The effect of plasmin-mediated degradation on fibrinolysis and tissue plasminogen activator diffusion.

We modify a three-dimensional multiscale model of fibrinolysis to study the effect of plasmin-mediated degradation of fibrin on tissue plasminogen activator (tPA) diffusion and fibrinolysis. We propose that tPA is released from a fibrin fiber by simple kinetic unbinding, as well as by "forced unbinding," which occurs when plasmin degrades fibrin to which tPA is bound. We show that, if tPA is bound to a small-enough piece of fibrin that it can diffuse into the clot, then plasmin can increase the effective diffusion of tPA. If tPA is bound to larger fibrin degradation products (FDPs) that can only diffuse along the clot, then plasmin can decrease the effective diffusion of tPA. We find that lysis rates are fastest when tPA is bound to fibrin that can diffuse into the clot, and slowest when tPA is bound to FDPs that can only diffuse along the clot. Laboratory experiments confirm that FDPs can diffuse into a clot, and they support the model hypothesis that forced unbinding of tPA results in a mix of FDPs, such that tPA bound to FDPs can diffuse both into and along the clot. Regardless of how tPA is released from a fiber, a tPA mutant with a smaller dissociation constant results in slower lysis (because tPA binds strongly to fibrin), and a tPA mutant with a larger dissociation constant results in faster lysis.

Synergism of red blood cells and tranexamic acid in the inhibition of fibrinolysis.

BACKGROUND: Postpartum hemorrhage (PPH) is the leading cause of maternal death worldwide. The World Maternal Antifibrinolytic trial showed that antifibrinolytic tranexamic acid (TXA) reduces PPH deaths. Maternal anemia increases the risk of PPH. The World Maternal Antifibrinolytic-2 trial is now assessing whether TXA can prevent PPH in women with anemia. Low red blood cell (RBC) counts promote fibrinolysis by altering fibrin structure and plasminogen activation. OBJECTIVES: We explored interactions between RBCs and TXA in inhibiting fibrinolysis. METHODS: We used global fibrinolytic assays (ball sedimentation and viscoelasticity) to monitor the lysis of fibrin containing plasminogen and tissue-type plasminogen activator. We applied a fluorogenic kinetic assay to measure plasmin generation in fibrin clots and scanning electron microscopy to study fibrin structure. RESULTS: According to parallel-line bioassay analysis of the fibrin lysis-time data, the antifibrinolytic potency of 4-128 µM TXA was increased in the presence of 10% to 40% (v/v) RBCs. Global fibrinolysis assays showed that the joint effect of RBCs and TXA was about 15% larger than the sum of their individual effects in the inhibition of fibrinolysis. In plasminogen activation, TXA added the same increment of inhibition to the effect of RBCs at any cell count in the fibrin clot. Regarding fibrin structure, TXA thickened fibrin fibers, which impaired plasminogen activation, whereas RBCs promoted fine fibers that were more resistant to plasmin. CONCLUSIONS: The antifibrinolytic potency of TXA is enhanced in fibrin formed in the presence of RBCs through inhibition of plasminogen activation and fibrin lysis, which correlates with modifications of fibrin structures.

Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal disease with limited therapeutic options. The infiltration of monocytes and fibroblasts into the injured lungs is implicated in IPF. Enolase-1 (ENO1) is a cytosolic glycolytic enzyme which could translocate onto the cell surface and act as a plasminogen receptor to facilitate cell migration via plasmin activation. Our proprietary ENO1 antibody, HL217, was screened for its specific binding to ENO1 and significant inhibition of cell migration and plasmin activation (patent: US9382331B2). METHODS: In this study, effects of HL217 were evaluated in vivo and in vitro for treating lung fibrosis. RESULTS: Elevated ENO1 expression was found in fibrotic lungs in human and in bleomycin-treated mice. In the mouse model, HL217 reduced bleomycin-induced lung fibrosis, inflammation, body weight loss, lung weight gain, TGF-ß upregulation in bronchial alveolar lavage fluid (BALF), and collagen deposition in lung. Moreover, HL217 reduced the migration of peripheral blood mononuclear cells (PBMC) and the recruitment of myeloid cells into the lungs. In vitro, HL217 significantly reduced cell-associated plasmin activation and cytokines secretion from primary human PBMC and endothelial cells. In primary human lung fibroblasts, HL217 also reduced cell migration and collagen secretion. CONCLUSIONS: These findings suggest multi-faceted roles of cell surface ENO1 and a potential therapeutic approach for pulmonary fibrosis.

