Multifunctional interaction of CihC/FbpC orthologs of relapsing fever spirochetes with host-derived proteins involved in adhesion, fibrinolysis, and complement evasion.

Introduction: Relapsing fever (RF) remains a neglected human disease that is caused by a number of diverse pathogenic Borrelia (B.) species. Characterized by high cell densities in human blood, relapsing fever spirochetes have developed plentiful strategies to avoid recognition by the host defense mechanisms. In this scenario, spirochetal lipoproteins exhibiting multifunctional binding properties in the interaction with host-derived molecules are known to play a key role in adhesion, fibrinolysis and complement activation. Methods: Binding of CihC/FbpC orthologs to different human proteins and conversion of protein-bound plasminogen to proteolytic active plasmin were examined by ELISA. To analyze the inhibitory capacity of CihC/FbpC orthologs on complement activation, a microtiter-based approach was performed. Finally, AlphaFold predictions were utilized to identified the complement-interacting residues. Results and discussion: Here, we elucidate the binding properties of CihC/FbpC-orthologs from distinct RF spirochetes including B. parkeri, B. hermsii, B. turicatae, and B. recurrentis to human fibronectin, plasminogen, and complement component C1r. All CihC/FbpC-orthologs displayed similar binding properties to fibronectin, plasminogen, and C1r, respectively. Functional studies revealed a dose dependent binding of plasminogen to all borrelial proteins and conversion to active plasmin. The proteolytic activity of plasmin was almost completely abrogated by tranexamic acid, indicating that lysine residues are involved in the interaction with this serine protease. In addition, a strong inactivation capacity toward the classical pathway could be demonstrated for the wild-type CihC/FbpC-orthologs as well as for the C-terminal CihC fragment of B. recurrentis. Pre-incubation of human serum with borrelial molecules except CihC/FbpC variants lacking the C-terminal region protected serum-susceptible Borrelia cells from complement-mediated lysis. Utilizing AlphaFold2 predictions and existing crystal structures, we mapped the putative key residues involved in C1r binding on the CihC/FbpC orthologs attempting to explain the relatively small differences in C1r binding affinity despite the substitutions of key residues. Collectively, our data advance the understanding of the multiple binding properties of structural and functional highly similar molecules of relapsing fever spirochetes proposed to be involved in pathogenesis and virulence.

Development of a chemiluminescence assay for tissue plasminogen activator inhibitor complex and its applicability to gastric cancer.

BACKGROUND: Venous thromboembolism (VTE), is a noteworthy complication in individuals with gastric cancer, but the current diagnosis and treatment methods lack accuracy. In this study, we developed a t-PAIC chemiluminescence kit and employed chemiluminescence to detect the tissue plasminogen activator inhibitor complex (t-PAIC), thrombin-antithrombin III complex (TAT), plasmin-α2-plasmin inhibitor complex (PIC) and thrombomodulin (TM), combined with D-dimer and fibrin degradation products (FDP), to investigate their diagnostic potential for venous thrombosis in gastric cancer patients. The study assessed variations in six indicators among gastric cancer patients at different stages. RESULTS: The t-PAIC reagent showed LOD is 1.2 ng/mL and a linear factor R greater than 0.99. The reagents demonstrated accurate results, with all accuracy deviations being within 5%. The intra-batch and inter-batch CVs for the t-PAIC reagent were both within 8%. The correlation coefficient R between this method and Sysmex was 0.979. Gastric cancer patients exhibited elevated levels of TAT, PIC, TM, D-D, FDP compared to the healthy population, while no significant difference was observed in t-PAIC. In the staging of gastric cancer, patients in III-IV stages exhibit higher levels of the six markers compared to those in I-II stages. The ROC curve indicates an enhancement in sensitivity and specificity of the combined diagnosis of four or six indicators. CONCLUSION: Our chemiluminescence assay performs comparably to Sysmex's method and at a reduced cost. The use of multiple markers, including t-PAIC, TM, TAT, PIC, D-D, and FDP, is superior to the use of single markers for diagnosing VTE in patients with malignant tumors. Gastric cancer patients should be screened for the six markers to facilitate proactive prophylaxis, determine the most appropriate treatment timing, ameliorate their prognosis, decrease the occurrence of venous thrombosis and mortality, and extend their survival.

Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13.

ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13, regulates the length of Von Willebrand factor (VWF) multimers and their platelet-binding activity. ADAMTS13 is constitutively secreted as an active protease and is not inhibited by circulating protease inhibitors. Therefore, the mechanisms that regulate ADAMTS13 protease activity are unknown. We performed an unbiased proteomics screen to identify ligands of ADAMTS13 by optimizing the application of BioID to plasma. Plasma BioID identified 5 plasma proteins significantly labeled by the ADAMTS13-birA* fusion, including VWF and plasminogen. Glu-plasminogen, Lys-plasminogen, mini-plasminogen, and apo(a) bound ADAMTS13 with high affinity, whereas micro-plasminogen did not. None of the plasminogen variants or apo(a) bound to a C-terminal truncation variant of ADAMTS13 (MDTCS). The binding of plasminogen to ADAMTS13 was attenuated by tranexamic acid or ε-aminocaproic acid, and tranexamic acid protected ADAMTS13 from plasmin degradation. These data demonstrate that plasminogen is an important ligand of ADAMTS13 in plasma by binding to the C-terminus of ADAMTS13. Plasmin proteolytically degrades ADAMTS13 in a lysine-dependent manner, which may contribute to its regulation. Adapting BioID to identify protein-interaction networks in plasma provides a powerful new tool to study protease regulation in the cardiovascular system.

Identification of plasminogen-binding sites in Streptococcus suis enolase that contribute to bacterial translocation across the blood-brain barrier.

Streptococcus suis is an emerging zoonotic pathogen that can cause invasive disease commonly associated with meningitis in pigs and humans. To cause meningitis, S. suis must cross the blood-brain barrier (BBB) comprising blood vessels that vascularize the central nervous system (CNS). The BBB is highly selective due to interactions with other cell types in the brain and the composition of the extracellular matrix (ECM). Purified streptococcal surface enolase, an essential enzyme participating in glycolysis, can bind human plasminogen (Plg) and plasmin (Pln). Plg has been proposed to increase bacterial traversal across the BBB via conversion to Pln, a protease which cleaves host proteins in the ECM and monocyte chemoattractant protein 1 (MCP1) to disrupt tight junctions. The essentiality of enolase has made it challenging to unequivocally demonstrate its role in binding Plg/Pln on the bacterial surface and confirm its predicted role in facilitating translocation of the BBB. Here, we report on the CRISPR/Cas9 engineering of S. suis enolase mutants eno261, eno252/253/255, eno252/261, and eno434/435 possessing amino acid substitutions at in silico predicted binding sites for Plg. As expected, amino acid substitutions in the predicted Plg binding sites reduced Plg and Pln binding to S. suis but did not affect bacterial growth in vitro compared to the wild-type strain. The binding of Plg to wild-type S. suis enhanced translocation across the human cerebral microvascular endothelial cell line hCMEC/D3 but not for the eno mutant strains tested. To our knowledge, this is the first study where predicted Plg-binding sites of enolase have been mutated to show altered Plg and Pln binding to the surface of S. suis and attenuation of translocation across an endothelial cell monolayer in vitro.

Dysregulated fibrinolysis and plasmin activation promote the pathogenesis of osteoarthritis.

Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here, we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator (uPA), and the uPA receptor (uPAR), was dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression after a destabilization of the medial meniscus in a mouse model whereas genetic deficiency of plasmin activator inhibitor, or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans and induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, 3'-phosphoinositide-dependent kinase-1, AKT, and ERK signaling cascades and activated matrix metalloproteinases to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.

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.

C5a-C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms.

