopen value, which is used to directly compare the effect of different ligands on Plg conformation. Based on our analysis using Plg GII, we have found that the Kopen of ε-aminocaproic acid (EACA) is approximately three times greater than that of tranexamic acid (TXA), which is widely recognized as a highly effective ligand. We demonstrated further that Plg undergoes a conformational change when it binds to the C-terminal peptides of the inhibitor α2-antiplasmin (α2AP) and receptor Plg-RKT. Our findings suggest that in addition to the C-terminal lysine, internal lysine(s) are also necessary for the formation of open Plg. Finally, we compared the conformational changes of Plg GI and GII directly and found that the closed form of GI, which has an N-linked glycosylation, is less stable. To summarize, we have successfully determined the response of Plg to various ligand/receptor peptides by directly measuring the kinetics of its conformational changes.

Correlation of D-dimer value with Lipid Levels in COVID-19 Patients at Tertiary Gresik Hospital

Introduction: Coronavirus disease 2019 (COVID-19) has infected millions of people worldwide, which is characterized by the manifestation of symptoms from coagulopathy to disseminated intravascular coagulation (DIC). This study aimed to investigated the correlation of D-dimer with lipoproteins values in COVID-19 patients. Methods: Observational cross-sectional analysis, using secondary data from medical records, based on the sample size formula it takes 78 samples, the data analysis method uses the SPSS program version 24. Results: From the results of the study, the number of patients with a D-dimer value of less than 0.5 ug/ml was 26%. While as many as 74% patients got the value of D-dimer increased by more than 0.5 ug/ml. Triglyceride and LDL levels were not associated with changes in D-dimer values. There is a correlation between the value of D-dimer with HDL (P = 0.024). High levels of D-dimer values are associated with the severity of symptoms and a poor prognosis. Excessive inflammatory processes will initiate coagulation via the extrinsic pathway, which progresses to disseminated intravascular coagulation (DIC) due to an imbalance between coagulation and fibrinolysis. Meanwhile, high HDL values reduce the risk of thrombotic events by initiating plasmin formation and are associated with low mortality rates. Conclusion: There is a statistically significant correlation between D-dimer values and HDL lipid levels. Suggestions, further research is needed to measure the correlation of D-dimer and HDL values in acute infection with the COVID-19 virus with a larger sample.

Tranexamic acid in a mouse model of cerebral amyloid angiopathy: setting the stage for a novel stroke treatment approach

BackgroundSymptomatic intracerebral hemorrhage (sICH) commonly occurs in patients with cerebral amyloid angiopathy (CAA). Amyloid also initiates plasminogen activation and might promote sICH. ObjectivesAs amyloid-driven plasmin formation can be blocked by tranexamic acid (TXA), we aimed to evaluate the biodistribution and long-term consequences of TXA on brain amyloid-beta (Aβ) levels, inflammation, and neurologic function in APP/PS1 mice. MethodsAPP/PS1 mice overexpressing the mutant human amyloid precursor protein and wild-type littermates were randomized to TXA (20 mg/mL) or placebo in the drinking water for 6 months. TXA in plasma and various organs was determined by liquid chromatography-mass spectrometry. Plasmin activity assays were performed to evaluate changes in fibrinolytic activity. Neurologic function was evaluated by Y-maze and parallel rod floor testing. Proximity ligation–based immunoassays were used to quantitate changes of 92 biomarkers of inflammation. Brain Aβ levels were assessed by immunohistochemistry. ResultsLong-term oral TXA administration inhibited fibrinolysis. TXA accumulated in the kidney (19.4 ± 11.2 μg/g) with 2- to 5-fold lower levels seen in the lung, spleen, and liver. TXA levels were lowest in the brain (0.28 ± 0.01 μg/g). Over 6 months, TXA had no discernible effect on motor coordination, novelty preference, or brain Aβ levels. TXA reduced plasma levels of epithelial cell adhesion molecule and increased CCL20. ConclusionLong-term TXA treatment does not alter brain Aβ levels or impact neurologic behavior in mice predisposed to amyloid deposition and had minor effects on the levels of inflammatory mediators. This finding supports the safety of TXA and lays the foundation for TXA as a novel treatment to reduce sICH in patients with CAA.

