Plasmin Activity Research Articles

Aprotinin – Conjugated biocompatible porous nanocomposite for dentine remineralization and biofilm degradation

A nanocomposite (141 nm and +12.46 mV surface charge at pH 2.4) that could provide a single treatment for S. mutans-associated dental carries has been developed. The composite had six components, viz., aprotinin (Apr) that enzymatically inhibits plasmin activity of the bacterium, chitosan (Chi) that inhibits bacterial cell adhesion and biofilm formation, calcium phosphate (CaP) for remineralization of dentine, dexamethasone (DEX) for odontoblast differentiation, collagen (Cen) for crosslinking, and chlorogenic acid (CGA) for bacterial cell death. The structure of nanocomposite (HR SEM) consisted of interconnected pores and was amorphous post incorporation of chitosan. A sustained release of calcium and phosphate ions, lasting from 1 to 20 days at pH 4, an appropriate pH to combat demineralization, was observed, which is essential for remineralization. A 68.79% biofilm degradation was observed after 24 h of treatment, indicating good inhibition of the bacteria. While S. mutans grew well on denture resin specimen, the adhesion was reduced when the specimen was treated with the nanocomposite, indicating deterrence to biofilm formation. 5 Day hemolysis assay revealed red blood cell compatibility of up to 100 μg/25 μL, indicating that the nanocomposite was compatible with RBC. Against L132 cells, cytotoxicity was not observed up to 40 μg/mL for 72 h, a clear indication of non-cytotoxic character of the nanocomposite. CaP(NP)@Chi@CGA@DEX@Cen@Apr nanocomposite thus helps remineralization at cariogenic pH and biofilm degradation of S. mutans.

Nephritis-Associated Plasmin Receptor (NAPlr): An Essential Inducer of C3-Dominant Glomerular Injury and a Potential Key Diagnostic Biomarker of Infection-Related Glomerulonephritis (IRGN).

Nephritis-associated plasmin receptor (NAPlr) was originally isolated from the cytoplasmic fraction of group A Streptococci, and was found to be the same molecule as streptococcal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and plasmin receptor (Plr) on the basis of nucleotide and amino acid sequence homology. Its main functions include GAPDH activity, plasmin-binding capacity, and direct activation of the complement alternative pathway (A-P). Plasmin trapped by deposited NAPlr triggers the degradation of extracellular matrix proteins, such as glomerular basement membranes and mesangial matrix, and the accumulation of macrophages and neutrophils, leading to the induction of plasmin-related endocapillary glomerular inflammation. Deposited NAPlr at glomerular endocapillary site directly activates the complement A-P, and the endocapillary release of complement-related anaphylatoxins, C3a and C5a, amplify the in situ endocapillary glomerular inflammation. Subsequently, circulating and in situ-formed immune complexes participate in the glomerular injury resulting in NAPlr-mediated glomerulonephritis. The disease framework of infection-related glomerulonephritis (IRGN) has been further expanded. GAPDH of various bacteria other than Streptococci have been found to react with anti-NAPlr antibodies and to possess plasmin-binding activities, allowing glomerular NAPlr and plasmin activity to be utilized as key biomarkers of IRGN.

Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by lactoferricin.

In addition to vaccines, there is an urgent need for supplemental antiviral therapeutics to dampen the persistent COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The transmembrane protease serine 2 (TMPRSS2), that is responsible for proteolytic priming of the SARS-CoV-2 spike protein, appears as a rational therapeutic target. Accordingly, selective inhibitors of TMPRSS2 represent potential tools for prevention and treatment of COVID-19. Previously, we identified the human milk glycoprotein lactoferrin as a natural inhibitor of plasminogen conversion to plasmin, a serine protease homologous to TMPRSS2. Here, we tested whether lactoferrin and lactoferricin, a biologically active natural peptide produced by pepsin-mediated digestion of lactoferrin, together with synthetic peptides derived from lactoferrin, were able to block TMPRSS2 and SARS-CoV-2 infection. Particularly, we revealed that both lactoferricin and the N-terminal synthetic peptide pLF1 significantly inhibited: i) proteolytic activity of TMPRSS2 and plasmin, ii) proteolytic processing of the SARS-CoV-2 spike protein, and iii) SARS-CoV-2 infection of SARS-CoV-2-permissive cells. Thus, natural and synthetic peptides derived from lactoferrin represent feasible candidates for supporting prevention and treatment of COVID-19.