Recent studies have indicated the involvement of neutrophil-mediated inflammatory responses in the process leading to intracranial aneurysm (IA) rupture. Receptors mediating neutrophil recruitment could thus be therapeutic targets of unruptured IAs. In this study, complement C5a receptor 1 (C5AR1) was picked up as a candidate that may cause neutrophil-dependent inflammation in IA lesions from comprehensive gene expression profile data acquired from rat and human samples. The induction of C5AR1 in IA lesions was confirmed by immunohistochemistry; the up-regulations of C5AR1/C5ar1 stemmed from infiltrated neutrophils, which physiologically express C5AR1/C5ar1, and adventitial fibroblasts that induce C5AR1/C5ar1 in human/rat IA lesions. In in vitro experiments using NIH/3T3, a mouse fibroblast-like cell line, induction of C5ar1 was demonstrated by starvation or pharmacological inhibition of mTOR signaling by Torin1. Immunohistochemistry and an experiment in a cell-free system using recombinant C5 protein and recombinant Plasmin indicated that the ligand of C5AR1, C5a, could be produced through the enzymatic digestion by Plasmin in IA lesions. In conclusion, we have identified a potential contribution of the C5a-C5AR1 axis to neutrophil infiltration as well as inflammatory responses in inflammatory cells and fibroblasts of IA lesions. This cascade may become a therapeutic target to prevent the rupture of IAs.

Amiloride Reduces Urokinase/Plasminogen-Driven Intratubular Complement Activation in Glomerular Proteinuria.

SIGNIFICANCE STATEMENT: Proteinuria predicts accelerated decline in kidney function in CKD. The pathologic mechanisms are not well known, but aberrantly filtered proteins with enzymatic activity might be involved. The urokinase-type plasminogen activator (uPA)-plasminogen cascade activates complement and generates C3a and C5a in vitro / ex vivo in urine from healthy persons when exogenous, inactive, plasminogen, and complement factors are added. Amiloride inhibits uPA and attenuates complement activation in vitro and in vivo . In conditional podocin knockout (KO) mice with severe proteinuria, blocking of uPA with monoclonal antibodies significantly reduces the urine excretion of C3a and C5a and lowers tissue NLRP3-inflammasome protein without major changes in early fibrosis markers. This mechanism provides a link to proinflammatory signaling in proteinuria with possible long-term consequences for kidney function. BACKGROUND: Persistent proteinuria is associated with tubular interstitial inflammation and predicts progressive kidney injury. In proteinuria, plasminogen is aberrantly filtered and activated by urokinase-type plasminogen activator (uPA), which promotes kidney fibrosis. We hypothesized that plasmin activates filtered complement factors C3 and C5 directly in tubular fluid, generating anaphylatoxins, and that this is attenuated by amiloride, an off-target uPA inhibitor. METHODS: Purified C3, C5, plasminogen, urokinase, and urine from healthy humans were used for in vitro / ex vivo studies. Complement activation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and ELISA. Urine and plasma from patients with diabetic nephropathy treated with high-dose amiloride and from mice with proteinuria (podocin knockout [KO]) treated with amiloride or inhibitory anti-uPA antibodies were analyzed. RESULTS: The combination of uPA and plasminogen generated anaphylatoxins C3a and C5a from intact C3 and C5 and was inhibited by amiloride. Addition of exogenous plasminogen was sufficient for urine from healthy humans to activate complement. Conditional podocin KO in mice led to severe proteinuria and C3a and C5a urine excretion, which was attenuated reversibly by amiloride treatment for 4 days and reduced by >50% by inhibitory anti-uPA antibodies without altering proteinuria. NOD-, LRR- and pyrin domain-containing protein 3-inflammasome protein was reduced with no concomitant effect on fibrosis. In patients with diabetic nephropathy, amiloride reduced urinary excretion of C3dg and sC5b-9 significantly. CONCLUSIONS: In conditions with proteinuria, uPA-plasmin generates anaphylatoxins in tubular fluid and promotes downstream complement activation sensitive to amiloride. This mechanism links proteinuria to intratubular proinflammatory signaling. In perspective, amiloride could exert reno-protective effects beyond natriuresis and BP reduction. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Increased Activity of a Renal Salt Transporter (ENaC) in Diabetic Kidney Disease, NCT01918488 and Increased Activity of ENaC in Proteinuric Kidney Transplant Recipients, NCT03036748 .

Comparative analysis of hyperfibrinolysis with activated coagulation between amniotic fluid embolism and severe placental abruption.

