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Fibrinolytic agents catalyze the conversion of the inactive proenzyme plasminogen into the active protease plasmin, degrading fibrin within the thrombus and recanalizing occluded vessels. The history of these medications dates to the discovery of the first fibrinolytic compound, streptokinase, from bacterial cultures in 1933. Over time, researchers identified two other plasminogen activators in human samples, namely urokinase and tissue plasminogen activator (tPA). Subsequently, tPA was cloned using recombinant DNA methods to produce alteplase. Several additional derivatives of tPA, such as tenecteplase and reteplase, were developed to extend the plasma half-life of tPA. Over the past decades, fibrinolytic medications have been widely used to manage patients with venous and arterial thromboembolic events. Currently, alteplase is approved by the U.S. Food and Drug Administration (FDA) for use in patients with pulmonary embolism with hemodynamic compromise, ST-segment elevation myocardial infarction (STEMI), acute ischemic stroke, and central venous access device occlusion. Reteplase and tenecteplase have also received FDA approval for treating patients with STEMI. This review provides an overview of the historical background related to fibrinolytic agents and briefly summarizes their approved indications across various thromboembolic diseases. [ABSTRACT FROM AUTHOR]

Ghrelin and its growth hormone secretagogue receptor (GHSR) have been found in the placenta, both in endothelial and trophoblast cells. Ghrelin has been shown to decrease blood pressure in several systems and improve endothelial function by stimulating VEGF production. Because locally increased Ghrelin was detected in the preeclamptic fetoplacental unit, we hypothesized its involvement in the fibrinolysis and vascular tone typically observed in preeclamptic patients. This study aimed to evaluate the synthesis of plasminogen activators (PAs), PA inhibitor-1 (PAI-1), and urokinase-type PA (uPA) receptor (uPAR) in human umbilical vein endothelial cells (HUVECs) since the components of the PA/plasmin system are vital players in the extracellular matrix remodeling process necessary for angiogenesis. HUVECs were treated for 24 h with increasing concentrations of Ghrelin (10−11–10−7 M) or IL-1β (0.1 ng/mL). PAs, PAI-1, and uPAR mRNAs were determined by real-time PCR and PA activity was determined by casein underlay. We demonstrated an increase in uPA, tissue-type PA (tPA), and uPAR mRNA; a reduction in PAI-1 mRNA in HUVECs treated with Ghrelin; and an increase in total uPA activity. In conclusion, our results suggest a potential compensatory physiological mechanism for Ghrelin in response to the maternal endothelial dysfunction observed in the preeclamptic fetoplacental unit.

Ghrelin and its growth hormone secretagogue receptor (GHSR) have been found in the placenta, both in endothelial and trophoblast cells. Ghrelin has been shown to decrease blood pressure in several systems and improve endothelial function by stimulating VEGF production. Because locally increased Ghrelin was detected in the preeclamptic fetoplacental unit, we hypothesized its involvement in the fibrinolysis and vascular tone typically observed in preeclamptic patients. This study aimed to evaluate the synthesis of plasminogen activators (PAs), PA inhibitor-1 (PAI-1), and urokinase-type PA (uPA) receptor (uPAR) in human umbilical vein endothelial cells (HUVECs) since the components of the PA/plasmin system are vital players in the extracellular matrix remodeling process necessary for angiogenesis. HUVECs were treated for 24 h with increasing concentrations of Ghrelin (10−11–10−7 M) or IL-1β (0.1 ng/mL). PAs, PAI-1, and uPAR mRNAs were determined by real-time PCR and PA activity was determined by casein underlay. We demonstrated an increase in uPA, tissue-type PA (tPA), and uPAR mRNA; a reduction in PAI-1 mRNA in HUVECs treated with Ghrelin; and an increase in total uPA activity. In conclusion, our results suggest a potential compensatory physiological mechanism for Ghrelin in response to the maternal endothelial dysfunction observed in the preeclamptic fetoplacental unit. [ABSTRACT FROM AUTHOR]

Physical activity‐induced mechanical stimuli play a crucial role in preserving bone mass and structure by promoting bone formation. While the Wnt pathway is pivotal for mediating the osteoblast response to loading, the exact mechanisms are not fully understood. Here, we found that mechanical stimulation induces osteoblastic Wnt1 expression, resulting in an upregulation of key osteogenic marker genes, including Runx2 and Sp7, while Wnt1 knockdown using siRNA prevented these effects. RNAseq analysis identified Plat as a major target through which Wnt1 exerts its osteogenic influence. This was corroborated by Plat depletion using siRNA, confirming its positive role in osteogenic differentiation. Moreover, we demonstrated that mechanical stimulation enhances Plat expression, which, in turn leads to increased expression of osteogenic markers like Runx2 and Sp7. Notably, Plat depletion by siRNA prevented this effect. We have established that Wnt1 regulates Plat expression by activating β‐Catenin. Silencing Wnt1 impairs mechanically induced β‐Catenin activation, subsequently reducing Plat expression. Furthermore, our findings showed that Wnt1 is essential for osteoblasts to respond to mechanical stimulation and induce Runx2 and Sp7 expression, in part through the Wnt1/β‐Catenin/Plat signaling pathway. Additionally, we observed significantly reduced Wnt1 and Plat expression in bones from ovariectomy (OVX)‐induced and age‐related osteoporotic mouse models compared with non‐OVX and young mice, respectively. Overall, our data suggested that Wnt1 and Plat play significant roles in mechanically induced osteogenesis. Their decreased expression in bones from OVX and aged mice highlights their potential involvement in post‐menopausal and age‐related osteoporosis, respectively. [ABSTRACT FROM AUTHOR]