Acute myocardial infarction and pulmonary embolism in a young serviceman with a mutation of the plasminogen activator inhibitor gene type 1

The clinical case of acute myocardial infarction and pulmonary embolism in a young soldier with the first occurred protracted attack of angina. The survey found the link between these conditions and gene mutation of type 1 plasminogen activator inhibitor that plays a key role in fibrinolysis by inhibiting the formation of plasmin and leading to slowdown in fibrinolysis processes and a longer-term persistence of blood clot. It is proven that homozygous 4G/4G mutation found in the patient results in the development of arterial and venous thrombosis at a young age and is associated with a tendency to relapse. It clearly shows that hereditary thrombophilia is characterized by the absence of obvious provocative factor and single universal diagnostic algorithm; the diagnosis is based on the comprehensive evaluation of laboratory data.

INFLUENCE OF AMINOCAPRONIC ACID ON THE REGRESSION OF NASAL POLYPS AND NASAL CONGESTION IN PATIENTS WITH POLYPOUS RHINOSINUSITIS

Chronic rhinosinusitis with nasal polyposis (CRS with NP) is a clinical syndrome including several clinical phenotypes and endotypes with differences in pathophysiology and is divided into neutrophilic (non-type 2) and eosinophilic (type 2) according to the dominant endotype, which is the most severe variant associated with therapy resistance and frequent recurrence. Such pheno-endotype requires personalized treatment, and pharmacotherapy based on endotyping is prospective. Aminocapronic acid (ACA) competitively inhibits plasminogen and plasmin formation, inhibits C3a and C5a, which can affect the pathophysiological mechanisms of eosinophilic inflammation and polypous growth, since, in addition to hemostatic properties, it also has desensitizing, anti-enzymatic, and anti-proliferative properties.
 The aim of the research: to evaluate the influence of additional administration of aminocapronic acid on the degree of regression of nasal polyps in comparison with patients receiving standard therapy of Chronic rhinosinusitis with nasal polyposis according to clinical recommendations.
 Material and methods. The study included 120 outpatients, divided into two groups: the main (n - 60) and control (n - 60) ones. The average age of the main group was 45.8 years; the control group was 47.0 years. Patients were prescribed basic treatment (irrigation therapy with saline solutions and mometasone furoate), and in the main group – additionally aminocapronic acid (ACA). The evaluation of treatment effectiveness was based on the analysis of the dynamics of nasal congestion and the regression of nasal polyps at V2 (day 5±1), V3 (day 10±1), V4 (day 20±1) and V5 (day 30±1) compared to V0. Indications for surgical treatment were determined at V3.
 Results. The use of aminocapronic acid in Chronic rhinosinusitis with nasal polyposis contributes to a reliable reduction in the severity of the main clinical symptoms (rhinorrhea, postnasal drip, nasal congestion, reduced sense of smell), nasal congestion starting from V2, regression of polyps – from V3 compared to patients of the control group. This ensured a statistically significant 25% reduction in the number of operated patients: 56.7% in the main group versus 81.7% in the control (p<0.05). Conservative treatment was continued by 43.3% of patients of the main group and by 18.3% of patients of the control group (p<0.05). After the operated patients were removed from the groups, they did not significantly differ in the dynamics of regression of nasal congestion and nasal polyps at V4 and V5 (p>0.05). Differences in the results of treatment are attributed to the clinical effects of Aminocapronic acid (ACA), since the group characteristics of the patients were commensurable.
 Conclusions:
 
 the additional appointment of Aminocapronic acid (ACA) to patients with Chronic rhinosinusitis with nasal polyposis contributes to a faster regression of nasal congestion associated with edematous changes of the mucous membrane of the nasal cavity, compared to the regression of polyps during the fifth day of treatment;
 there is a significant difference in the severity of symptoms in the main group during the 10th day of treatment compared to patients in the control group;
 the reliable positive dynamics of clinical symptoms correlates with a reliable, at 25%, decrease in the number of operative interventions in the main group.

