Search Results (71)

Background: Fibrinolytic impairment is one of the key factors involved in the pathogenesis of hemostasis disturbances in sepsis, significantly contributing to microthrombosis, organ dysfunction, and mortality rates. While hemostatic assessment in sepsis typically focuses on coagulation activation, evaluating fibrinolytic activity remains challenging due to methodological limitations and a lack of standardization of the currently available methods. Objectives: This comprehensive review examines current methods for assessing fibrinolytic activity in bacterial sepsis, their clinical applications, strengths and limitations, and future perspectives for improved diagnostic approaches. Methods: We conducted a systematic literature search and identified 52 studies that investigated fibrinolysis assessment in adult patients with bacterial sepsis using biomarkers or global tests. Studies included mainly observational cohorts examining various fibrinolytic assessment methods. Results: Fibrinolytic shutdown, primarily mediated by the overproduction of plasminogen activator inhibitor-1 (PAI-1), occurs early in sepsis and correlates with disease severity and mortality. Current assessment methods include plasma biomarker measurements (PAI-1, plasmin-antiplasmin complexes, D-dimer), global plasma-based tests (clot lysis time, plasmin generation assays), and whole-blood viscoelastic testing (rotational thromboelastometry, ROTEM; thromboelastography, TEG). Modified viscoelastic tests incorporating tissue plasminogen activators demonstrate enhanced sensitivity for detecting fibrinolytic resistance. Despite efforts, standardization is still limited, and routine clinical implementation has not been achieved yet. Conclusions: Fibrinolytic assessment provides important prognostic information in sepsis, despite methodological challenges. The integration of point-of-care viscoelastic testing with modified protocols shows promise for real-time evaluation. Future research should focus on developing standardized, automated assays suitable for routine clinical practice, enabling personalized therapeutic interventions that target fibrinolytic dysfunction in sepsis. Full article

Alpha2-plasmin inhibitor (α2PI) has a heterogeneous structure due to proteolytic cleavages in the circulation. The C-terminally cleaved form loses the plasminogen binding site and is, therefore, a slow plasmin inhibitor (NPB-α2PI). As FXIII primarily crosslinks the plasminogen-binding intact form (PB-α2PI) to fibrin, the effect of NPB-α2PI on fibrinolysis has been less studied. Herein, we investigated the effect of C-terminal truncation. Total-, PB-, and NPB-α2PI antigen levels and α2PI incorporation were measured by ELISAs from samples of 80 healthy individuals. Clot lysis parameters of the same subjects were investigated using an in vitro clot lysis assay. α2PI incorporation into the clot was demonstrated by Western blotting. Clot lysis and clot structure were also analyzed using an α2PI-deficient plasma substituted with recombinant PB- and NPB-α2PI. Both plasma and clot-bound levels of total- and NPB-α2PI showed a significant positive correlation with clot lysis parameters. NPB-α2PI was detected in the clot due to non-covalent binding. Regardless of the type of binding, both forms affected the clot structure by increasing the thickness of the fibrin fibers and reducing the pore size. In conclusion, we found that NPB-α2PI can bind non-covalently to fibrin, and this binding contributes to changes in clot structure and inhibition of fibrinolysis. Full article