Plasmin-Induced Lens Epithelial Cells Detachment for the Reduction of Posterior Capsular Opacification.

Purpose: We sought to evaluate the efficacy and safety of plasmin injection in the capsular bag during the cataract operation for the prevention of posterior capsule opacification. Methods: Thirty-seven anterior capsular flaps taken from phacoemulsification surgery were immersed in either 1 µg/mL plasmin (plasmin group, n = 27) or phosphate-buffered saline (control group, n = 10) for 2 minutes and photographed after fixation and nuclear staining to compare the numbers of residual lens epithelial cells. In the animal experiments, the plasmin solution was injected into the capsular bag and remained for 5 minutes during hydrodissection or after lens extraction. The degree of posterior capsular opacity of the rabbits at 2 months were photographed by slit lamp biomicroscopy. In HLE-B3 cell culture, the cell detachment rate, proliferation, and apoptosis after the plasmin digestion were analyzed. Results: The residual lens epithelial cell numbers on the capsule after plasmin treatment were 168 ± 190.7/mm2 in the 1 µg/mL plasmin group, which was significantly lower than that of the control (1012 ± 798.8/mm2; P < 0.0001). In a rabbit model, the treatment of plasmin resulted in a significantly clearer posterior capsule compared with that of the control group at 2 months postoperatively. Conclusions: This study suggested that plasmin injection can induce effective lens epithelial cell detachment, which could be a promising adjunctive treatment to further improve the success rate in posterior capsule opacification prevention. Translational Relevance: Plasmin injection for lens epithelial cell detachment could significantly decrease the number of residual lens epithelial cells. This approach could be a promising treatment incorporating the current treatment approach to further improve the success rate in posterior capsule opacification prevention.

The serine protease plasmin plays detrimental roles in epithelial sodium channel activation and podocyte injury in Dahl salt-sensitive rats.

Salt-sensitive hypertension is associated with poor clinical outcomes. The epithelial sodium channel (ENaC) in the kidney plays pivotal roles in sodium reabsorption and blood pressure regulation, in which its γ subunit is activated by extracellular serine proteases. In proteinuric nephropathies, plasmin filtered through injured glomeruli reportedly activates γENaC in the distal nephron and causes podocyte injury. We previously reported that Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet developed hypertension and proteinuria along with γENaC activation and that a synthetic serine protease inhibitor, camostat mesilate, mitigated these changes. However, the role of plasmin in DS rats remained unclear. In this study, we evaluated the relationship between plasmin and hypertension as well as podocyte injury and the effects of plasmin inhibitors in DS rats. Five-week-old DS rats were divided into normal-salt diet, HS diet, and HS+plasmin inhibitor (either tranexamic acid [TA] or synthetic plasmin inhibitor YO-2) groups. After blood pressure measurement and 24 h urine collection over 5 weeks, rats were sacrificed for biochemical analyses. The HS group displayed severe hypertension and proteinuria together with activation of plasmin in urine and γENaC in the kidney, which was significantly attenuated by YO-2 but not TA. YO-2 inhibited the attachment of plasmin(ogen) to podocytes and alleviated podocyte injury by inhibiting apoptosis and inflammatory/profibrotic cytokines. YO-2 also suppressed upregulation of protease-activated receptor-1 and phosphorylated ERK1/2. These results indicate an important role of plasmin in the development of salt-sensitive hypertension and related podocyte injury, suggesting plasmin inhibition as a potential therapeutic strategy.

Absence of hyperfibrinolysis may explain lack of efficacy of tranexamic acid in hypoproliferative thrombocytopenia.