DYNAMICS OF CLINICAL SYMPTOMS UNDER THE INFLUENCE OF ENDOTYPE-ORIENTED PHARMACOTHERAPY OF CHRONIC RHINOSINUSITIS WITH NASAL POLYPOSIS

Chronic rhinosinusitis (CRS) is a clinical syndrome including several clinical phenotypes and endotypes with differences in pathophysiology. Chronic rhinosinusitis with nasal polyposis (NP) is the most severe CRS phenotype associated with treatment resistance and frequent recurrence after surgery. Among patients with CRS with NP, up to 96% have radiological changes that demonstrate damage to the paranasal sinuses and indicate a diffuse lesion. Pharmacotherapy based on endotyping using aminocapronic acid (ACA) competitively inhibits plasminogen and plasmin formation, inhibits C3a and C5a, which can affect the pathophysiological mechanisms of polypus growth, is promising.
 The aim of the study: to evaluate the clinical effectiveness of the additional appointment of ACA in comparison with patients receiving standard therapy of CRS with NP according to clinical recommendations.
 Material and methods. The study included 120 outpatients, divided into two groups: the main (n-60) and the control (n-60) ones. The main group (n-60) included 35 (58.3%) men and 25 (41.7%) women, the control group (n-60) included 32 (53.3%) men and 28 (46.7%) of women. The average age of the main group was 45.8 years; the control group was 47.0 years. Patients were prescribed basic treatment, but patients in the main group were additionally prescribed aminocapronic acid (ACA). The evaluation of the treatment effectiveness was based on the analysis of the dynamics of clinical symptoms: rhinorrhea, postnasal drip, nasal congestion, reduced sense of smell according to the SNOT 22 scale (from 0 to 5 points for each symptom) at V2 (5±1), V3 (10±1), V4 (20±1) and V5 (30±1) compared to V0, as well as the presence of indications for surgical treatment were determined at V3.
 Results. The use of aminocapronic acid in CRS with NP contributes to a reliable reduction in the severity of the main clinical symptoms (rhinorrhea, postnasal drip, nasal congestion, reduced sense of smell) at V2 and V3 in comparison with patients of the control group. This provided a statistically significant difference at 25% in the reduction of operated patients: 56.7% in the main group versus 81.7% in the control group (p<0.05). Conservative treatment was continued for 43.3% of patients in the main group and for 18.3% of patients in the control group (p<0.05). Such a difference in the dynamic of symptoms regression can be explained by the peculiarities of the biological action of ACA, which can influence the pathophysiological features that characterize the endotype of diffuse eosinophilic CRS with NP. After the removal of the operated patients, the groups did not differ significantly differ in the dynamics of the regression of rhinorrhea, postnasal drip, nasal congestion, decreased sense of smell at V4 and V5 (p>0.05). Differences in the results of treatment are attributed to the clinical effects of ACA, since the group characteristics of the patients were commensurable.
 Conclusions:
 
 the use of ACA in addition to the basic therapy in patients suffering from CRS with NP contributes to a significant reduction in the severity of the main clinical symptoms in the first 10 days of treatment compared to patients in the control group;
 the positive dynamics of clinical symptoms correlates with a reliable, at 25%, reduction in the number of surgical interventions.

PREDICTORS OF THROMBOGENICITY AND DEVELOPMENT OF ENDOTHELIITIS IN PATIENTS WITH СOVID-19 — ASSOCIATED CARDIAC AND VASCULAR SURGICAL PATHOLOGY IN THE ACUTE AND POST-COVID PERIODS

PREDICTORS OF THROMBOGENICITY AND DEVELOPMENT OF ENDOTHELIITIS IN PATIENTS WITH СOVID-19 — ASSOCIATED CARDIAC AND VASCULAR SURGICAL PATHOLOGY IN THE ACUTE AND POST-COVID PERIODS