Background/Objectives: We define severe postpartum hemorrhage (PPH) with macroscopic hematuria as clinical disseminated intravascular coagulation (DIC), a life-threatening condition. We also report a methodology using machine learning, a subtype of artificial intelligence, for developing the boundary criterion for predicting hematuria on the fibrinogen–fibrin/fibrinogen degradation product (FDP) plane. A positive FDP–fibrinogen/3–60 (mg/dL) value indicates hematuria; otherwise, non-hematuria is observed. We aimed to validate this criterion using severe placental abruption (PA), and to examine the activation of the coagulation–fibrinolytic system in clinical DIC. Methods: Of 17,285 deliveries across nine perinatal centers in Japan between 2020 and 2024, 13 had severe PA without hematuria, 18 had severe PPH without hematuria, and 3 had severe PPH with hematuria, i.e., clinical DIC. We calculated the values of the criterion formula for 13 cases of severe PA to validate the boundary criterion and compared the laboratory tests for coagulation–fibrinolytic activation among the three groups. Results: The calculated values using the criterion for the 13 PA without hematuria ranged from −108.91 to −5.87 and all were negative. In cases of clinical DIC, fibrinogen levels (median, 62 mg/dL) were lower (p < 0.05), while levels of FDP (96 mg/dL), the thrombin–antithrombin complex (120 ng/mL), and the plasmin-α₂–plasmin inhibitor complex (28.4 μg/mL) were significantly higher than in the other two groups. Conclusions: This study demonstrated the validity of the boundary criterion for predicting hematuria using severe PA. The coagulation–fibrinolytic test results suggested that PPH cases with hematuria were assumed to have clinical DIC, indicating that this criterion may be considered for diagnosing DIC during delivery. However, further additional patient data are needed to confirm the usefulness of this criterion because of the very low number of hematuria cases. Full article

The role of the plasminogen activation system is to regulate the activity of the extracellular protease plasmin. It comprises the urokinase plasminogen activator (uPA), a specific extracellular protease which activates plasminogen, its inhibitor PAI1, and the urokinase plasminogen activator receptor, uPAR, which localizes the urokinase activity. The plasminogen activation system is involved in tissue remodeling through extracellular matrix degradation, and therefore participates in numerous physiological and pathological processes, which make it a potential biomarker. To investigate the role of these molecules in the cellular processes, we cloned human uPA, PAI1, and uPAR and overexpressed them in two cell lines, the glioblastoma line A1235 and the transformed human embryonal kidney cells HEK 293. We analyzed the urokinase activity and the expression of plasminogen activation system elements on the protein and RNA level by Western blot analysis and RTqPCR. Cell proliferation was followed up by cell counting, cell migration and invasion by wound-healing and the transwell assays, respectively, and cell adhesion and dispersal by spheroid formation. The cells transfected with urokinase sequence had increased urokinase activity and uPA expression, while the PAI1-transfected cells decreased urokinase activity, increased PAI1 expression, and decreased cell migration. HEK 293 cells expressing PAI formed only small spheroids. The effects of the uPA system molecules depended on their interactions with each other and with other molecules in the microenvironment, as well as on the cell-type-specific signaling. Full article

Neuroendocrine neoplasms (NENs) are a diverse group originating from endocrine cells/their precursors in pancreas, small intestine, or lung. The key serum marker is chromogranin A (CgA). While commonly elevated in patients with NEN, its prognostic value is still under discussion. Secretion/posttranslational proteolytic cleavage of CgA results in multiple bioactive fragments, which are essential regulators of the cardiovascular and immune system. miR-223, regulator of Nrlp3 inflammasome and neutrophil activation, was recently found to have decreased in patients with NEN. We performed flow cytometry of circulating neutrophils in a patient cohort (n = 10) with NEN, microdissection and histology of tumor tissue. Subsequently, in vitro transfections using the well-established human pancreatic NEN cell line (BON), and co-culture experiments with primary macrophages and neutrophils were performed. Serum miR-223 in patients correlated with the expression of the neutrophil activation marker CD15 in circulating cells. Neutrophilic CD62L/CD63 showed good discrimination compared to healthy controls. Immune cell-derived miR-155, miR-193 and miR-223 colocalize with neutrophil in the extra-tumoral tissue alongside Nlrp3-associated caspase-1 activation. miR-223 knockdown in BON decreased the CgA intracellularly, increased in cellular granularity and caspase-1 activation. Plasmin inhibitor a2-aP reverted those effects. Western Blot showed fragmented CgA following miR-223 knockdown, which altered the inflammatory potential of neutrophils. Our data hence provide initial insights into an immunoregulatory mechanism via miR-223 and CgA in NEN cells, as regulation of miR-223 in NEN may affect tumor-associated inflammation. Full article