Premature delivery impacts the concentration of plasminogen activators and a plasminogen activator inhibitor and the plasmin activity in human milk.

Background and aimsPlasmin in human milk partially hydrolyzes milk proteins within the mammary gland and may enhance the hydrolysis of milk proteins within the infant’s stomach. This study examined the effects of extremely preterm (EP)-, very preterm (VP)-, and term-delivery on plasmin activity and the concentrations of plasminogen activators [urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA)], plasminogen activator inhibitor type 1 (PAI-1) and the complexes of PAI-1/uPA and PAI-1/tPA in human milk.Materials and methodsHuman milk samples were collected from mothers who delivered extremely preterm infants [24–27 weeks gestational age (GA), n = 20], very preterm infants (28–32 weeks GA, n = 12), and term infants (38–39 weeks GA, n = 8) during 2–72 days postnatally. Plasmin activity was determined using fluorometric substrate assay, whereas concentrations of uPA, tPA, PAI-1, the PAI-1/uPA complex and the PAI-1/tPA complex were quantified by ELISA.ResultsPlasmin activity, uPA and tPA were detected in all human milk samples, PAI-1 and the PAI-1/uPA complex were present in 42.5 and 32.5% of milk samples, respectively, and the PAI-1/tPA complex was not detected. Plasmin activity was correlated negatively with postnatal age and postmenstrual age (PMA) in the VP group and positively with postnatal age in the term group. uPA and tPA concentrations decreased with increasing postnatal age in both EP and VP groups but did not correlate in the term group. uPA concentration was correlated positively with GA in the VP group and tended to be elevated with increasing GA in the combined three groups. In contrast, tPA concentrations were correlated negatively with GA and PMA in the combined three groups (P < 0.008) and with PMA in the EP and VP groups. PAI-1 concentration tended to be correlated positively with postnatal age in the combined three groups. No correlation was detected with the PAI-1/uPA complex.ConclusionPremature delivery impacted the plasmin activity and the concentrations of uPA, tPA, and PAI-1 in human milk. Whether these changes in milk plasminogen activators and inhibitors have a role in balancing the proteolytic digestion of premature infants remains to be investigated.

Improving the microbial and nutritional quality of skim milk using microfiltration combined with thermal and nonthermal techniques

The effectiveness of microfiltration (MF), alone or combined with high temperature short time (HTST) pasteurisation, ultraviolet-C (UV-C), and ultrasonication (US) in improving the microbial and nutritional quality of skim milk was evaluated in comparison with commercial ultra-pasteurisation (UP). Compared with untreated skim milk, MF combined with UV-C and US retained more bioactive proteins, which made these techniques superior to MF combined with HTST. Almost no bioactive proteins survived after UP. MF alone reduced the bacterial load by 2.5 log; however, MF combined with HTST (MH), UV-C (MUV), or US (MUS) can reduce the bacterial load to an undetectable level and extend the microbial shelf-life of skim milk to 40 days. In addition, MUV and MUS did not induce significant protein oxidation. Even though the skim milk after MUV or MUS showed only minor microbial growth, the shelf-life was limited by proteolysis through plasmin activity.

Exogenous Tetranectin Alleviates Pre-formed-fibrils-induced Synucleinopathies in SH-SY5Y Cells by Activating the Plasminogen Activation System.