Amniotic fluid embolism (AFE) and placental abruption (PA) are typical obstetric diseases associated with disseminated intravascular coagulation (DIC). AFE is more likely to be complicated with enhanced fibrinolysis than PA. AFE may have an additional mechanism activating fibrinolytic cascade. We aimed to compare the coagulation/fibrinolysis factors among AFE, PA, and peripartum controls. We assessed AFE cases registered in the Japanese AFE Registry, and PA cases complicated with DIC (severe PA) and peripartum controls recruited at our hospital. The following factors in plasma were compared: prothrombin fragment 1 + 2 (PF1 + 2), plasmin α2-plasmin inhibitor complex (PIC), tissue factor (TF), tissue plasminogen activator (tPA), annexin A2 (AnnA2), total thrombin activatable fibrinolysis inhibitor (TAFI) including its activated form (TAFIa), and plasminogen activator inhibitor-type 1 (PAI-1). PF1 + 2 and PIC were markedly increased in both AFE (n = 27) and severe PA (n = 12) compared to controls (n = 23), without significant difference between those disease groups; however, PIC in AFE showed a tendency to elevate relative to PF1 + 2, compared with severe PA. AFE had significantly increased tPA and decreased total TAFI levels compared with severe PA and controls, which might be associated with further plasmin production in AFE and underlie its specific fibrinolytic activation pathway.

Plasma from patients with vaccine-induced immune thrombotic thrombocytopenia displays increased fibrinolytic potential and enhances tissue-type plasminogen activator but not urokinase-mediated plasminogen activation.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenovirus vector-based COVID-19 vaccines. VITT is associated with markedly raised levels of D-dimer; yet, how VITT modulates the fibrinolytic system is unknown. OBJECTIVES: We aimed to compare changes in fibrinolytic activity in plasma from patients with VITT, patients diagnosed with venous thromboembolism (VTE) after vaccination but without VITT (VTE-no VITT), and healthy vaccinated controls. METHODS: Plasma levels of plasmin-antiplasmin (PAP) complexes, plasminogen, and alpha-2-antiplasmin (α2AP) from 10 patients with VITT, 10 patients with VTE-no VITT, and 14 healthy vaccinated controls were evaluated by enzyme-linked immunosorbent assay and/or Western blotting. Fibrinolytic capacity was evaluated by quantitating PAP levels at baseline and after ex vivo plasma stimulation with 50-nM tissue-type plasminogen activator (tPA) or urokinase for 5 minutes. RESULTS: Baseline PAP complex levels in control and VTE-no VITT individuals were similar but were ∼7-fold higher in plasma from patients with VITT (P < .0001). VITT samples also revealed consumption of α2AP and fibrinogenolysis consistent with a hyperfibrinolytic state. Of interest, VITT plasma produced significantly higher PAP levels after ex vivo treatment with tPA, but not urokinase, compared to the other groups, indicative of increased fibrinolytic potential. This was not due to D-dimer as addition of D-dimer to VTE-no VITT plasma failed to potentiate tPA-induced PAP levels. CONCLUSION: A marked hyperfibrinolytic state occurs in patients with VITT, evidenced by marked elevations in PAP, α2AP consumption, and fibrinogenolysis. An unidentified plasma cofactor that selectively potentiates tPA-mediated plasminogen activation also appears to exist in the plasma of patients with VITT.

Identification of potent anti-fibrinolytic compounds against plasminogen and tissue-type plasminogen activator employing in silico approaches.