Coagulation factor XIa is a new serine-protease family drug target for next-generation anticoagulants. With the snake venom Kunitz-type peptide BF9 as the scaffold, we obtained a highly active XIa inhibitor BF9-N17K in our previous work, but it also inhibited the hemostatic target plasmin. Here, in order to enhance the selectivity of BF9-N17K toward XIa, four mutants, BF9-N17K-L19A, BF9-N17K-L19S, BF9-N17K-L19D, and BF9-N17K-L19K, were further designed using the P2′ amino acid classification scanning strategy. The anticoagulation assay showed that the four P2′ single-point mutants still had apparent inhibitory anticoagulation activity that selectively inhibited the human intrinsic coagulation pathway and had no influence on the extrinsic coagulation pathway or common coagulation pathway, which indicated that the single-point mutants had minimal effects on the anticoagulation activity of BF9-N17K. Interestingly, the enzyme inhibitor assay experiments showed that the XIa and plasmin inhibitory activities were significantly changed by the P2′ amino acid replacements. The XIa inhibitory activity of BF9-N17K-L19D was apparently enhanced, with an IC₅₀ of 19.28 ± 2.53 nM, and its plasmin inhibitory was significantly weakened, with an IC₅₀ of 459.33 ± 337.40 nM. BF9-N17K-L19K was the opposite to BF9-N17K-L19D, which had enhanced plasmin inhibitory activity and reduced XIa inhibitory activity. For BF9-N17K-L19A and BF9-N17K-L19S, no apparent changes were found in the serine protease inhibitory activity, and they had similar XIa and plasmin inhibitory activities to the template peptide BF9-N17K. These results suggested that the characteristics of the charge of the P2′ site might be associated with the drug selectivity between the anticoagulant target XIa and hemostatic target plasmin. In addition, according to the molecular diversity and sequence conservation, a common motif GR/PCR/KA/SXIP-XYGGC is proposed in the XIa-inhibitory Kunitz-type peptides, which might provide a new clue for further peptide engineering. In conclusion, through P2′ amino acid classification scanning with the snake venom Kunitz-type peptide scaffold, a new potent and selective XIa inhibitor, BF9-N17K-L19D, was discovered, which provides a new XIa-targeting lead drug template for the treatment of thrombotic-related diseases. Full article

The structure of aspirated coronary thrombus in ST-segment elevation myocardial infarction (STEMI) is still being studied. Our aims were to characterize coronary thrombi of different ages, focusing on the appearance of activated protein C (APC/PC) and its relation to the elements of neutrophil extracellular traps (NETs), and the factors closely related to fibrin as factor XIII (FXIII) and α2 plasmin inhibitor (α2-PI). The thrombi of n = 24 male patients with atherosclerotic coronary plaque rupture related to native coronary artery occlusion were selected for histopathology analysis. Thrombus age was distinguished as fresh, lytic, and organized, and then analyzed by immunofluorescent staining and confocal microscopy. FXIII was present at a high level and showed a high degree of co-localization with fibrin in all stages of thrombus evolution. The amount of α2-PI was low in the fresh thrombi, which increased significantly to the lytic phase. It was evenly distributed and consistently associated with fibrin. APC/PC appeared in the fresh thrombus and remained constant during its evolution. The presence of NET marker and CD66b was most dominant in the lytic phase. APC/PC co-localization with the elements of NET formation shows its role in NET degradation. These observations suggest the importance of searching for further targeted therapeutic strategies in STEMI patients. Full article