Intracerebral haemorrhage is an unmet medical need affecting more than 3 million people worldwide every year and leading to the formation of an intracerebral haematoma. Updated guidelines (2022) for the management of intracerebral haemorrhage patients recognize that minimally invasive approaches for the evacuation of supratentorial intracerebral haemorrhage have demonstrated reductions in mortality compared with medical management alone. However, improvement of functional outcome with a procedure involving thrombolytic therapy was neutral in the last large phase 3 clinical trial and requires a more effective and safer thrombolytic agent than those currently available. Here, we demonstrate that O2L-001 allows for the extended release of W253R/R275S recombinant tissue-type plasminogen activator (rtPA). A new rtPA variant, called optimized tPA (OptPA), offers improved efficacy for haematoma evacuation as well as improved safety. OptPA was produced in a Chinese hamster ovary cell line before purification, nanoprecipitation using the NANOp2Lysis® technological platform followed by suspension in a solution of 17% poloxamer 407 to obtain O2L-001. Plasmin generation assays were performed to demonstrate O2L-001 safety. Ex vivo haematoma models using human blood were used to demonstrate O2L-001 thrombolysis properties and efficacy. For the best translational significance, a clinical sized haematoma was used to ensure catheter placement and to allow administration of the thrombolytic agent into the core of the haematoma via a minimally invasive procedure. The capacity of OptPA to convert plasminogen into plasmin is strongly decreased compared to rtPA, thereby reducing potential bleeding events. However, a clot lysis assay showed that OptPA had the same fibrinolytic activity as rtPA. We demonstrated that long-term exposure to a thrombolytic agent was essential to achieve high thrombolysis efficacy. Indeed, 24 h continuous exposure to 0.1 µg/ml rtPA had similar efficacy than repeated short exposure to 30 µg/ml rtPA. This finding led to the development of O2L-001, allowing the extended release of OptPA in the first 6 h following injection. An ex vivo model using human blood was used to demonstrate O2L-001 efficacy. Interestingly, unlike rtPA, O2L-001 was able to induce the complete lysis of the 5 ml haematoma. In clinical sized haematomas (obtained from 30 ml of human blood), a single injection of O2L-001 at 1 mg/ml into the core of the haematoma led to a 44% increase in thrombolysis compared to rtPA. Taken together, these results demonstrate that O2L-001 provides new hope for haematoma evacuation and the treatment of patients with intracerebral haemorrhage. [ABSTRACT FROM AUTHOR]

Failure of hemostasis and the formation of blood clots in the arteries are the main reasons that provoke the onset of cardiovascular diseases (CVDs) such as myocardial infarction and ischemic stroke. Cardiovascular diseases have become the primary cause of deaths and disabilities across the globe. Therefore, this problem needs to be addressed with urgency. The disintegration of blood clots requires fibrinolytic agents, which are involved in thrombolysis. Streptokinase and urokinase are fibrinolytic enzymes; the former is primarily produced from microbial sources and the latter is isolated from urine, respectively. Streptokinase and urokinase have been in use for a long time to treat cardiovascular diseases. This review explains in detail the comparison of employing streptokinase and urokinase for the said purpose in a cost-effective manner. The recombinant production of both the agents has been discussed in detail. Furthermore, the efficacy of both the agents has been compared based upon their side effects and retention time in the body. A thorough study has been made to compare the influence of using both the agents on the health of cardiovascular patients in the last decade. [ABSTRACT FROM AUTHOR]

DSPAα1 is a potent rude thrombolytic protein with high medicative value. DSPAα1 has two natural N-glycan sites (N153Q-S154-S155, N398Q-K399-T400) that may lead to immune responses when administered in vivo. We aimed to study the effect of its N-glycosylation sites on DSPAα1 in vitro and in vivo by mutating these N-glycosylation sites. In this experiment, four single mutants and one double mutant were predicted and expressed in Pichia pastoris. When the N398Q-K399-T400 site was mutated, the fibrinolytic activity of the mutant was reduced by 75%. When the N153Q-S154-S155 sites were inactivated as described above, the plasminogen activating activity of its mutant was reduced by 40%, and fibrin selectivity was significantly reduced by 21-fold. The introduction of N-glycosylation on N184-G185-A186T and K368N-S369-S370 also considerably reduced the activity and fibrin selectivity of DSPAα1. The pH tolerance and thermotolerance of all mutants did not change significantly. In vivo experiments also confirmed that N-glycosylation mutations can reduce the safety of DSPAα1, lead to prolonged bleeding time, non-physiological reduction of coagulation factor (α2-AP, PAI) concentration, and increase the risk of irregular bleeding. This study ultimately demonstrated the effect of N-glycosylation mutations on the activity and safety of DSPAα1. [ABSTRACT FROM AUTHOR]