The American Trial Using Tranexamic Acid (TXA) in Thrombocytopenia (A-TREAT, NCT02578901) demonstrated no superiority of TXA over placebo in preventing World Health Organization (WHO) grade 2 or higher bleeding in patients with severe thrombocytopenia requiring supportive platelet transfusion following myeloablative therapy for hematologic disorders. In this ancillary study, we sought to determine whether this clinical outcome could be explained on the basis of correlative assays of fibrinolysis. Plasma was collected from A-TREAT participants (n = 115) before the initiation of study drug (baseline) and when TXA was at steady-state trough concentration (follow-up). Global fibrinolysis was measured by 3 assays: euglobulin clot lysis time (ECLT), plasmin generation (PG), and tissue-type plasminogen activator (tPA)-challenged clot lysis time (tPA-CLT). TXA was quantified in follow-up samples by tandem mass spectrometry. Baseline samples did not demonstrate fibrinolytic activation by ECLT or tPA-CLT. Furthermore, neither ECLT nor levels of plasminogen activator inhibitor-1, tPA, plasminogen, alpha2-antiplasmin, or plasmin-antiplasmin complexes were associated with a greater risk of WHO grade 2+ bleeding. TXA trough concentrations were highly variable (range, 0.7-10 µg/mL) and did not correlate with bleeding severity, despite the fact that plasma TXA levels correlated strongly with pharmacodynamic assessments by PG (Spearman r, -0.78) and tPA-CLT (r, 0.74). We conclude that (1) no evidence of fibrinolytic activation was observed in these patients with thrombocytopenia, (2) trough TXA concentrations varied significantly between patients receiving the same dosing schedule, and (3) tPA-CLT and PG correlated well with TXA drug levels.

Cryoprecipitate transfusion in trauma patients attenuates hyperfibrinolysis and restores normal clot structure and stability: Results from a laboratory sub-study of the FEISTY trial.

BACKGROUND: Fibrinogen is the first coagulation protein to reach critical levels during traumatic haemorrhage. This laboratory study compares paired plasma samples pre- and post-fibrinogen replacement from the Fibrinogen Early In Severe Trauma studY (FEISTY; NCT02745041). FEISTY is the first randomised controlled trial to compare the time to administration of cryoprecipitate (cryo) and fibrinogen concentrate (Fg-C; Riastap) in trauma patients. This study will determine differences in clot strength and fibrinolytic stability within individuals and between treatment arms. METHODS: Clot lysis, plasmin generation, atomic force microscopy and confocal microscopy were utilised to investigate clot strength and structure in FEISTY patient plasma. RESULTS: Fibrinogen concentration was significantly increased post-transfusion in both groups. The rate of plasmin generation was reduced 1.5-fold post-transfusion of cryo but remained unchanged with Fg-C transfusion. Plasminogen activator inhibitor 1 activity and antigen levels and Factor XIII antigen were increased post-treatment with cryo, but not Fg-C. Confocal microscopy analysis of fibrin clots revealed that cryo transfusion restored fibrin structure similar to those observed in control clots. In contrast, clots remained porous with stunted fibres after infusion with Fg-C. Cryo but not Fg-C treatment increased individual fibre toughness and stiffness. CONCLUSIONS: In summary, our data indicate that cryo transfusion restores key fibrinolytic regulators and limits plasmin generation to form stronger clots in an ex vivo laboratory study. This is the first study to investigate differences in clot stability and structure between cryo and Fg-C and demonstrates that the additional factors in cryo allow formation of a stronger and more stable clot.

The suboptimal fibrinolytic response in COVID-19 is dictated by high PAI-1.

BACKGROUND: Severe COVID-19 disease is associated with thrombotic complications and extensive fibrin deposition. This study investigates whether the hemostatic complications in COVID-19 disease arise due to dysregulation of the fibrinolytic system. METHODS: This prospective study analyzed fibrinolytic profiles of 113 patients hospitalized with COVID-19 disease with 24 patients with non-COVID-19 respiratory infection and healthy controls. Antigens were quantified by Ella system or ELISA, clot lysis by turbidimetric assay, and plasminogen activator inhibitor-1 (PAI-1)/plasmin activity using chromogenic substrates. Clot structure was visualized by confocal microscopy. RESULTS: PAI-1 and its cofactor, vitronectin, are significantly elevated in patients with COVID-19 disease compared with those with non-COVID-19 respiratory infection and healthy control groups. Thrombin activatable fibrinolysis inhibitor and tissue plasminogen activator were elevated in patients with COVID-19 disease relative to healthy controls. PAI-1 and tissue plasminogen activator (tPA) were associated with more severe COVID-19 disease severity. Clots formed from COVID-19 plasma demonstrate an altered fibrin network, with attenuated fiber length and increased branching. Functional studies reveal that plasmin generation and clot lysis were markedly attenuated in COVID-19 disease, while PAI-1 activity was elevated. Clot lysis time significantly correlated with PAI-1 levels. Stratification of COVID-19 samples according to PAI-1 levels reveals significantly faster lysis when using the PAI-1 resistant (tPA) variant, tenecteplase, over alteplase lysis. CONCLUSION: This study shows that the suboptimal fibrinolytic response in COVID-19 disease is directly attributable to elevated levels of PAI-1, which attenuate plasmin generation. These data highlight the important prognostic potential of PAI-1 and the possibility of using pre-existing drugs, such as tenecteplase, to treat COVID-19 disease and potentially other respiratory diseases.