Background: The thrombin-activatable fibrinolysis inhibitor (TAFI) is an important regulator in the balance between blood clot formation (coagulation) and dissolution (fibrinolysis), which is mainly activated by thrombin bonded with thrombomodulin (TM). Methods: In this study, the investigation focused on the unique target TAFI of fungi fibrinolytic compound 1 (FGFC1), a novel fibrinolytic compound sourced from the deep sea. In this sense, the regulation of TAFI by FGFC1, in comparison to established TAFI inhibitors such as DS-1040 and PCTI in hPPP, was investigated, which was validated through the molecular docking of FGFC1 to TAFI. The inhibitory effect of FGFC1 on TAFI-mediating coagulation (ex vivo and in vitro) and its fibrinolytic effect (ex vivo) were investigated in hPPP and hCMEC/D3 cells, respectively, followed by SEM. Results: FGFC1 solutions ranging from 0.023 to 0.736 mM effectively inhibited TAFI activation. Notably, the 0.023 mM concentration demonstrated significant suppression, comparable to DS-1040 and PCTI. These inhibitory effects of FGFC1 (0.023–0.368 mM) were further validated through the enhancement in TAFI (TAFIa) activation by fibrins in the coagulum prior to proteolysis, resulting in the cleavage of TAFIa from 33 kDa to 28 kDa. Furthermore, these regulatory effects of FGFC1 on TAFI were demonstrated to have minimal association with TM-mediated control, as confirmed through a molecular docking analysis. FGFC1 (0.023–0.092 mM) was suggested to have obstructive effects on TAFI-mediated coagulation in the hPPP, which was demonstrated by the inhibition of clot aggregation, protein crystallization, and platelet anchoring, as observed through SEM. Simultaneously, FGFC1 (0.023 to 0.368 mM) significantly enhanced TAFI-mediated fibrinolysis, which was also supported by increased levels of t-PA, u-PA, and plasmin. Conclusions: From the above findings, FGFC1 is identified as a novel dual-target bioactive compound participating in blood formation/dissolution that demonstrates anti-coagulation and fibrinolytic effects by regulating TAFI activation, inhibiting TAFIa–fibrin combination, and initiating proteolysis. It also provided convincing evidence that TAFI plays a critical role in thrombolysis as a molecular link between coagulation and fibrinolysis. Furthermore, the application of FGFC1 was indicated as a potential therapeutic strategy in thromboembolic and hemorrhagic diseases. Full article

Plasmin is a serine protease induced by UV-irradiation in skin that contributes to inflammation. We showed that plasmin is upregulated in photo-exposed facial skin and that this correlates with increased transepidermal water loss (TEWL). Plasmin activity upregulates downstream pathways such as pro-inflammatory cytokines and matrix-metalloproteinases (MMPs). In addition, the plasminogen system modulates cutaneous melanogenesis. In this study, we investigated potential skin-aging effects of plasmin with a dual inhibitor of plasmin and its activator urokinase (uPA). We established a range of in vitro and ex vivo assays to investigate inflammation, MMP-9 activation, and collagen modulation, and the melanogenesis modulation activity of plasmin. A specific plasmin inhibitor, Amidinobenzyl Benzylsulfonyl D-Seryl Homophenylalaninamide Acetate (ABSHA), was used in these assays to downregulate these effects. We found that ABSHA was able to down-regulate UV-irradiation-induced MMP-9 expression, and subsequent collagen IV degradation, ex vivo. In addition, the increased melanin synthesis in epidermal melanocytes was reduced significantly by ABSHA. Furthermore, dermal fibroblasts treated with the plasmin inhibitor showed increased collagen I synthesis. We further investigated these effects in a two-month, monocentric, placebo-controlled human study on female Chinese volunteers. We found a significant increase in collagen density by ultrasound measurement and an increase in elasticity by cutometer assessment in the group using a formulation consisting of a 10 ppm ABSHA solution. This resulted in decreased wrinkle volumes on both the forehead and crow’s feet as shown by Primos CR. Looking at age spots, there was a decrease in overall ITA° and melanin density as well as in the total age spot area. Our results establish plasmin as a skin-aging enzyme. Using specific inhibitors against plasmin shows promise against age-induced skin conditions. Full article