Bat plasminogen activators α2 (DSPAα2) has extremely high medicinal value as a powerful natural thrombolytic protein. However, wild-type DSPAα2 has two N-glycosylation sites (N185 and N398) and its non-human classes of high-mannose-type N-glycans may cause immune responses in vivo. By mutating the N-glycosylation sites, we aimed to study the effect of its N-glycan chain on plasminogen activation, fibrin sensitivity, and to observe the physicochemical properties of DSPAα2. A logical structure design was performed in this study. Four single mutants and one double mutant were constructed and expressed in Pichia pastoris. When the N398 site was eliminated, the plasminogen activator in the mutants had their activities reduced to ~40%. When the N185 site was inactivated, there was a weak decrease in the plasminogen activation of its mutant, while the fibrin sensitivity significantly decreased by ~10-fold. Neither N-glycosylation nor deglycosylation mutations changed the pH resistance or heat resistance of DSPAα2. This study confirms that N-glycosylation affects the biochemical function of DSPAα2, which provides a reference for subsequent applications of DSPAα2.

Background: The aim of this study is based on the effects of the treatment, the recombinant tissue plasminogen activator (rTPA), applied to stroke patients in Emergency Departments (ED) by intravenous, to optic nerve sheath diameter (ONSD) measurements. Material and Methods: The study was designed as prospective and observational. A total of 41 patients were included in this study with the following diagnoses: an acute clinical stroke, known well-being within 4.5 hours, and who had a finding for rTPA. Ultrasonographic (USG) ONSD measurements have been performed on a voluntary basis with the consent of the patients and their relatives. Results: A statistically significant difference has been found between the ONSDs measured at the 0th, 15th, 30th and 60th minutes of the patients who were admitted to the ED and were on rTPA treatment due to stroke (p

Modern published data and the generalized results of our own study of the mechanisms of action of vasopressin in the body are presented. A wide range of physiological effects of this neuropeptide, its structure, synthesis, and receptors are considered. The connection between the synthesis and release of vasopressin and the activation of the cholinergic system is emphasized. Particular attention is paid to the effects of this neuropeptide on the blood coagulation system. Emphasis is placed on elucidating the degree of participation of central and peripheral cholinergic receptors in the reactions of the hemostasis system to vasopressin. The leading role of these receptors in enhancing fibrinolysis and the activity of tissue plasminogen activator under the action of the peptide is described. The data summarized in this review indicate the need for further study of the mechanisms that ensure the realization of this effect of vasopressin for its use in clinical practice in order to prevent and treat thrombotic complications with the highest efficiency and minimal side effects. [ABSTRACT FROM AUTHOR]

DSPAα1 is a potent rude thrombolytic protein with high medicative value. DSPAα1 has two natural N-glycan sites (N153Q-S154-S155, N398Q-K399-T400) that may lead to immune responses when administered in vivo. We aimed to study the effect of its N-glycosylation sites on DSPAα1 in vitro and in vivo by mutating these N-glycosylation sites. In this experiment, four single mutants and one double mutant were predicted and expressed in Pichia pastoris. When the N398Q-K399-T400 site was mutated, the fibrinolytic activity of the mutant was reduced by 75%. When the N153Q-S154-S155 sites were inactivated as described above, the plasminogen activating activity of its mutant was reduced by 40%, and fibrin selectivity was significantly reduced by 21-fold. The introduction of N-glycosylation on N184-G185-A186T and K368N-S369-S370 also considerably reduced the activity and fibrin selectivity of DSPAα1. The pH tolerance and thermotolerance of all mutants did not change significantly. In vivo experiments also confirmed that N-glycosylation mutations can reduce the safety of DSPAα1, lead to prolonged bleeding time, non-physiological reduction of coagulation factor (α2-AP, PAI) concentration, and increase the risk of irregular bleeding. This study ultimately demonstrated the effect of N-glycosylation mutations on the activity and safety of DSPAα1.

Objectives: Hypoxia is a common feature of extravillous trophoblast (EVT) cells that promote invasion during the early stages of human placentation. This study aimed to examine whether hypoxia-induced an invasive phenotype in EVT cells in vitro and explore the underlying molecular mechanisms. Design: The invasiveness of primary EVT cells isolated from the first trimester placental tissues during weeks 5–8 of gestation was examined under hypoxic (5% O2) and normoxic (20% O2) conditions. Methods: Invasiveness was determined by transwell and wound-healing invasion assays using the IncuCyte ZOOM™ Live-Cell Imaging System. Protein expression of the urokinase plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor of hypoxia or normoxia-treated cells was measured using Western blot analysis. Knockdown of hypoxia-inducible factor-1 alpha (HIF-1α) was assessed using small interfering RNA (siRNA). Results: Hypoxia enhanced EVT cell invasion but did not affect apoptosis. The stimulatory effect of hypoxia on EVT cell invasiveness was associated with induction of the uPA-uPAR pathway. The synthetic inhibitor of uPAR significantly inhibited hypoxia-induced EVT cell invasion. Silencing of HIF-1α by siRNA abolished the stimulatory effect of hypoxia and inhibited the upregulation of uPAR expression, suggesting that the HIF-1α-uPAR signal is the key mediator for hypoxia-induced EVT cell invasion. Further experiments need to be performed to elucidate the HIF-1α-uPAR signal pathways. Conclusions: The low oxygen-regulated early events of EVT invasion may be mediated by the HIF-1α-uPAR pathway. [ABSTRACT FROM AUTHOR]