Tranexamic acid: Beyond antifibrinolysis.

Tranexamic acid (TXA) is a popular antifibrinolytic drug widely used in hemorrhagic trauma patients and cardiovascular, orthopedic, and gynecological surgical patients. TXA binds plasminogen and prevents its maturation to the fibrinolytic enzyme plasmin. A number of studies have demonstrated the broad life-saving effects of TXA in trauma, superior to those of other antifibrinolytic agents. Besides preventing fibrinolysis and blood loss, TXA has been reported to suppress posttraumatic inflammation and edema. Although the efficiency of TXA transcends simple inhibition of fibrinolysis, little is known about its mechanisms of action besides the suppression of plasmin maturation. Understanding the broader effects of TXA at the cell, organ, and organism levels are required to elucidate its potential mechanisms of action transcending antifibrinolytic activity. In this article, we provide a brief review of the current clinical use of TXA and then focus on the effects of TXA beyond antifibrinolytics such as its anti-inflammatory activity, protection of the endothelial and epithelial monolayers, stimulation of mitochondrial respiration, and suppression of melanogenesis.

Preparation of thermo-responsive polymer encapsulated exosomes and its role as a therapeutic agent for blood clot lysis.

Tissue plasminogen activators induce enzymatic activation of plasminogen to plasmin that cleaves fibrin strands in blood clots. In the present study, extracellular vesicles such as exosomes from fibrosarcoma cell line HT1080 were utilized as clot-busting agents. These exosomes were being used for clot lysis of whole blood which showed 28% lysis within 10 h, which was comparable to that of the streptokinase (commercial plasmin activator) with no significant difference. These exosomes were able to facilitate the migration of endothelial cells in a scratch wound assay where normalized wound area remaining was 7.5% at 18 h. Also, exosomes aided in attenuation of oxidative stress generated on the cells, thereby maintaining cell viability. These exosomes were further encapsulated in a thermo-responsive polymer for better localized delivery that showed no cytotoxic effects, and sustained delivery was achieved up to a concentration of 117 µg/mL in 25 days, which corresponds to around 65% of the total amount of exosomes added. When a combination of exosomes and thermo-responsive polymer was utilized, the clot lysis activity reached to around 22% in 72 h. Thus, it proves the potential of this combinatorial approach which can be effectively used for thrombus degradation and healing of endothelium lining in damaged blood vessels.

Urokinase plasminogen activator induces epithelial-mesenchymal and metastasis of pancreatic cancer through plasmin/MMP14/TGF-ß axis, which is inhibited by 4-acetyl-antroquinonol B treatment.

BACKGROUND: The current standard therapy for metastatic pancreatic cancer is ineffective, necessitating a new treatment approach for prognosis improvement. The urokinase-plasmin activator (uPA) is a critical factor in epithelial-mesenchymal transition (EMT) and cancer metastasis, but its underlying mechanisms in pancreatic cancer remains elusive. METHODS: We investigated uPA expression in our pancreatic cancer cohort. A bioinformatics approach was used to further determine the role of uPA in pancreatic cancer. We employed MiaPaCa-2 and PANC-1 cell lines to investigate how uPA regulates EMT and metastasis in pancreatic cancer and present a novel approach aimed at inhibiting uPA in pancreatic cancer. RESULTS: We observed that higher uPA mRNA expression was significantly associated with overall-poor survival and progression-free survival in pancreatic cancer. uPA was highly expressed in tumor tissue. Gene set enrichment analysis revealed a positive association between uPA mRNA expression and EMT and transforming growth factor ß (TGF-ß) signaling pathways. Moreover, shRNA-mediated uPA gene knockdown reduced plasmin, MMP14, and TGF-ß activation, leading to the inhibition of PANC-1 cells' EMT marker expression, migration, invasion, and cell viability. Notably, 4-acetyl-antroquinonol B (4-AAQB) treatment suppressed MiaPaCa-2 and PANC-1 cell migratory and invasive abilities by inhibiting the uPA/MMP14/TGF-ß axis through upregulation of miR-181d-5p. In the xenograft mouse model of orthotropic pancreatic cancer, 4-AAQB treatment has reduced tumor growth and metastasis rate by deactivating uPA and improving the survival of the mice model. CONCLUSION: Accordingly, to extent of our knowledge and previous studies, we demonstrated that 4-AAQB is an anti Pan-Cancer drug, and may inhibit pancreatic cancer EMT and metastasis and serve as a new therapeutic approach for patients with late-stage pancreatic cancer.