A REVIEW ON TRANEXAMIC ACID

The objective is to study the effectiveness of hemostatic therapy with tranexamic acid in pregnant women with miscarriage that started. Tranexamic acid works by slowing the breakdown of blood clots, which helps to prevent prolonged bleeding. It belongs to a class of drugs known as antibrinolytics. Tranexamic acid is a synthetic derivative of the amino acid lysine and binds the 5 lysine binding sites on plasminogen. This inhibits plasmin formation and displaces plasminogen from the brin surface. It may also directly inhibit plasmin and partially inhibit brinogenolysis at higher concentrations. The investigators hypothesize that tranexamic acid as an adjunct to misoprostol will be more effective than misoprostol alone in stopping postpartum bleeding without recourse to further treatment in signicantly more women. The use of tranexamic acid, as hemostatic therapy in pregnant women with a miscarriage, signicantly reduces the duration of bleeding, promotes the accelerating the organization and resorption of intrauterine hematomas, reduces the duration of inpatient treatment. The rst trimester is associated with the highest risk for miscarriage. Most miscarriages occur in the rst trimester before the 12th week of pregnancy. A miscarriage in the second trimester (between 13 and 19 weeks) happens in 1% to 5% of pregnancies. Tranexamic acid appeared safe and effective for the prevention and management of bleeding during pregnancy.

Deficiency of Urokinase-Type Plasminogen Activator Receptor Is Associated with the Development of Perivascular Fibrosis in Mouse Heart

It was suggested that the urokinase system plays a certain role in the regulation of activity of the endothelial-mesenchymal transition and in the development of perivascular fibrosis. Urokinase (uPA), the key component of the urokinase system, is a serine protease that binds to its receptor on the cell surface (uPAR) and affects the cell microenvironment components through the formation of plasmin, remodeling of the extracellular matrix, release of growth factors, and initiation of intracellular signals. The heart of PLAUR gene knockout C57BL/129 (uPAR-/-) mice showed signs of vasculopathy: reduced number of capillaries/arterioles, signs of endothelial-mesenchymal transition in endothelial cells, vascular wall remodeling, and deposition of extracellular matrix components. These changes were combined with enhanced expression of urokinase and active forms of TGF-β1. Apparently, uPAR is a part of a multicomponent system that provides multifaceted regulatory effects on the components of forming vessels and vascular wall cells, which allows considering it as a possible target for targeted antifibrotic therapy.

Repurposing Tranexamic Acid as an Anticancer Agent.

Tranexamic Acid (TA) is a clinically used antifibrinolytic agent that acts as a Lys mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic Lys and Arg, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. Further, TA inhibited uptake of Lys and Arg by cancer cells. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.

Tenecteplase use in the management of acute ischemic stroke: Literature review and clinical considerations.

Several research articles have been published within the last decade comparing the use of tenecteplase to alteplase in ischemic stroke management. Prior reporting on the comparative therapeutic efficacy and safety profiles of tenecteplase and alteplase is reviewed. Tenecteplase is a variant of native tissue-type plasminogen activator, which rapidly promotes thrombolysis by catalyzing formation of the serine protease plasmin. Tenecteplase has theoretical advantages over alteplase as it has greater fibrin specificity and has a longer half-life than alteplase. This allows the administration of a single bolus over 5 to 10 seconds, as opposed to a bolus followed by a 1-hour infusion with alteplase. While currently approved by the Food and Drug Administration for the treatment of ST-segment elevation myocardial infarction, tenecteplase has also been studied in the treatment of acute ischemic stroke and has extensive data for this off-label indication. The most comprehensive trials to date evaluating the use of tenecteplase in acute ischemic stroke include the TNK-S2B, Australian TNK, ATTEST, Nor-Test, and EXTEND-IA TNK trials. Findings from these randomized controlled studies suggest that tenecteplase is at least as efficacious as alteplase in terms of neurological outcomes. The majority of these studies also reported a trend toward improved safety profiles with the use of tenecteplase. Current clinical evidence shows that tenecteplase is not inferior to alteplase for the treatment of ischemic stroke and suggests that tenecteplase may have a superior safety profile. Furthermore, tenecteplase also has practical advantages in terms of its administration. This can potentially lead to a decrease in medication errors and improvement in door to thrombolytic time.

Schistosoma mansoni phosphoglycerate mutase: a glycolytic ectoenzyme with thrombolytic potential.