The fibrinolytic system is composed of the protease plasmin, its precursor plasminogen and their respective activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), counteracted by their inhibitors, plasminogen activator inhibitor type 1 (PAI-1), plasminogen activator inhibitor type 2 (PAI-2), protein C inhibitor (PCI), thrombin activable fibrinolysis inhibitor (TAFI), protease nexin 1 (PN-1) and neuroserpin. The action of plasmin is counteracted by α2-antiplasmin, α2-macroglobulin, TAFI, and other serine protease inhibitors (antithrombin and α2-antitrypsin) and PN-1 (protease nexin 1). These components are essential regulators of many physiologic processes. They are also involved in the pathogenesis of many disorders. Recent advancements in our understanding of these processes enable the opportunity of drug development in treating many of these disorders. [ABSTRACT FROM AUTHOR]

Failure of hemostasis and the formation of blood clots in the arteries are the main reasons that provoke the onset of cardiovascular diseases (CVDs) such as myocardial infarction and ischemic stroke. Cardiovascular diseases have become the primary cause of deaths and disabilities across the globe. Therefore, this problem needs to be addressed with urgency. The disintegration of blood clots requires fibrinolytic agents, which are involved in thrombolysis. Streptokinase and urokinase are fibrinolytic enzymes; the former is primarily produced from microbial sources and the latter is isolated from urine, respectively. Streptokinase and urokinase have been in use for a long time to treat cardiovascular diseases. This review explains in detail the comparison of employing streptokinase and urokinase for the said purpose in a cost-effective manner. The recombinant production of both the agents has been discussed in detail. Furthermore, the efficacy of both the agents has been compared based upon their side effects and retention time in the body. A thorough study has been made to compare the influence of using both the agents on the health of cardiovascular patients in the last decade. [ABSTRACT FROM AUTHOR]

The aims of the present study were to confirm that regulation of the PA and environment via TGF-β regulation of sperm by Percoll-separated in porcine uterine epithelial cells. And, it was performed to identify the cytokines (TGF-β1, 2 and 3, TGF-β receptor1 and 2; interleukin, IL-6, IL-8) and PA-related genes (urokinase-PA, uPA; tissue- PA, tPA; PA inhibitor, PAI; uPA-receptor, uPAR) by spermatozoa. The experiment used porcine uterus epithelial cells (pUECs) and uterine tissue epithelial cells, Boar sperm were separated by discontinuous Percoll density gradient (45/90%), and tissues were co-incubated with spermatozoa, followed by real-time PCR. PA activity was measured of sperm by discontinuous Percoll density gradient (45/90%) for 24 hours. To measure viability and acrosome damage of sperm double stained propidium iodide (PI) and SYBR- 14 or FITC-PNA were used. In results, binding ratio of Percoll-separated sperm was found no differences, but sperms isolated from 90% Percoll layer reduced PA activity (p < 0.05). when co-cultured sperm selected Percoll in porcine uterus tissues epithelial cells, 90% layer sperm increased TGF-β R1, contrastively tPA and PAI-1 in comparison with control (p < 0.05). 45% sperm was decreased the expression of uPA (p < 0.05). TGF-β decreased PA activity in the supernatant collected from pUECs (p < 0.05). Especially, The group including uPA, PAI-1 were induce sperm intact, while it was reduced in sperm damage when compared to control (p < 0.05). Also, there was no significant difference group of tPA and tPA+I in the dead sperm and acrosome damage compared to control. The expression of tPA and PAI showed a common response. Percoll-separated spermatozoa in 90% layer reduced tPA and IL-related gene mRNA expression. Thus, Percoll-sparated sperm in 90% layer show that it can suppress inflammation through increased expression of TGF-β and downregulation of PA and IL in epithelial cells compared to 45% layer Percoll.

Dermatomyositis (DM), an inflammatory disorder, is often associated with interstitial lung disease (ILD). However, the underlying mechanism remains unclear. Our study performed RNA sequencing (RNA-seq) and integrative bioinformatics analysis of differentially expressed genes (DEGs) in patients with dermatomyositis-associated interstitial lung disease (DM-ILD) and healthy controls. A total of 2,018 DEGs were identified between DM-ILD and healthy blood samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that DEGs were mainly involved in immune- and inflammatory-related biological processes and pathways. Disease ontology (DO) enrichment analysis identified 35 candidate key genes involved in both skin and lung diseases. Meanwhile, a total of 886 differentially expressed alternative splicing (AS) events were found between DM-ILD and healthy blood samples. After overlapping DEGs with differential AS genes, the plasminogen activator and urokinase receptor (PLAUR) involved in immune-related biological processes and complement and coagulation cascades was screened and identified as the most important gene associated with DM-ILD. The protein–protein interaction (PPI) network revealed that PLAUR had interactions with multiple candidate key genes. Moreover, we observed that there were significantly more neutrophils and less naive B cells in DM-ILD samples than in healthy samples. And the expression of PLAUR was significantly positively correlated with the abundance of neutrophils. Significant higher abundance of PLAUR in DM-ILD patients than healthy controls was validated by RT-qPCR. In conclusion, we identified PLAUR as an important player in regulating DM-ILD by neutrophil-associated immune response. These findings enrich our understanding, which may benefit DM-ILD patients. [ABSTRACT FROM AUTHOR]