Plasmin-resistant PSD-95 inhibitors resolve effect-modifying drug-drug interactions between alteplase and nerinetide in acute stroke.

Neuroprotection for acute ischemic stroke is achievable with the eicosapeptide nerinetide, an inhibitor of the protein-protein interactions of the synaptic scaffolding protein PSD-95. However, nerinetide is subject to proteolytic cleavage if administered after alteplase, a standard-of-care thrombolytic agent that nullifies nerinetide's beneficial effects. Here, we showed, on the basis of pharmacokinetic data consistent between rats, primates, and humans, that in a rat model of embolic middle cerebral artery occlusion (eMCAO), nerinetide maintained its effectiveness when administered before alteplase. Because of its short plasma half-life, it can be followed by alteplase within minutes without reducing its neuroprotective effectiveness. In addition, the problem of protease sensitivity is solved by substituting cleavage-prone amino acids from their l- to their d-enantiomeric form. Treatment of rats subjected to eMCAO with such an agent, termed d-Tat-l-2B9c, eliminated protease sensitivity and maintained neuroprotective effectiveness. Our data suggest that both the clinical-stage PSD-95 inhibitor nerinetide and protease-resistant agents such as d-Tat-l-2B9c may be practically integrated into existing stroke care workflows and standards of care.

A unique group of scabies mite pseudoproteases promotes cutaneous blood coagulation and delays plasmin-induced fibrinolysis.

BACKGROUND: Scabies, a highly contagious skin disease affecting more than 200 million people worldwide at any time, is caused by the parasitic mite Sarcoptes scabiei. In the absence of molecular markers, diagnosis requires experience making surveillance and control challenging. Superficial microthrombi in the absence of vasculitis in scabies-affected skin are a recognised, yet unexplained histopathological differential of scabies infection. This study demonstrates that a family of Scabies Mite Inactivated Cysteine Protease Paralogues (SMIPP-Cs) excreted by the mites plays a role in formation of scabies-induced superficial microthrombi. METHODOLOGY/PRINCIPAL FINDINGS: A series of in vitro and ex vivo experiments involving two representative recombinant SMIPP-Cs was carried out. In the presence of SMIPP-Cs, the thrombin clotting time (TCT), fibrin formation and plasmin induced fibrinolysis were monitored in vitro. The ultrastructure of the SMIPP-C-modulated fibrin was analysed by Scanning Electron Microscopy (SEM). Immuno-histological analyses were performed ex vivo, to localise the SMIPP-C proteins within scabies infected skin biopsies. SMIPP-Cs displayed pro-coagulant properties. They bound calcium ions, reduced the thrombin clotting time, enhanced the fibrin formation rate and delayed plasmin-induced fibrinolysis. The SMIPP-Cs associated with fibrin clots during fibrinogen polymerisation and did not bind to preformed fibrin. Scanning electron microscopy revealed that the fibrin clots formed in the presence of SMIPP-Cs were aberrant and denser than normal fibrin clots. SMIPP-Cs were detected in microthrombi which are commonly seen in scabietic skin. CONCLUSIONS/SIGNIFICANCE: The SMIPP-Cs are the first scabies mite proteins found in sub-epidermal skin layers and their pro-coagulant properties promote superficial microthrombi formation in scabetic skin. Further research is needed to evaluate their potential as diagnostic or therapeutic target.

Application of a plasmin generation assay to define pharmacodynamic effects of tranexamic acid in women undergoing cesarean delivery.