Schistosomiasis is a debilitating parasitic disease caused by intravascular flatworms called schistosomes (blood flukes) that affects >200 million people worldwide. Proteomic analysis has revealed the surprising presence of classical glycolytic enzymes – typically cytosolic proteins – located on the extracellular surface of the parasite tegument (skin). Immunolocalization experiments show that phosphoglycerate mutase (PGM) is widely expressed in parasite tissues and is highly expressed in the tegument. We demonstrate that live Schistosoma mansoni parasites express enzymatically active PGM on their tegumental surface. Suppression of PGM using RNA interference (RNAi) diminishes S. mansoni PGM (SmPGM) gene expression, protein levels, and surface enzyme activity. Sequence comparisons place SmPGM in the cofactor (2,3-bisphosphoglycerate)-dependent PGM (dPGM) family. We have produced recombinant SmPGM (rSmPGM) in an enzymatically active form in Escherichia coli. The Michaelis-Menten constant (Km) of rSmPGM for its glycolytic substrate (3-phosphoglycerate) is 0.85 mM ± 0.02. rSmPGM activity is inhibited by the dPGM-specific inhibitor vanadate. Here, we show that rSmPGM not only binds to plasminogen but also promotes its conversion to an active form (plasmin) in vitro. This supports the hypothesis that host-interactive tegumental proteins (such as SmPGM), by enhancing plasmin formation, may help degrade blood clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo.

Involvement of Urokinase-Type Plasminogen Activator Receptor in the Formation of a Profibrotic Microenvironment in the Epicardial Region

The study of the mechanisms of development and progression of fibrosis is one of the key directions of modern cardiology. Our work suggests that the urokinase receptor (uPAR) is involved in the regulation of mesothelial cell activity and epicardial fibrosis development, which, when interacting with specific ligands and intermediate proteins, can activate intracellular signaling, trigger the cascade of proteolytic reactions, including local plasmin formation and activation of matrix metalloproteinases, providing matrix remodeling.Objective: to perform a comparative study of fibrogenic activity of the epicardium in the hearts of uPAR-/- and wild-type animals and evaluate the effect of cardiac microenvironment factors on the migration activity of epicardial mesothelial cells.Material and methods. We used histological and immunofluorescent staining, microarray analysis of proinflammatory cytokine levels, and a method for assessing the migratory properties of epicardial cells.Results. Results. We found that compared to wild-type animals, uPAR-/- animals show significant thickening of the epicardial area (2.46+0.77 (uPAR-/- mice) and 1.02+0.17 (Wt mice) relative units, P=0.033) accompanied by accumulation of extracellular matrix proteins. Deficiency of uPAR gene leads to formation of proinflammatory microenvironment in the heart (increased levels of proinflammatory factors such as IL-1, IL-13, IL-17, RANTES and MIP1), increased migratory activity of epicardial mesothelial cells, accumulation of TCF21+fibroblast/myofibroblast precursors (29.8+13.7 (uPAR-/- mouse) and 3.03+0.8 (Wt mouse) cells per visual field,P=0.02), as well as development of subepicardial fibrosis.Conclusion. These findings suggest that uPAR is a promising candidate for the developing targeted agents to prevent the development and progression of cardiac fibrosis.

Peroxide-Induced Damage to Plasminogen Molecules.

Plasminogen is a zymogenic form of plasmin, an enzyme that plays a fundamental role in the dissolution of fibrin clots as well as in many other physiological processes. For the first time, by the method of gas chromatography-mass spectrometry, post-translational modifications in the primary structure of plasminogen treated with physiologically relevant amounts of hydrogen peroxide were identified. It was found that methionine and tryptophan residues located in different structural regions of plasminogen served as targets of the oxidant. Plasminogen oxidation caused a dose-dependent effect in decreasing the fibrinogenolytic activity of plasmin evidenced by the formation of fibrinogen degradation products. The possible antioxidant role of methionines in the oxidative modification of plasminogen is discussed.

Fibrinolysis: A Primordial System Linked to the Immune Response.