Cancer patients are characterized by hypercoagulable state and an increased rate of thrombotic events, the most common being venous thromboembolism. Several hemostatic pathways that are significantly implicated in mechanisms of thromboembolic disease are also involved in growth, invasion, and metastatic spread of malignant cells as well in tumor-induced neo-angiogenesis. This close connection between cancer and the hemostatic system has prompted numerous studies on the role of alterations in the level plasma biomarkers of the different compartments of hemostasis in predicting cancer prognosis. In this review, we collect the results of several exemplificative studies that have evaluated clotting activation biomarkers in relation to different cancer outcomes with a final emphasis on current research and forthcoming directions in this field. [ABSTRACT FROM AUTHOR]

Dermatomyositis (DM), an inflammatory disorder, is often associated with interstitial lung disease (ILD). However, the underlying mechanism remains unclear. Our study performed RNA sequencing (RNA-seq) and integrative bioinformatics analysis of differentially expressed genes (DEGs) in patients with dermatomyositis-associated interstitial lung disease (DM-ILD) and healthy controls. A total of 2,018 DEGs were identified between DM-ILD and healthy blood samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that DEGs were mainly involved in immune- and inflammatory-related biological processes and pathways. Disease ontology (DO) enrichment analysis identified 35 candidate key genes involved in both skin and lung diseases. Meanwhile, a total of 886 differentially expressed alternative splicing (AS) events were found between DM-ILD and healthy blood samples. After overlapping DEGs with differential AS genes, the plasminogen activator and urokinase receptor (PLAUR) involved in immune-related biological processes and complement and coagulation cascades was screened and identified as the most important gene associated with DM-ILD. The protein–protein interaction (PPI) network revealed that PLAUR had interactions with multiple candidate key genes. Moreover, we observed that there were significantly more neutrophils and less naive B cells in DM-ILD samples than in healthy samples. And the expression of PLAUR was significantly positively correlated with the abundance of neutrophils. Significant higher abundance of PLAUR in DM-ILD patients than healthy controls was validated by RT-qPCR. In conclusion, we identified PLAUR as an important player in regulating DM-ILD by neutrophil-associated immune response. These findings enrich our understanding, which may benefit DM-ILD patients.

Sepsis is a life-threatening condition which develops as a dysregulated immune response in the face of infection and which is associated with profound hemostatic disturbances and in the most extreme cases disseminated intravascular coagulation (DIC). In addition, the fibrinolytic system is subject to alterations during infection and sepsis, and impaired fibrinolysis is currently considered a key player in sepsis-related microthrombus formation and DIC. However, we still lack reliable biomarkers to assess fibrinolysis in the clinical setting. Furthermore, drugs targeting the fibrinolytic system have potential value in sepsis patients with severe fibrinolytic disturbances, but these are still being tested in the preclinical stage. The present review provides an overview of key fibrinolytic changes in sepsis, reviews the current literature on potential laboratory markers of altered fibrinolysis in adult sepsis patients, and discusses future perspectives for diagnosis and treatment of fibrinolytic disturbances in sepsis patients. [ABSTRACT FROM AUTHOR]

The fibrinolytic system is composed of the protease plasmin, its precursor plasminogen and their respective activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), counteracted by their inhibitors, plasminogen activator inhibitor type 1 (PAI-1), plasminogen activator inhibitor type 2 (PAI-2), protein C inhibitor (PCI), thrombin activable fibrinolysis inhibitor (TAFI), protease nexin 1 (PN-1) and neuroserpin. The action of plasmin is counteracted by α2-antiplasmin, α2-macroglobulin, TAFI, and other serine protease inhibitors (antithrombin and α2-antitrypsin) and PN-1 (protease nexin 1). These components are essential regulators of many physiologic processes. They are also involved in the pathogenesis of many disorders. Recent advancements in our understanding of these processes enable the opportunity of drug development in treating many of these disorders.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. It is typically detected at an advanced stage, at which the therapeutic options are very limited. One remarkable feature of PDAC that contributes to its resilience to treatment is the extreme stromal activation seen in these tumors. Often, the vast majority of tumor bulk consists of non-tumor cells that together provide a tumor-promoting environment. One of the signals that maintains and activates the stroma is the developmental protein Sonic Hedgehog (SHH). As the disease progresses, tumor cells produce increasing amounts of SHH, which activates the surrounding stroma to aid in tumor progression. To better understand this response and identify targets for inhibition, we aimed to elucidate the proteins that mediate the SHH-driven stromal response in PDAC. For this a novel mixed-species coculture model was set up in which the cancer cells are human, and the stroma is modeled by mouse fibroblasts. In conjunction with next-generation sequencing we were able to use the sequence difference between these species to genetically distinguish between the epithelial and stromal responses to SHH. The stromal SHH-dependent genes from this analysis were validated and their relevance for human disease was subsequently determined in two independent patient cohorts. In non-microdissected tissue from PDAC patients, in which a large amount of stroma is present, the targets were confirmed to associate with tumor stroma versus normal pancreatic tissue. Patient survival analysis and immunohistochemistry identified CDA, EDIL3, ITGB4, PLAUR and SPOCK1 as SHH-dependent stromal factors that are associated with poor prognosis in PDAC patients. Summarizing, the presented data provide insight into the role of the activated stroma in PDAC, and how SHH acts to mediate this response. In addition, the study has yielded several candidates that are interesting therapeutic targets for a disease for which treatment options are still inadequate.