Essentials Tranexamic acid (TXA) is an antifibrinolytic drug used to reduce bleeding. Assaying plasmin generation (PG) in plasma detects clinically relevant TXA levels in vitro and ex vivo. 3.1-16.2 µg/mL TXA half-maximally inhibits PG in plasma from women undergoing cesarean delivery. PG velocity shows the strongest dose-relationship at low TXA concentrations (≤10 µg/mL). ABSTRACT: Background Tranexamic acid (TXA) is used to reduce bleeding. TXA inhibits plasmin(ogen) binding to fibrin and reduces fibrinolysis. TXA antifibrinolytic activity is typically measured by clot lysis assays; however, effects on plasmin generation (PG) are unclear due to a lack of tools to measure PG in plasma. Aims Develop an assay to measure PG kinetics in human plasma. Determine effects of TXA on PG and compare with fibrinolysis measured by rotational thromboelastometry (ROTEM). Methods We characterized effects of plasminogen, tissue plasminogen activator, fibrinogen, and α2 -antiplasmin on PG in vitro. We also studied effects of TXA on PG in plasma from 30 pregnant women administered intravenous TXA (5, 10, or 15 mg/kg) during cesarean delivery. PG was measured by calibrated fluorescence. PG parameters were compared with TXA measured by mass spectrometry and ROTEM of whole blood. Results The PG assay is specific for plasmin and sensitive to tissue plasminogen activator, fibrin(ogen), and α2 -antiplasmin. Addition of TXA to plasma in vitro dose dependently prolonged the clot lysis time and delayed and reduced PG. For all doses of TXA administered intravenously, the PG assay detected delayed time-to-peak (≤3 hours) and reduced the velocity, peak, and endogenous plasmin potential (≤24 hours) in plasma samples obtained after infusion. The PG time-to-peak, velocity, and peak correlated significantly with TXA concentration and showed less variability than the ROTEM lysis index at 30 minutes or maximum lysis. Conclusions The PG assay detects pharmacologically relevant concentrations of TXA administered in vitro and in vivo, and demonstrates TXA-mediated inhibition of PG in women undergoing cesarean delivery.

Exogenous activation and inhibition of plasminogen/plasmin activity during in vitro maturation of bovine cumulus-oocyte complexes: A biological and spectroscopic approach.

This study deals with the effect of plasminogen/plasmin on the in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs). Exogenous plasminogen activator streptokinase (SK) added to the IVM medium revealed similar values of cumulus expansion and oocyte nuclear maturation compared to controls (standard IVM medium). However, a decrease in both determinations was observed in COCs matured with the supplementation of ɛ-aminocaproic acid (ɛ-ACA), a specific plasmin inhibitor. After in vitro fertilization, no differences were observed in either cleavage or blastocyst rates between SK and control groups; however, ε-ACA treatment caused a decrease in both developmental rates. Zona pellucida (ZP) digestion time decreased in the SK group while it increased in the ε-ACA group. Raman microspectroscopy revealed an increase in the intensity of the band corresponding to the glycerol group of sialic acid in the ZP of oocytes matured with SK, whereas ZP spectra of oocytes treated with ɛ-ACA presented similarities with immature oocytes. The results indicate that although treatment with SK did not alter oocyte developmental competence, it induced modifications in the ZP of oocytes that could modify the folding of glycoproteins. Plasmin inhibition impairs oocyte maturation and has an impact on embryo development, thus evidencing the importance of this protease during IVM.

Plasminogen/plasmin affects expression of glycolysis regulator TIGAR and induces autophagy in lung adenocarcinoma A549 cells.