A novel fibrinolytic enzyme was produced by the liquid fermentation of Coprinus comatus. The enzyme was purified from the culture supernatant by hydrophobic interactions, gel filtration, and ion exchange chromatographies. It was purified by 241.02-fold, with a specific activity of 3619 U/mg and a final yield of 10.02%. SDS-PAGE analysis confirmed the purity of the enzyme, showing a single band with a molecular weight of 19.5 kDa. The first nine amino acids of the N-terminal of the purified enzyme were A-T-Y-T-G-G-S-Q-T. The enzyme exhibited optimal activity at a temperature of 42 °C and pH 7.6. Its activity was significantly improved by Zn²⁺, K⁺, Ca²⁺, Mn²⁺, and Mg²⁺ while being inhibited by Fe²⁺, Fe³⁺, Al²⁺, and Ba²⁺. The activity of the enzyme was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and it was also dose-dependently inhibited by phenylmethylsulfonyl fluoride (PMSF) and soy trypsin inhibitor (SBTI). However, inhibitors such as N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK), aprotinin, and pepstatin did not significantly affect its activity, suggesting that the enzyme was a serine-like metalloproteinase. The enzyme acted as both a plasmin-like fibrinolytic enzyme and a plasminogen activator, and it also exhibited the capability to hydrolyze fibrinogen and fibrin. In vitro, it demonstrated the ability to dissolve blood clots and exhibit anticoagulant properties. Furthermore, it was found that the enzyme prolonged activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT), and reduced the levels of fibrinogen (FIB) and prothrombin activity (PA). Based on these studies, the enzyme has great potential to be developed as a natural agent for the prevention and treatment of thrombotic diseases. Full article

Background/Objectives: The coagulation cascade due to tissue damage is considered to be one of the causes of poor prognostic outcomes in patients with acute exacerbations of interstitial lung disease (AE-ILD). This study aimed to confirm coagulopathy in AE-ILD by evaluating the differences in the clinical characteristics of coagulation/fibrinolysis markers between stable ILD and AE-ILD. Methods: Overall, 81 patients were enrolled in this retrospective study and categorized into the following two groups: a chronic ILD group comprising 63 outpatients and an acute ILD group comprising 18 inpatients diagnosed with AE-ILD. Serum markers, including thrombin–antithrombin III complex (TAT), D-dimer, plasmin–α2 plasmin inhibitor complex (PIC), and surfactant protein D (SP-D), were compared between the groups. Results: Among the 18 patients with acute ILD, 17 did not meet the International Society of Thrombosis and Hemostasis scoring system for disseminated intravascular coagulation. In acute ILD, the SP-D levels were statistically significantly positively correlated with TAT, D-dimer, and PIC levels, while the Krebs von den Lungen 6 (KL-6) levels showed no correlation with any of these coagulation/fibrinolytic markers. A positive correlation was observed between SP-D levels and TAT, D-dimer, and PIC levels in acute ILD. Serum TAT, D-dimer, and PIC all showed good area under the receiver operating characteristic (ROC) curve (AUC) values in ROC analysis for the diagnosis of acute ILD. Conclusions: In the clinical setting of AE-ILD, it may be important to focus not only on alveolar damage markers such as SP-D but also on coagulation/fibrinolytic markers including TAT, D-dimer, and PIC. Full article