Parkinson's disease (PD) is a common neurodegenerative disease. Previously we identified tetranectin (TN) as a differentially expressed protein in the cerebrospinal fluid of PD patients, and we were surprised to find that TN knockout mice developed PD features. However, the specific role of TN in PD has not been clarified. In this study, we aimed to determine the effect of exogenous TN on cellular PD models and elucidate the underlying mechanisms. We found that exogenous TN could alleviate pre-formed-fibrils (PFFs)-induced synucleinopathies in SH-SY5Y cells and reduce the cell-to-cell transmission of α-synuclein (SYN). We also found that TN can promote the degradation of SYN by plasmin, which may account for its effect on cellular PD models. Moreover, administration of SYN/PFFs decreased the expression of TN and increased the expression of plasminogen activator inhibitor-1 (PAI-1) in SH-SY5Y cells, thereby reducing plasmin activity. Our findings depict a possible SYN-TN-plasmin interaction in which elevated levels of extracellular SYN monomers and aggregates in PD diminish the production of TN and PAI-1. Such changes lead to a reduced plasmin activity, which in turn reduces the degradation of extracellular SYN, thus forming a vicious cycle.

TRPM7 restrains plasmin activity and promotes transforming growth factor-β1 signaling in primary human lung fibroblasts

Sustained exposure of the lung to various environmental or occupational toxins may eventually lead to pulmonary fibrosis, a devastating disease with no cure. Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix (ECM) proteins such as fibronectin and collagens. The peptidase plasmin degrades the ECM, but protein levels of the plasmin activator inhibitor-1 (PAI-1) are increased in fibrotic lung tissue, thereby dampening plasmin activity. Transforming growth factor-β1 (TGF-β1)-induced activation of SMAD transcription factors promotes ECM deposition by enhancing collagen, fibronectin and PAI-1 levels in pulmonary fibroblasts. Hence, counteracting TGF-β1-induced signaling is a promising approach for the therapy of pulmonary fibrosis. Transient receptor potential cation channel subfamily M Member 7 (TRPM7) supports TGF-β1-promoted SMAD signaling in T-lymphocytes and the progression of fibrosis in kidney and heart. Thus, we investigated possible effects of TRPM7 on plasmin activity, ECM levels and TGF-β1 signaling in primary human pulmonary fibroblasts (pHPF). We found that two structurally unrelated TRPM7 blockers enhanced plasmin activity and reduced fibronectin or PAI-1 protein levels in pHPF under basal conditions. Further, TRPM7 blockade strongly inhibited fibronectin and collagen deposition induced by sustained TGF-β1 stimulation. In line with these data, inhibition of TRPM7 activity diminished TGF-β1-triggered phosphorylation of SMAD-2, SMAD-3/4-dependent reporter activation and PAI-1 mRNA levels. Overall, we uncover TRPM7 as a novel supporter of TGF-β1 signaling in pHPF and propose TRPM7 blockers as new candidates to control excessive ECM levels under pathophysiological conditions conducive to pulmonary fibrosis.

A Case of Disseminated Intravascular Coagulation Syndrome following a Twenty Week Missed Abortion

Disseminated Intravascular Coagulation Syndrome (DIC) is defined as the pathological disruption of the balanced process of hemostasis that is characterized by systemic activation of the blood coagulation cascade, which results in generation and deposition of fibrin . This leads to formation of microvascular thrombi in small blood vessels and activation of plasmin causing fibrinolysis. This process combined leads to multiple organ dysfunction due to depleted platelets and clotting factors that are needed for hemostasis, thus presenting as excessive bleeding . While DIC in pregnancy is rare, only accounting for approximately 1-5% of all DIC cases in the general population, it occurs in 0.3-3.5% of delivery hospitalizations. The process itself can stem from a multitude of sources during pregnancy including, but not limited to, placental abruption, postpartum hemorrhage, Preeclampsia, Eclampsia, HELLP syndrome, acute fatty liver, amniotic fluid embolism, and pregnancy-related sepsis. Each individual diagnosis has distinct features and treatment regimens to prevent progression to DisseminatedIntravascular Coagulation Syndrome. DIC is a major contributor to maternal mortality with approximately 20-25%of maternal deaths being related to hemorrhage, amniotic fluid embolism, or hypertensive disorders, all of which can be related to coagulopathies. Distinguishing features of hemodynamically stable and unstable patients guide the managementand determine how quickly the team must react when actively managing Disseminated Intravascular Coagulation Syndromein the pregnant population. The objective of this case study is to review a unique patient presentation that is consistent witha benign diagnosis that quickly progressed to a potentially life-threatening condition. By reviewing this case study, I intend to discuss the initial disease presentation, diagnosis, and treatment options of DIC in pregnancy.