Pericellular plasmin generation triggers apoptosis/anoikis in normal adherent cells. However, cancer cells are notoriously resistant to anoikis, enabling metastasis and new tumor growth beyond their original environment. Autophagy can be a major contributor to anoikis resistance in cancer. AIM: To investigate if protective autophagy can be induced in lung adenocarcinoma cells in response to plasminogen treatment. MATERIALS AND METHODS: Human lung adenocarcinoma A549 cells were incubated with Glu-plasminogen (0.1-1.0 µM) for 24 h. Pericellular plasmin activity was monitored spectrophotometrically by a cleavage of the specific chromogenic- substrate S-2251. Cell survival was assessed by 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)-test. Degradation of fibronectin, levels of autophagy markers (beclin-1 and light chain 3 (LC3)) and glycolysis regulator (TIGAR) were evaluated by western blot. Intracellular localization of LC-3 was visualized by immunocytochemistry. RESULTS: It was shown that plasminogen is converted into plasmin on the surface of adenocarcinoma cells in a dose-dependent manner. Plasmin disrupted cellular adhesive contacts resulting in cell detachment. A549 cells did not loss their viability after plasminogen treatment for 24 h, while 1.0 µM plasminogen was cytotoxic for non-transformed fibroblasts. Plasminogen 0.1, 0.5, and 1.0 µM induced 7.08-, 5.18-, and 3.78-fold elevation of TIGAR expression (p < 0.05), respectively. Enhanced TIGAR expression indicates switch on pentose phosphate pathway, protection against oxidative stress to prevent apoptosis, facilitation of DNA repair and the degradation of their own organelles (autophagy). Exposure of adenocarcinoma cells to plasminogen in concentrations of 0.1 and 0.5 µM caused 1.74- and 2.19-fold elevation of beclin-1 expression vs untreated cells (p < 0.05), respectively. Unlike K1-3 fragment, plasminogen treatment (0.1-0.5 µM) resulted in increased expression of LC3-I and stimulated rapid conversion of LC3-I to LC3-II. Up-regulation of beclin-1 levels and enhanced LC3-I/II conversion in plasminogen-treated A549 cells are the hallmarks of autophagy induction. According to immunocytochemistry data, increased LC3 puncta and autophagosome formation after exposure to plasminogen could reflect autophagy activation. CONCLUSIONS: Therefore, we showed stimulation of prosurvival signals and induction of autophagy in plasminogen-treated adenocarcinoma cells rendering them resistant to apoptosis/anoikis. Based on the obtained data, autophagy has a great potential for novel targets that affect cancer cell death, in addition to the current cytotoxic agents.

Vitreomacular traction quantitative cutoffs for the assessment of resolution after ocriplasmin intravitreal treatment.

This study aimed to assess optical coherence tomography (OCT) parameters associated with vitreomacular traction (VMT) resolution after ocriplasmin intravitreal injection and also associated with the development of vitreomacular complications. Study designed was a retrospective case series. Structural OCT images were acquired at baseline and over the follow-up after treatment. We developed a mathematical model to provide quantitative parameters associated with VMT resolution. Moreover, we adopted the same model to assess the quantitative parameters associated with development of further vitreomacular complications or with the worsening of the coexisting condition. Main outcome measures were BCVA, central macular thickness (CMT), VMT reflectivity, VMT size, VMT resolution, epiretinal membrane (ERM), macular holes. 73 eyes of 73 VMT patients (mean age 73 ± 9 years) were recruited. The mean follow-up duration was 2.6 ± 1.1 years. Mean baseline BCVA was 0.38 ± 0.18 LogMAR, improving to 0.26 ± 0.20 at the end of the follow-up (p < 0.01). Baseline CMT was 431 ± 118 µm, improving to 393 ± 122 µm at the end of the follow-up (p < 0.01). 38/73 eyes (52%) showed only VMT, whereas 35/73 eyes (48%) also showed coexisting alterations at baseline. VMT resolved in 40/73 eyes (55% of cases). Our model disclosed VMT reflectivity as the most involved parameter in VMT resolution. VMT size showed less influence on the success of ocriplasmin treatment. ERM was negatively associated with VMT resolution. Moreover, VMT reflectivity values and ERM represented the most important parameters for the onset of vitreomacular complications.

Plasmin inhibition by bacterial serpin: Implications in gum disease.

Tannerella forsythia is a periodontopathogen that expresses miropin, a protease inhibitor in the serpin superfamily. In this study, we show that miropin is also a specific and efficient inhibitor of plasmin; thus, it represents the first proteinaceous plasmin inhibitor of prokaryotic origin described to date. Miropin inhibits plasmin through the formation of a stable covalent complex triggered by cleavage of the Lys368-Thr369 (P2-P1) reactive site bond with a stoichiometry of inhibition of 3.8 and an association rate constant (kass) of 3.3 × 105 M-1s-1. The inhibition of the fibrinolytic activity of plasmin was nearly as effective as that exerted by α2-antiplasmin. Miropin also acted in vivo by reducing blood loss in a mice tail bleeding assay. Importantly, intact T. forsythia cells or outer membrane vesicles, both of which carry surface-associated miropin, strongly inhibited plasmin. In intact bacterial cells, the antiplasmin activity of miropin protects envelope proteins from plasmin-mediated degradation. In summary, in the environment of periodontal pockets, which are bathed in gingival crevicular fluid consisting of 70% of blood plasma, an abundance of T. forsythia in the bacterial biofilm can cause local inhibition of fibrinolysis, which could have possible deleterious effects on the tooth-supporting structures of the periodontium.