The zymogen protease Plasminogen (Plg) and its active form plasmin (Plm) carry out important functions in the blood clot disintegration (breakdown of fibrin fibers) process. Inhibition of plasmin effectively reduces fibrinolysis to circumvent heavy bleeding. Currently, available Plm inhibitor tranexamic acid (TXA) used for treating severe hemorrhages is associated with an increased incidence of seizures which in turn were traced to gamma-aminobutyric acid antagonistic activity (GABAa) in addition to having multiple side effects. Fibrinolysis can be suppressed by targeting the three important protein domains: the kringle-2 domain of tissue plasminogen activator, the kringle-1 domain of plasminogen, and the serine protease domain of plasminogen. In the present study, one million molecules were screened from the ZINC database. These ligands were docked to their respective protein targets using Autodock Vina, Schrödinger Glide, and ParDOCK/BAPPL+. Thereafter, the drug-likeness properties of the ligands were evaluated using Discovery Studio 3.5. Subsequently, we subjected the protein-ligand complexes to molecular dynamics simulation of 200 ns in GROMACS. The identified ligands P76(ZINC09970930), C97(ZINC14888376), and U97(ZINC11839443) for each protein target are found to impart higher stability and greater compactness to the protein-ligand complexes. Principal component analysis (PCA) implicates, that the identified ligands occupy smaller phase space, form stable clusters, and provide greater rigidity to the protein-ligand complexes. Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis reveals that P76, C97, and U97 exhibit better binding free energy (ΔG) when compared to that of the standard ligands. Thus, our findings can be useful for the development of promising anti-fibrinolytic agents.Communicated by Ramaswamy H. Sarma.

ALDH1A3 promotes invasion and metastasis in triple-negative breast cancer by regulating the plasminogen activation pathway.

Aldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell marker that promotes metastasis. Triple-negative breast cancer (TNBC) progression has been linked to ALDH1A3-induced gene expression changes. To investigate the mechanism of ALDH1A3-mediated breast cancer metastasis, we assessed the effect of ALDH1A3 on the expression of proteases and the regulators of proteases that degrade the extracellular matrix, a process that is essential for invasion and metastasis. This revealed that ALDH1A3 regulates the plasminogen activation pathway; it increased the levels and activity of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). This resulted in a corresponding increase in the activity of serine protease plasmin, the enzymatic product of tPA and uPA. The ALDH1A3 product all-trans-retinoic acid similarly increased tPA and plasmin activity. The increased invasion of TNBC cells by ALDH1A3 was plasminogen-dependent. In patient tumours, ALDH1A3 and tPA are co-expressed and their combined expression correlated with the TNBC subtype, high tumour grade and recurrent metastatic disease. Knockdown of tPA in TNBC cells inhibited plasmin generation and lymph node metastasis. These results identify the ALDH1A3-tPA-plasmin axis as a key contributor to breast cancer progression.

The probable role of tissue plasminogen activator/neuroserpin axis in Alzheimer's disease: a new perspective.

Alzheimer's disease (AD) is the most common type of dementia associated with amyloid beta (Aß) deposition. Dysfunction of the neuronal clearance pathway promotes the accumulation of Aß. The plasminogen-activating system (PAS) is controlled by various enzymes like tissue plasminogen activators (tPA). Neuronal tPA enhances the conversion of plasminogen to plasmin, which cleaves Aß; this function is controlled by many inhibitors of PAS, including a plasminogen-activating inhibitor (PAI-1) and neuroserpin. Therefore, the objective of the present narrative review was to explore the potential role of tPA/neuroserpin in the pathogenesis of AD. PAI-1 activity is increased in AD, which is involved in accumulating Aß. Progressive increase of Aß level during AD neuropathology is correlated with the over-production of PAI-1 with subsequent reduction of plasmin and tPA activities. Reducing plasmin and tPA activities promote Aß by reducing Aß clearance. Neuroserpin plays a critical role in the pathogenesis of AD as it regulates the expression and accumulation of Aß. Higher expression of neuroserpin inhibits the neuroprotective tPA and the generation of plasmin with subsequent reduction in the clearance of Aß. These observations raise conflicting evidence on whether neuroserpin is neuroprotective or involved in AD progression. Thus, neuroserpin over-expression with subsequent reduction of tPA may propagate AD neuropathology.

Identification and characterization of calreticulin as a novel plasminogen receptor.