A Case Of Disseminated Intravascular Coagulation Syndrome following a Twenty Week Missed Abortion

Disseminated Intravascular Coagulation Syndrome (DIC) is defined as the pathological disruption of the balanced process of hemostasis that is characterized by systemic activation of the blood coagulation cascade, which results in generation and deposition of fibrin. This leads to formation of microvascular thrombi in small blood vessels and activation of plasmin causing fibrinolysis. This process combined leads to multiple organ dysfunction due to depleted platelets and clotting factors that are needed for hemostasis, thus presenting as excessive bleeding. While DIC in pregnancy is rare, only accounting for approximately 1-5% of all DIC cases in the general population, it occurs in 0.3-3.5% of delivery hospitalizations. The process itself can stem from a multitude of sources during pregnancy including, but not limited to, placental abruption, postpartum hemorrhage, Preeclampsia, Eclampsia, HELLP syndrome, acute fatty liver, amniotic fluid embolism, and pregnancy-related sepsis. Each individual diagnosis has distinct features and treatment regimens to prevent progression to Disseminated Intravascular Coagulation Syndrome. DIC is a major contributor to maternal mortality with approximately 20-25% of maternal deaths being related to hemorrhage, amniotic fluid embolism, or hypertensive disorders, all of which can be related to coagulopathies. Distinguishing features of hemodynamically stable and unstable patients guide the management and determine how quickly the team must react when actively managing Disseminated Intravascular Coagulation Syndrome in the pregnant population. The objective of this case study is to review a unique patient presentation that is consistent with a benign diagnosis that quickly progressed to a potentially life-threatening condition. By reviewing this case study, I intend to discuss the initial disease presentation, diagnosis, and treatment options of DIC in pregnancy.

A Novel Enolase-1 Antibody Targets Multiple Interacting Players in the Tumor Microenvironment of Advanced Prostate Cancer.

Prostate cancer is one of the most common causes of cancer death in men worldwide, and the treatment options are limited for patients with advanced stages of prostate cancer. Upon oncogenic or inflammatory stimulation, tumor cells or immune cells express cell surface enolase-1 (ENO1) as plasminogen receptor to facilitate their migration via plasmin activation. Little is known about the roles of ENO1 in prostate cancer, especially in the tumor microenvironment (TME). We hypothesized that targeting surface ENO1 with specific mAbs would exert multifactorial therapeutic potentials against prostate cancer. In vivo, we showed ENO1 mAb (HuL227) reduced the growth of subcutaneous PC-3 xenograft, monocytes recruitment, and intratumoral angiogenesis. In a PC-3 intratibial implantation model, HuL227 reduced tumor growth and osteoclast activation in the bone. To investigate the antitumor mechanism of ENO1 mAb, we found that blocking surface ENO1 significantly reduced VEGF-A-induced tube formation of endothelial cells in vitro. Furthermore, HuL227 inhibited inflammation-enhanced osteoclasts activity and the secretion of invasion-related cytokines CCL2 and TGFβ from osteoclasts. In addition, inflammation-induced migration and chemotaxis of androgen-independent prostate cancer cells were dose-dependently inhibited by HuL227. In summary, we showed that, ENO1 mAb targets multiple TME niches involved in prostate cancer progression and bone metastasis via a plasmin-related mechanism, which may provide a novel immunotherapy approach for men with advanced prostate cancer.

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell–bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.