Preferential Reactivity of Dipeptidyl Peptidase-IV Inhibitor-Associated Bullous Pemphigoid Autoantibodies to the Processed Extracellular Domains of BP180.

Bullous pemphigoid (BP) is a common autoimmune blistering disease in which autoantibodies target the hemidesmosomal components BP180 and/or BP230 in basal keratinocytes. In BP, 80 to 90% of autoantibodies target the juxtamembranous extracellular non-collagenous 16th A (NC16A) domain of BP180. Recently, the administration of dipeptidyl peptidase-IV inhibitors (DPP4i), which are widely used as antihyperglycemic drugs, has been recognized to be a causative factor for BP. DPP4i-associated BP (DPP4i-BP) autoantibodies tend to target epitopes on non-NC16A regions of BP180, and the pathomechanism for the development of the unique autoantibodies remains unknown. To address the characteristics of DPP4i-BP autoantibodies in detail, we performed epitope analysis of 18 DPP4i-BP autoantibodies targeting the non-NC16A domains of BP180 using various domain-specific as well as plasmin-digested polypeptides derived from recombinant BP180. Firstly, Western blotting showed that only one DPP4i-BP serum reacted with the epitopes on the intracellular domain of BP180, and no sera reacted with the C-terminal domain of the molecule. In addition, only 2 DPP4i-BP sera reacted with BP230 as determined by enzyme-linked immunosorbent assay. Thus, DPP4i-BP autoantibodies were found to mainly target the non-NC16A mid-portion of the extracellular domain of BP. Interestingly, Western blotting using plasmin-digested BP180 as a substrate revealed that all of the DPP4i-BP sera reacted more intensively with the 97-kDa processed extracellular domain of BP180, which is known as the LABD97 autoantigen, than full-length BP180 did. All of the DPP4i-BP autoantibodies targeting the LABD97 autoantigen were IgG1, and IgG4 was observed to react with the molecule in only 7 cases (38.9%). In summary, the present study suggests that IgG1-class autoantibodies targeting epitopes on the processed extracellular domain of BP180, i.e., LABD97, are the major autoantibodies in DPP4i-BP.

A direct oral anticoagulant edoxaban accelerated fibrinolysis via enhancement of plasmin generation in human plasma: dependent on thrombin-activatable fibrinolysis inhibitor.

A direct oral anticoagulant, edoxaban, is as effective as vitamin K antagonists for the treatment of venous thromboembolism (VTE). However, the mechanism underlying the treatment effect on VTE remains to be determined. The aims of this study were to evaluate the effect of edoxaban on tissue plasminogen activator (t-PA)-induced clot lysis in human plasma and to determine the roles of plasmin and thrombin-activatable fibrinolysis inhibitor (TAFI) in the profibrinolytic effect by edoxaban. Pooled human normal plasma or TAFI-deficient plasma (containing 180 ng/mL t-PA and 0.1 nM thrombomodulin) was mixed with edoxaban or an activated TAFI inhibitor, potato tuber carboxypeptidase inhibitor (PCI). Clot was induced by adding tissue factor and phospholipids. Clot lysis time and plasma plasmin-α2 antiplasmin complex (PAP) concentration were determined. Clot structure was imaged with a scanning electron microscope. In normal plasma, edoxaban at clinically relevant concentrations (75, 150, and 300 ng/mL) and PCI significantly shortened clot lysis time. PCI increased PAP concentration and a correlation between PAP concentration and percent of clot lysis was observed. Edoxaban also dose-dependently elevated PAP concentration. In TAFI-deficient plasma, the effects of edoxaban and PCI on clot lysis and PAP concentration were markedly diminished as compared with normal plasma. Fibrin fibers were thinner in clots formed in the presence of edoxaban. In conclusion, edoxaban at clinically relevant concentrations accelerates t-PA-induced fibrinolysis via increasing plasmin generation in human plasma. The effects of edoxaban is mainly dependent on TAFI. The profibrinolytic effect of edoxaban might contribute to the efficacy for the treatment of VTE.