We explored the association of type 2 diabetes related blood markers with brain amyloid accumulation on PiB-PET scans in 41 participants from the FINGER PET sub-study. We built logistic regression models for brain amyloid status with12 plasma markers of glucose and lipid metabolism, controlled for diabetes and APOEɛ4 carrier status. Lower levels of insulin, insulin resistance index (HOMA-IR), C-peptide, and plasminogen activator (PAI-1) were associated with amyloid positive status, although the results were not significant after adjusting for multiple testing. None of the models found evidence for associations between amyloid status and fasting glucose or HbA1c. [ABSTRACT FROM AUTHOR]

Pathogens that colonize deep tissues and spread systemically encounter the innate host resistance mechanism of complement‐mediated lysis and complement opsonization leading to engulfment and degradation by phagocytic cells. Yersinia and Salmonella species have developed numerous strategies to block the antimicrobial effects of complement. These include recruitment of complement regulatory proteins factor H, C4BP, and vitronectin (Vn) as well as interference in late maturation events such as assembly of C9 into the membrane attack complex that leads to bacterial lysis. This review will discuss the contributions of various surface structures (proteins, lipopolysaccharide, and capsules) to evasion of complement‐mediated immune clearance of the systemic pathogens Yersiniae and Salmonellae. Bacterial proteins required for recruitment of complement regulatory proteins will be described, including the details of their interaction with host regulatory proteins, where known. The potential role of the surface proteases Pla (Yersinia pestis) and PgtE (Salmonella species) on the activity of complement regulatory proteins will also be addressed. Finally, the implications of complement inactivation on host cell interactions and host cell targeting for type 3 secretion will be discussed. [ABSTRACT FROM AUTHOR]

Blood coagulation and the fibrinolytic system contribute to vascular lesions. Fibrinolysis in normal circulating blood strongly depends on the balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) secreted from vascular endothelial cells; however, the mechanisms by which endothelial fibrinolysis is regulated remain to be fully understood. In the present study, human vascular endothelial EA.hy926 cells were transfected with small interfering RNA for nuclear factor erythroid 2-related factor 2 (NRF2) and the expression of t-PA and PAI-1 and fibrinolytic activity in the conditioned medium were examined. EA.hy926 cells were also treated with sulforaphane, an NRF2 activator, and fibrinolytic activity was examined to confirm the NRF2 signaling pathway's effect. Enhanced fibrinolytic activity in the conditioned medium was observed in association with increased expression and secretion levels of t-PA in NRF2 knockdown EA.hy926 cells. However, sulforaphane inhibited fibrinolytic activity and t-PA synthesis in EA.hy926 cells without any cell damage. The expression level of PAI-1 did not change in either NRF2 knockdown or sulforaphane treated cells. These results suggest that transcription factor NRF2 may play a role in down-regulating endothelial t-PA synthesis and fibrinolytic activity. [ABSTRACT FROM AUTHOR]

Vascular occlusion is one of the major causes of mortality and morbidity. Blood vessel blockage can lead to thrombotic complications such as myocardial infarction, stroke, deep venous thrombosis, peripheral occlusive disease, and pulmonary embolism. Thrombolytic therapy currently aims to rectify this through the administration of recombinant tissue plasminogen activator. Research is underway to design an ideal thrombolytic drug with the lowest risk. Despite the potent clot lysis achievable using approved thrombolytic drugs such as alteplase, reteplase, streptokinase, tenecteplase, and some other fibrinolytic agents, there are some drawbacks, such as high production cost, systemic bleeding, intracranial hemorrhage, vessel re-occlusion by platelet-rich and retracted secondary clots, and non-fibrin specificity. In comparison, bacterial staphylokinase, is a new, small-size plasminogen activator, unlike bacterial streptokinase, it hinders the systemic degradation of fibrinogen and reduces the risk of severe hemorrhage. A fibrin-bound plasmin–staphylokinase complex shows high resistance to a2-antiplasmin-related inhibition. Staphylokinase has the potential to be considered as a promising thrombolytic agent with properties of cost-effective production and the least side effects. [ABSTRACT FROM AUTHOR]