Calreticulin (CRT) was originally identified as a key calcium-binding protein of the endoplasmic reticulum. Subsequently, CRT was shown to possess multiple intracellular functions, including roles in calcium homeostasis and protein folding. Recently, several extracellular functions have been identified for CRT, including roles in cancer cell invasion and phagocytosis of apoptotic and cancer cells by macrophages. In the current report, we uncover a novel function for extracellular CRT and report that CRT functions as a plasminogen-binding receptor that regulates the conversion of plasminogen to plasmin. We show that human recombinant or bovine tissue-derived CRT dramatically stimulated the conversion of plasminogen to plasmin by tissue plasminogen activator or urokinase-type plasminogen activator. Surface plasmon resonance analysis revealed that CRT-bound plasminogen (KD = 1.8 µM) with moderate affinity. Plasminogen binding and activation by CRT were inhibited by ε-aminocaproic acid, suggesting that an internal lysine residue of CRT interacts with plasminogen. We subsequently show that clinically relevant CRT variants (lacking four or eight lysines in carboxyl-terminal region) exhibited decreased plasminogen activation. Furthermore, CRT-deficient fibroblasts generated 90% less plasmin and CRT-depleted MDA MB 231 cells also demonstrated a significant reduction in plasmin generation. Moreover, treatment of fibroblasts with mitoxantrone dramatically stimulated plasmin generation by WT but not CRT-deficient fibroblasts. Our results suggest that CRT is an important cellular plasminogen regulatory protein. Given that CRT can empower cells with plasmin proteolytic activity, this discovery may provide new mechanistic insight into the established role of CRT in cancer.

Plasminogen activation and plasmin inhibition during in vitro fertilization in bovine: implications for fertilization parameters and early embryo development.

Components of the plasminogen/plasmin system, known to be present in the oocyte, play a key role in maturation and fertilization. The objective of this study was to examine the effect of plasminogen activation and plasmin inhibition by exogenous supplementation of the IVF medium with streptokinase (SK) or ɛ-aminocaproic acid (ε-ACA), respectively, on fertilization parameters and preimplantation embryo development. After in vitro maturation, bovine cumulus-oocyte complexes (COCs) were inseminated in the presence of SK or ε-ACA. The addition of SK to the IVF medium facilitated the adhesion of the spermatozoa to the zona pellucida without affecting the percentages of monospermy. Cleavage rates and blastocyst yield were similar between the SK and Control groups while they were lower with the ε-ACA treatment. Additionally, we found that the expression levels of embryo quality-related genes (SDHA and DNMT3A) could be modified in blastocysts by the addition of SK or ε-ACA during IVF. The results obtained indicate that supplementation of the IVF medium with SK did not greatly alter the embryonic developmental parameters related to embryo quality in blastocysts. Moreover, we noticed that ε-ACA treatment compromises the success of in vitro embryo development, thus highlighting the importance of the plasminogen/plasmin activity during the early stages of embryogenesis in bovine.

Proteoform-Resolved Profiling of Plasminogen Activation Reveals Novel Abundant Phosphorylation Site and Primary N-Terminal Cleavage Site.

Plasminogen (Plg), the zymogen of plasmin (Plm), is a glycoprotein involved in fibrinolysis and a wide variety of other physiological processes. Plg dysregulation has been implicated in a range of diseases. Classically, human Plg is categorized into two types, supposedly having different functional features, based on the presence (type I) or absence (type II) of a single N-linked glycan. Using high-resolution native mass spectrometry, we uncovered that the proteoform profiles of human Plg (and Plm) are substantially more extensive than this simple binary classification. In samples derived from human plasma, we identified up to 14 distinct proteoforms of Plg, including a novel highly stoichiometric phosphorylation site at Ser339. To elucidate the potential functional effects of these post-translational modifications, we performed proteoform-resolved kinetic analyses of the Plg-to-Plm conversion using several canonical activators. This conversion is thought to involve at least two independent cleavage events: one to remove the N-terminal peptide and another to release the active catalytic site. Our analyses reveal that these processes are not independent but are instead tightly regulated and occur in a step-wise manner. Notably, N-terminal cleavage at the canonical site (Lys77) does not occur directly from intact Plg. Instead, an activation intermediate corresponding to cleavage at Arg68 is initially produced, which only then is further processed to the canonical Lys77 product. Based on our results, we propose a refined categorization for human Plg proteoforms. In addition, we reveal that the proteoform profile of human Plg is more extensive than that of rat Plg, which lacks, for instance, the here-described phosphorylation at Ser339.

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.

The fibrinolysis renaissance.