The fibrinolytic system provides an essential means to remove fibrin deposits and blood clots. The actual protease responsible for this is plasmin, formed from its precursor, plasminogen. Fibrin is heralded as it most renowned substrate but for many years plasmin has been known to cleave many other substrates, and to also activate other proteolytic systems. Recent clinical studies have shown that the promotion of plasmin can lead to an immunosuppressed phenotype, in part via its ability to modulate cytokine expression. Almost all immune cells harbor at least one of a dozen plasminogen receptors that allows plasmin formation on the cell surface that in turn modulates immune cell behavior. Similarly, a multitude of pathogens can also express their own plasminogen activators, or contain surface proteins that provide binding sites host plasminogen. Plasmin formed under these circumstances also empowers these pathogens to modulate host immune defense mechanisms. Phylogenetic studies have revealed that the plasminogen activating system predates the appearance of fibrin, indicating that plasmin did not evolve as a fibrinolytic protease but perhaps has its roots as an immune modifying protease. While its fibrin removing capacity became apparent in lower vertebrates these primitive under-appreciated immune modifying functions still remain and are now becoming more recognised.

Assessing Plasmin Generation in Health and Disease.

Fibrinolysis is an important process in hemostasis responsible for dissolving the clot during wound healing. Plasmin is a central enzyme in this process via its capacity to cleave fibrin. The kinetics of plasmin generation (PG) and inhibition during fibrinolysis have been poorly understood until the recent development of assays to quantify these metrics. The assessment of plasmin kinetics allows for the identification of fibrinolytic dysfunction and better understanding of the relationships between abnormal fibrin dissolution and disease pathogenesis. Additionally, direct measurement of the inhibition of PG by antifibrinolytic medications, such as tranexamic acid, can be a useful tool to assess the risks and effectiveness of antifibrinolytic therapy in hemorrhagic diseases. This review provides an overview of available PG assays to directly measure the kinetics of plasmin formation and inhibition in human and mouse plasmas and focuses on their applications in defining the role of plasmin in diseases, including angioedema, hemophilia, rare bleeding disorders, COVID-19, or diet-induced obesity. Moreover, this review introduces the PG assay as a promising clinical and research method to monitor antifibrinolytic medications and screen for genetic or acquired fibrinolytic disorders.

Tissue-Type Plasminogen Activator and Tenecteplase-Mediated Increase in Blood Brain Barrier Permeability Involves Cell Intrinsic Complement.

Background: Tissue-type plasminogen activator (t-PA) has been the mainstay of therapeutic thrombolysis for patients with acute ischaemic stroke (AIS). However, t-PA can cause devastating intracerebral hemorrhage. t-PA can also influence the CNS in part by modulation of BBB permeability. Complement activation also occurs after AIS and has also been reported to increase BBB permeability. The complement components, C3 and C5, can also be activated by t-PA via plasmin formation and cell intrinsic complement may be involved in this process. Tenecteplase (TNK-tPA) is a t-PA variant with a longer plasma half-life, yet the ability of TNK-tPA to modulate the BBB and complement is less clear.Aim: To evaluate the effect of C5 and C5a-receptor 1 (C5aR1) inhibitors on t-PA- and TNK-tPA-mediated opening of the BBB.Methods: We used an in vitro model of the BBB where human brain endothelial cells and human astrocytes were co-cultured on the opposite sides of a porous membrane assembled in transwell inserts. The luminal (endothelial) compartment was stimulated with t-PA or TNK-tPA together with plasminogen, in the presence of PMX205 (a non-competitive C5aR1 antagonist), Avacopan (a competitive C5aR1 antagonist) or Eculizumab (a humanized monoclonal inhibitor of human C5). BBB permeability was assessed 5 and 24 h later. Immunofluorescence was also used to detect changes in C5 and C5aR1 expression in endothelial cells and astrocytes.Results: PMX205, but not Avacopan or Eculizumab, blocked t-PA-mediated increase in BBB permeability at both the 5 and 24 h time points. PMX205 also blocked TNK-tPA-mediated increase in BBB permeability. Immunofluorescence analysis revealed intracellular staining of C5 in both cell types. C5aR1 expression was also detected on the cell surfaces and also located intracellularly in both cell types.Conclusion: t-PA and TNK-tPA-mediated increase in BBB permeability involves C5aR1 receptor activation from cell-derived C5a. Selective inhibitors of C5aR1 may have therapeutic potential in AIS.