There has been an increasing prevalence of cardiovascular diseases such as myocardial infarction and stroke in modern societies because of multiple lifestyle related issues like sedentariness and obesity, alcohol consumption and many more "life-style"factors. The FDA-approved thrombolytics such as Tissue Plasminogen Activator, Streptokinase etc. are used to lyse the clots in thrombotic disorders such as myocardial infarction, stroke etc. but re-occlusion and bleeding that are co-incident to their clinical usage are not addressed. Hence, there is need to develop thrombolytics having properties like increased fibrin clot specificity and thrombin inhibition capability to prevent re-occlusion. In the present work, a fusion protein construct containing two components i.e. Staphylokinase (SAK) and Epidermal Growth Factor (EGF) 4, 5, 6-like domains of human thrombomodulin (THBD) was expressed in Pichia pastoris after genetic optimization. SAK isolated from Staphylococcus aureus is a fibrin-specific plasminogen activator while EGF 4, 5, 6-like domains are reported to be responsible for imparting thrombin inhibition to human thrombomodulin, and therefore, expected could help prevent re-occlusion in the novel construct - SAK_EGF, which is a 43 kDa protein. After expression, it was purified (approx. 13-fold) using two-step purification protocol involving ion-exchange followed by Gel Filtration Chromatography (GFC). The functional characterization including plasminogen activation and thrombin inhibition showed that both the fusion partners viz. SAK and 4,5,6 EGF-like domains retained their respective activities after fusion, confirming it to be a bio-active construct. Thus, this engineered protein could be clinically promising due to the combinatorial effect of fibrin-specific thrombus lysis and prevention of re-occulusion. • Staphylokinase (SAK) and selected Epidermal Growth Factor like domains (EGF) of Thrombomodulin were fused at genetic level. • To prevent glycosylation in Staphylokinase, a potential glycosylation site was mutated. • Hyper producer clones were screened using increasing concentrations of the antibiotic Geneticin (G418). • The SAK_EGF fusion protein was purified by a simple two-step chromatography process. • Both fusion partners were found to be functionally active. [ABSTRACT FROM AUTHOR]

Abstract Background: Lachesis muta rhombeata is one of the venomous snakes of medical importance in Brazil whose envenoming is characterized by local and systemic effects which may produce even shock and death. Its venom is mainly comprised of serine and metalloproteinases, phospholipases A2 and bradykinin-potentiating peptides. Based on a previously reported fractionation of L. m. rhombeata venom (LmrV), we decided to perform a subproteome analysis of its major fraction and investigated a novel component present in this venom. Methods: LmrV was fractionated through molecular exclusion chromatography and the main fraction (S5) was submitted to fibrinogenolytic activity assay and fractionated by reversed-phase chromatography. The N-terminal sequences of the subfractions eluted from reversed-phase chromatography were determined by automated Edman degradation. Enzyme activity of LmrSP-4 was evaluated upon chromogenic substrates for thrombin (S-2238), plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) and upon fibrinogen. All assays were carried out in the presence or absence of possible inhibitors. The fluorescence resonance energy transfer substrate Abz-KLRSSKQ-EDDnp was used to determine the optimal conditions for LmrSP-4 activity. Molecular mass of LmrSP-4 was determined by MALDI-TOF and digested peptides after trypsin and Glu-C treatments were analyzed by high resolution MS/MS using different fragmentation modes. Results: Fraction S5 showed strong proteolytic activity upon fibrinogen. Its fractionation by reversed-phase chromatography gave rise to 6 main fractions (S5C1-S5C6). S5C1-S5C5 fractions correspond to serine proteinases whereas S5C6 represents a C-type lectin. S5C4 (named LmrSP-4) had its N-terminal determined by Edman degradation up to the 53rd amino acid residue and was chosen for characterization studies. LmrSP-4 is a fibrinogenolytic serine proteinase with high activity against S-2302, being inhibited by PMSF and benzamidine, but not by 1,10-phenantroline. In addition, this enzyme exhibited maximum activity within the pH range from neutral to basic and between 40 and 50 °C. About 68% of the LmrSP-4 primary structure was covered, and its molecular mass is 28,190 Da. Conclusions: Novel serine proteinase isoforms and a lectin were identified in LmrV. Additionally, a kallikrein-like serine proteinase that might be useful as molecular tool for investigating bradykinin-involving process was isolated and partially characterized.

The emerging concept of a crosstalk between hemostasis, inflammation, and immune system prompt recent works on coagulation cascade in multiple sclerosis (MS). Studies on MS pathology identified several coagulation factors since the beginning of the disease pathophysiology: fibrin deposition with breakdown of blood brain barrier, and coagulation factors within active plaques may exert pathogenic role, especially through the innate immune system. Studies on circulating coagulation factors showed complex imbalance involving several components of hemostasis cascade (thrombin, factor X, factor XII). To analyze the role of the coagulation process in connection with other pathogenic pathways, we implemented a systematic matching of genome-wide association studies (GWAS) data with an informative and unbiased network of coagulation pathways. Using MetaCore (version 6.35 build 69300, 2018) we analyzed the connectivity (i.e., direct and indirect interactions among two networks) between the network of the coagulation process and the network resulting from feeding into MetaCore the MS GWAS data. The two networks presented a remarkable over-connectivity: 958 connections vs. 561 expected by chance; z-score = 17.39; p-value < 0.00001. Moreover, genes coding for cluster of differentiation 40 (CD40) and plasminogen activator, urokinase (PLAU) shared both networks, pointed to an integral interplay between coagulation cascade and main pathogenic immune effectors. In fact, CD40 pathways is especially operative in B cells, that are currently a major therapeutic target in MS field. The potential interaction of PLAU with a signal of paramount importance for B cell pathogenicity, such as CD40, suggest new lines of research and pave the way to implement new therapeutic targets.