Two anti-fibrotic drugs, pirfenidone (PFD) and nintedanib (NTD), are currently used to treat idiopathic pulmonary fibrosis (IPF). Peripheral blood mononuclear cells (PBMCs) are immunocompetent cells that could orchestrate cell–cell interactions associated with IPF pathogenesis. We employed RNA sequencing to examine the transcriptome signature in the bulk PBMCs of patients with IPF and the effects of anti-fibrotic drugs on these signatures. Differentially expressed genes (DEGs) between “patients with IPF and healthy controls” and “before and after anti-fibrotic treatment” were analyzed. Enrichment analysis suggested that fatty acid elongation interferes with TGF-β/Smad signaling and the production of oxidative stress since treatment with NTD upregulates the fatty acid elongation enzymes ELOVL6. Treatment with PFD downregulates COL1A1, which produces wound-healing collagens because activated monocyte-derived macrophages participate in the production of collagen, type I, and alpha 1 during tissue damage. Plasminogen activator inhibitor-1 (PAI-1) regulates wound healing by inhibiting plasmin-mediated matrix metalloproteinase activation, and the inhibition of PAI-1 activity attenuates lung fibrosis. DEG analysis suggested that both the PFD and NTD upregulate SERPINE1, which regulates PAI-1 activity. This study embraces a novel approach by using RNA sequencing to examine PBMCs in IPF, potentially revealing systemic biomarkers or pathways that could be targeted for therapy. Full article

Chronic inflammatory diseases, such as rheumatoid arthritis, spondyloarthritis, systemic lupus erythematosus, Crohn’s disease, periodontitis, and carcinoma metastasis frequently result in bone destruction. Pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-17 are known to influence bone loss by promoting the differentiation and activation of osteoclasts. Fibrinolytic factors, such as plasminogen (Plg), plasmin, urokinase-type plasminogen activator (uPA), its receptor (uPAR), tissue-type plasminogen activator (tPA), α2-antiplasmin (α2AP), and plasminogen activator inhibitor-1 (PAI-1) are expressed in osteoclasts and osteoblasts and are considered essential in maintaining bone homeostasis by regulating the functions of both osteoclasts and osteoblasts. Additionally, fibrinolytic factors are associated with the regulation of inflammation and the immune system. This review explores the roles of fibrinolytic factors in bone destruction caused by inflammation. Full article

Neural regeneration and neuroprotection represent strategies for future management of neurodegenerative disorders such as Alzheimer’s disease (AD) or glaucoma. However, the complex molecular mechanisms that are involved in neuroprotection are not clearly understood. A promising candidate that maintains neuroprotective signaling networks is neuroserpin (Serpini1), a serine protease inhibitor expressed in neurons which selectively inhibits extracellular tissue-type plasminogen activator (tPA)/plasmin and plays a neuroprotective role during ischemic brain injury. Abnormal function of this protein has been implicated in several conditions including stroke, glaucoma, AD, and familial encephalopathy with neuroserpin inclusion bodies (FENIB). Here, we explore the potential biochemical roles of Serpini1 by comparing proteome changes between neuroserpin-deficient (NS^−/−) and control mice, in the retina (RE), optic nerve (ON), frontal cortex (FC), visual cortex (VC), and cerebellum (CB). To achieve this, a multiple-plex quantitative proteomics approach using isobaric tandem mass tag (TMT) technology was employed followed by functional enrichment and protein–protein interaction analysis. We detected around 5000 proteins in each tissue and a pool of 6432 quantified proteins across all regions, resulting in a pool of 1235 differentially expressed proteins (DEPs). Principal component analysis and hierarchical clustering highlighted similarities and differences in the retina compared to various brain regions, as well as differentiating NS^−/− proteome signatures from control samples. The visual cortex revealed the highest number of DEPs, followed by cerebellar regions. Pathway analysis unveiled region-specific changes, including visual perception, focal adhesion, apoptosis, glutamate receptor activation, and supramolecular fiber organization in RE, ON, FC, VC, and CB, respectively. These novel findings provide comprehensive insights into the region-specific networking of Serpini1 in the central nervous system, further characterizing its potential role as a neuroprotective agent. Data are available via ProteomeXchange with identifier PXD046873. Full article

Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease–serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease–serpin in the framework of heparin that we review includes the following: thrombin–antithrombin III, plasmin–anti-plasmin, C1 esterase/kallikrein–C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)–alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer. Full article