The Effect of Alpha s1 Genotype on Some Physiological and Chemical Milk Characteristics in Garganica Goat

In the present study individual milk samples were collected from 80 Garganica goats and the population was genotyped for CSN1S1. Seven different alleles were observed with the A allele found as predominant, followed by the F and B allele, and 14 different genotypes were observed. A proteomic approach was used to classify the individual milk on the basis of protein abundance: four levels of αs1-casein (CN) expression were identified as high (HAS-1), intermediate (IAS-1), low (LAS-1), and null (NAS-1). The present paper aimed to investigate the influence of the level of expression of αs1-casein fraction in milk on proteolytic activities, chemical composition, and renneting properties of individual Garganica goat milk. Results from the present study evidenced that the level of expression of αs1-casein fraction in Garganica goat milk affected chemical composition, curd firmness, and indigenous proteolytic enzymes. In particular, fat, protein, and casein content were higher in HAS-1 and IAS-1 compared to LAS-1 and NAS-1. The SCC decreased passing from the HAS-1 to the NAS-1 and the curd firmness displayed the highest value in HAS-1. The activity of plasmin (PL) was higher in HAS-1 and IAS-1 groups compared to the LAS-1 and NAS-1; whereas, PL plasminogen-derived (PG) activity was the highest in NAS-1 group. On the contrary, the activity of elastase was the highest in LAS-1, intermediate in the IAS-1 group, and the lowest in HAS-1 and NAS-1 groups. Present data demonstrated that the level of synthesis of αs1-CN reflects the efficiency of the mammary gland in the activation of the cellular mechanisms and influences the proteolytic activity in milk, especially the PL-PG system.

Relationships Between Plasminogen-Binding M-Protein and Surface Enolase for Human Plasminogen Acquisition and Activation in Streptococcus pyogenes.

The proteolytic activity of human plasmin (hPm) is utilized by various cells to provide a surface protease that increases the potential of cells to migrate and disseminate. Skin-trophic Pattern D strains of Streptococcus pyogenes (GAS), e.g., GAS isolate AP53, contain a surface M-protein (PAM) that directly and strongly interacts (Kd ~ 1 nM) with human host plasminogen (hPg), after which it is activated to hPm by a specific coinherited bacterial activator, streptokinase (SK2b), or by host activators. Another ubiquitous class of hPg binding proteins on GAS cells includes “moonlighting” proteins, such as the glycolytic enzyme, enolase (Sen). However, the importance of Sen in hPg acquisition, especially when PAM is present, has not been fully developed. Sen forms a complex with hPg on different surfaces, but not in solution. Isogenic AP53 cells with a targeted deletion of PAM do not bind hPg, but the surface expression of Sen is also greatly diminished upon deletion of the PAM gene, thus confounding this approach for defining the role of Sen. However, cells with point deletions in PAM that negate hPg binding, but fully express PAM and Sen, show that hPg binds weakly to Sen on GAS cells. Despite this, Sen does not stimulate hPg activation by SK2b, but does stimulate tissue-type plasminogen activator-catalyzed activation of hPg. These data demonstrate that PAM plays the dominant role as a functional hPg receptor in GAS cells that also contain surface enolase.

ADAPTOR PROTEIN Ruk/CIN85REGULATES REDOX BALANCE IN 4T1MOUSE BREAST CANCER CELLS EXPOSED TO PLASMIN(OGEN).

Cell surface plasmin is involved in tumor growth and metastatic dissemination by regulating cancer cells adhesion, migration and invasion. Plasmin-induced cell detachment is accompanied by an increased rate of reactive oxygen species (ROS) generation and cell death. However, cancer cells acquire the ability to develop adaptive mechanisms to resist ROS-mediated apoptosis. To establish the role of adaptor protein Ruk/CIN85in the control of viability and redox balance in breast adenocarcinoma cells exposed to plasmin(ogen). Mouse 4T1cells with the stable overexpression of adaptor protein Ruk/CIN85 (RukUp subline) and corresponding control (Mock subline) were treated with Glu-plasminogen (1-100nM). Plasminogen to plasmin conversion was monitored spectrophotometrically by cleavage of the specific chromogenic substrate S2251. Specific uPA inhibitor BC11was used to verify the uPA-mediated mechanism of plasminogen pericellular activation by 4T1cells. Cell survival rate was assessed by MTT-test and cell proliferation was estimated by colony formation assay. Enzymatic activities of catalase, glutathione peroxidase, superoxide dismutase, as well as hydrogen peroxide (H2O2) levels were measured by spectrophotomertric and fluorometric assays. The intracellular ROS generation was monitored by flow cytometry using H2DCF-DA fluorescent probe. Plasminogen was shown to be converted into an active proteinase plasmin on the surface of carcinoma cells in uPA-dependent manner. Plasmin(ogen) suppressed proliferation and affected survival of both studied 4T1sublines. However, RukUp cells displayed higher resistance to plasmin(ogen)-induced cytotoxicity than Mock cells. Plasmin(ogen) promoted significant elevation in ROS generation rate in cells with the basal level of Ruk/CIN85expression. In contrast, RukUp cells appear to be more effective in counteracting prooxidant changes due to the activation of some enzymes of the glutathione system, in particular glutathione peroxidase, and a concomitant decrease of H2O2accumulation. Adaptor protein Ruk/CIN85is involved in the regulation of redox homeostasis in cancer cells to maintain levels of ROS, thus promoting redox adaptation in cancer cells exposed to plasmin(ogen). Thus, Ruk/CIN85may represent one of the relevant targets in order to diminish the resistance of cancer cells to ROS-mediated apoptosis.