The emerging concept of a crosstalk between hemostasis, inflammation, and immune system prompt recent works on coagulation cascade in multiple sclerosis (MS). Studies on MS pathology identified several coagulation factors since the beginning of the disease pathophysiology: fibrin deposition with breakdown of blood brain barrier, and coagulation factors within active plaques may exert pathogenic role, especially through the innate immune system. Studies on circulating coagulation factors showed complex imbalance involving several components of hemostasis cascade (thrombin, factor X, factor XII). To analyze the role of the coagulation process in connection with other pathogenic pathways, we implemented a systematic matching of genome-wide association studies (GWAS) data with an informative and unbiased network of coagulation pathways. Using MetaCore (version 6.35 build 69300, 2018) we analyzed the connectivity (i.e., direct and indirect interactions among two networks) between the network of the coagulation process and the network resulting from feeding into MetaCore the MS GWAS data. The two networks presented a remarkable over-connectivity: 958 connections vs. 561 expected by chance; z -score = 17.39; p -value < 0.00001. Moreover, genes coding for cluster of differentiation 40 (CD40) and plasminogen activator, urokinase (PLAU) shared both networks, pointed to an integral interplay between coagulation cascade and main pathogenic immune effectors. In fact, CD40 pathways is especially operative in B cells, that are currently a major therapeutic target in MS field. The potential interaction of PLAU with a signal of paramount importance for B cell pathogenicity, such as CD40, suggest new lines of research and pave the way to implement new therapeutic targets. [ABSTRACT FROM AUTHOR]

The Gram-negative bacterium Yersinia pestis causes plague, a fatal flea-borne anthropozoonosis, which can progress to aerosol-transmitted pneumonia. Y. pestis overcomes the innate immunity of its host thanks to many pathogenicity factors, including plasminogen activator, Pla. This factor is a broad-spectrum outer membrane protease also acting as adhesin and invasin. Y. pestis uses Pla adhesion and proteolytic capacity to manipulate the fibrinolytic cascade and immune system to produce bacteremia necessary for pathogen transmission via fleabite or aerosols. Because of microevolution, Y. pestis invasiveness has increased significantly after a single amino-acid substitution (I259T) in Pla of one of the oldest Y. pestis phylogenetic groups. This mutation caused a better ability to activate plasminogen. In paradox with its fibrinolytic activity, Pla cleaves and inactivates the tissue factor pathway inhibitor (TFPI), a key inhibitor of the coagulation cascade. This function in the plague remains enigmatic. Pla (or pla) had been used as a specific marker of Y. pestis, but its solitary detection is no longer valid as this gene is present in other species of Enterobacteriaceae. Though recovering hosts generate anti-Pla antibodies, Pla is not a good subunit vaccine. However, its deletion increases the safety of attenuated Y. pestis strains, providing a means to generate a safe live plague vaccine.

Yersinia pestis plasminogen activator, a surface protease (Pla) is encoded by species-specific plasmid pPla. This enzyme belongs to outer membrane protein family and has a ß-barrel structure. Pla protease interacts complexly with homeostasis system of a hostorganism and is an important factor of plague agent virulence. Represented are the literature data on the part of plasminogen activator in the development of bubonic and pneumonic plague. Described is the role of R-form lipopolysaccharide in protease Pla activity manifestation. Plasminogen activator directly contributes to fibrinolysis by activating plasminogen and inactivating plasminogen-1 activator inhibitor (PAI-1) and ά2-anti-plasmin, as well as promoted by thrombin fibrinolysis inhibitor (TAFI). Pro-coagulant Pla protein activity is caused by the destruction of tissue factor pathway inhibitor (TFPI). In addition, given is the information on non-proteolytic functions of plasminogen activator.

Abstract Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) act in the proteolysis of basement membrane and extracellular matrix structures, facilitating tumor invasion. The purpose of this study was to evaluate the relationship between these proteins and clinicopathological parameters in squamous cell carcinoma of the oral tongue (SCCOT). Sixty cases of SCCOT were submitted to immunohistochemistry and analyzed semiquantitatively at the invasion front and in the tumor core. The results were associated with lymph node metastasis, clinical stage, locoregional recurrence, clinical outcome and histological grade of malignancy. A higher expression of uPA was observed in cases of tumors of high-grade versus low-grade malignancy (p = 0.010). Moreover, the cases with the worst pattern of invasion presented an overexpression of uPA (p = 0.011). The presence of locoregional recurrence was associated with uPAR (p = 0.039), and the expression of both biomarkers was much higher at the invasion front than in the tumor core (p < 0.001). The results suggest uPA and uPAR are involved in the progression and aggressiveness of SCCOT, mainly at the tumor-host interface.

Background Soluble urokinase plasminogen activator receptor (suPAR) is a proinflammatory biomarker associated with immune activation and fibrinolysis inhibition. Plasminogen activator inhibitor (PAI‐1) is associated with excessive fibrin accumulation, thrombus formation, and atherosclerosis. The relationship between cross‐coronary suPAR and PAI‐1 production and endothelial dysfunction remains unknown. Methods and Results Seventy‐nine patients (age 53±10 years, 75% women) with angina and normal coronary arteries or mild coronary artery disease (20% after acetylcholine) and mircovascular endothelial dysfunction (coronary blood flow change