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.

Abstract 131: Plasminogen Oxidized Phospholipid Adducts Drive Macrophage Activation In Through TLR2

Oxidized phospholipids (OxPL) are potent proinflammatory mediators that can trigger toll-like receptor 2 (TLR2) signaling and inflammasome activation in macrophages. Plasminogen (PLG), a key enzyme in the fibrinolytic pathway, has been shown to harbor OxPL adducts. We hypothesized that PLG can drive inflammasome activity in macrophages through OxPL activation of TLR2 in a fibrin-independent mechanism. To assess PLG-induced macrophage activation, total RNA sequencing was used to quantify gene changes in response to human PLG treatments, and we observed robust induction of pro-inflammatory genes, including Il1b, Il6, Nos2, Il12 , and Nlrp3 in primary mouse macrophages. In addition, a 400-fold increase in IL-6 protein was observed in the media. To determine if this activation is specifically via TLR2, a HEK293-NF-kB reporter system was used to screen PLG-induced activation of human TLRs, and we found that human PLG significantly-induced NF-kB signaling and transactivation in reporter cells expressing TLR2, TL1/2, TLR2/6, but failed to activate TLRs1/3/4/5/6/9. PLG activation of TLR2 was found to be blocked by pre-treatment of C29, a small molecule inhibitor of TLR2. In addition, when Tlr2-/- primary macrophages were treated with human PLG a 30% reduction in Il1b, Il6 and Tnf gene (mRNA) expression was observed compared to wild-type controls. Furthermore, we found PLG-TLR2 activation to be fibrin-independent, as there was no reduction in PLG activation by the lysine analogs tranexamic acid and e-aminocaproic acid or the plasmin activity inhibitor - VPLCK in our HEK293-NF-kB reporter system. To characterize the effect of PLG on inflammation in vivo , wild-type and Plg-/- mice were injected with AAV8-PCSK9 and treated with an atherogenic diet for 16 weeks. Remarkably, the Plg-/- mice demonstrated reduced plaque mass and aortic lipid content compared to wild-type control mice, with a 40% reduction in en face Sudan IV staining and a 10% reduction in lesion area by Oil-Red-O staining of neutral lipids. Collectively these results support that PLG-OxPL likely activate inflammasomes in macrophages through TLR2 and suggest this is likely to be involved in atherogenesis in vivo .

Application of Multiharmonic QCM-D for Detection of Plasmin at Hydrophobic Surfaces Modified by β-Casein

Plasmin protease plays an important role in many processes in living systems, including milk. Monitoring plasmin activity is important for control of the nutritional quality of milk and other dairy products. We designed a biosensor to detect the proteolytic activity of plasmin, using multiharmonic quartz crystal microbalance with dissipation (QCM-D). The β-casein immobilized on the hydrophobic surface of 1-dodecanethiol on the AT-cut quartz crystal was used to monitor plasmin activity. We demonstrated detection of plasmin in a concentration range of 0.1–20 nM, with the limit of detection about 0.13 ± 0.01 nM. The analysis of viscoelastic properties of the β-casein layer showed rapid changes of shear elasticity modulus, μ, and coefficient of viscosity, η, at plasmin sub-nanomolar concentrations, followed by modest changes at nanomolar concentrations, indicating multilayer architecture β-casein. A comparative analysis of viscoelastic properties of β-casein layers following plasmin and trypsin cleavage showed that the higher effect of trypsin was due to larger potential cleavage sites of β-casein.

Screening of Surface-Exposed Lipoproteins of Leptospira Involved in Modulation of Host Innate Immune Response.

Leptospira, a zoonotic pathogen, is capable of causing both chronic and acute infection in a susceptible host. Surface-exposed lipoproteins play a major role in modulating the host immune response by activating the innate cells like macrophages and dendritic cells or evading complement attack and killing by phagocytes like neutrophils to favor pathogenesis and establish infection. In this study, we screened some surface-exposed lipoproteins known to be involved in pathogenesis to assess their possible role in immune modulation (innate immune activation or evasion). Surface proteins of the Len family (LenB, LenD, and LenE), Lsa30, Loa22, and Lipl21 were purified in recombinant form and then tested for their ability to activate macrophages of the different host (mouse, human, and bovine). These proteins were tested for binding with complement regulators like Factor H (FH), C4 Binding Protein (C4BP), and host protease Plasminogen (PLG) and also as nucleases to access their possible role in innate immune evasion. Our results show that, of various proteins tested, Loa22 induced strong innate activation and Lsa30 was least stimulatory, as evident from the production of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor–α) and expression of surface markers [CD80, CD86, and major histocompatibility complex class II (MHCII)]. All the tested proteins were able to bind to FH, C4BP, and PLG; however, Loa22 showed strong binding to PLG correlating to plasmin activity. All the proteins except Loa22 showed nuclease activity, albeit with a requirement of different metal ions. The nuclease activity of these proteins correlated to in vitro degradation of neutrophil extracellular trap (NET). In conclusion, our results indicate that these surface proteins are involved in innate immune modulation and may play a critical role in assisting the bacteria in invading and colonizing the host tissue for persistent infection.

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

BackgroundThe 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. MethodsWe 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. ResultsWe 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 β (TGF-β) signaling pathways. Moreover, shRNA-mediated uPA gene knockdown reduced plasmin, MMP14, and TGF-β 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-β 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. ConclusionAccordingly, 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.

Variable Response to Antifibrinolytics Correlates with Blood-loss and Transfusion in Posterior Spinal Fusion.

Posterior spinal fusion (PSF) activates the fibrinolytic protease plasmin, which is implicated in blood loss and transfusion. While antifibrinolytic drugs have improved blood loss and reduced transfusion, variable blood loss has been observed in similar PSF procedures treated with the same dose of antifibrinolytics. However, both the cause of this and the appropriate measures to determine antifibrinolytic efficacy during high-blood-loss spine surgery are unknown, making clinical trials to optimize antifibrinolytic dosing in PSF difficult. We hypothesized that patients undergoing PSF respond differently to antifibrinolytic dosing, resulting in variable blood loss, and that specific diagnostic markers of plasmin activity will accurately measure the efficacy of antifibrinolytics in PSF. A prospective study of 17 patients undergoing elective PSF with the same dosing regimen of TXA was conducted. Surgery-induced plasmin activity was exhaustively analyzed in perioperative blood samples and correlated to measures of inflammation, bleeding, and transfusion. While markers of in vivo plasmin activation (PAP and D-dimer) suggested significant breakthrough plasmin activation and fibrinolysis (P < 0.01), in vitro plasmin assays, including TEG, did not detect plasmin activation. In vivo measures of breakthrough plasmin activation correlated with blood loss (R2 = 0.400, 0.264; P < 0.01), transfusions (R2 = 0.388; P < 0.01), and complement activation (R2 = 0.346, P < 0.05). Despite all patients receiving a high dose of TXA, its efficacy among patients was variable, indicated by notable intra-operative plasmin activity. Markers of in vivo plasmin activation best correlated with clinical outcomes. These findings suggest that the efficacy of antifibrinolytic therapy to inhibit plasmin in PSF surgery should be determined by markers of in vivo plasmin activation in future studies. Level II-diagnostic.