Fibrinolysis is the system primarily responsible for removal of fibrin deposits and blood clots in the vasculature. The terminal enzyme in the pathway, plasmin, is formed from its circulating precursor, plasminogen. Fibrin is by far the most legendary substrate, but plasmin is notoriously prolific and is known to cleave many other proteins and participate in the activation of other proteolytic systems. Fibrinolysis is often overshadowed by the coagulation system and viewed as a simplistic poorer relation. However, the primordial plasminogen activators evolved alongside the complement system, approximately 70 million years before coagulation saw the light of day. It is highly likely that the plasminogen activation system evolved with its roots in primordial immunity. Almost all immune cells harbor at least one of a dozen plasminogen receptors that allow plasmin formation on the cell surface that in turn modulates immune cell behavior. Similarly, numerous pathogens express their own plasminogen activators or contain surface proteins that provide binding sites for host plasminogen. The fibrinolytic system has been harnessed for clinical medicine for many decades with the development of thrombolytic drugs and antifibrinolytic agents. Our refined understanding and appreciation of the fibrinolytic system and its alliance with infection and immunity and beyond are paving the way for new developments and interest in novel therapeutics and applications. One must ponder as to whether the nomenclature of the system hampered our understanding, by focusing on fibrin, rather than the complex myriad of interactions and substrates of the plasminogen activation system.

Fingerprinting of Proteases, Protease Inhibitors and Indigenous Peptides in Human Milk.

The presence of proteases and their resulting level of activity on human milk (HM) proteins may aid in the generation of indigenous peptides as part of a pre-digestion process, of which some have potential bioactivity for the infant. The present study investigated the relative abundance of indigenous peptides and their cleavage products in relation to the abundance of observed proteases and protease inhibitors. The proteomes and peptidomes in twelve HM samples, representing six donors at lactation months 1 and 3, were profiled. In the proteome, 39 proteases and 29 protease inhibitors were identified in 2/3 of the samples. Cathepsin D was found to be present in higher abundance in the proteome compared with plasmin, while peptides originating from plasmin cleavage were more abundant than peptides from cathepsin D cleavage. As both proteases are present as a system of pro- and active- forms, their activation indexes were calculated. Plasmin was more active in lactation month 3 than month 1, which correlated with the total relative abundance of the cleavage product ascribed to plasmin. By searching the identified indigenous peptides in the milk bioactive peptide database, 283 peptides were ascribed to 10 groups of bioactivities. Antimicrobial peptides were significantly more abundant in month 1 than month 3; this group comprised 103 peptides, originating from the ß-CN C-terminal region.

Dupuytren's Disease Is Mediated by Insufficient TGF-ß1 Release and Degradation.

Dupuytren's disease (DD) is a fibroproliferative disorder affecting the palmar fascia, causing functional restrictions of the hand and thereby limiting patients' daily lives. The disturbed and excessive myofibroblastogenesis, causing DD, is mainly induced by transforming growth factor (TGF)-ß1. But, the extent to which impaired TGF-ß1 release or TGF-ß signal degradation is involved in pathologically altered myofibroblastogenesis in DD has been barely examined. Therefore, the complex in which TGF-ß1 is secreted in the extracellular matrix to elicit its biological activity, and proteins such as plasmin, integrins, and matrix metalloproteinases (MMPs), which are involved in the TGF-ß1 activation, were herein analyzed in DD-fibroblasts (DD-FBs). Additionally, TGF-ß signal degradation via caveolin-1 was examined with 5-fluoruracil (5-FU) in detail. Gene expression analysis was performed via Western blot, PCR, and immunofluorescence analyses. As a surrogate parameter for disturbed myofibroblastogenesis, 𝛼-smooth-muscle-actin (𝛼-SMA) expression was evaluated. It was demonstrated that latency-associated peptide (LAP)-TGF-ß and latent TGF-ß-binding protein (LTBP)-1 involved in TGF-ß-complex building were significantly upregulated in DD. Plasmin a serinprotease responsible for the TGF-ß release was significantly downregulated. The application of exogenous plasmin was able to inhibit disturbed myofibroblastogenesis, as measured via 𝛼-SMA expression. Furthermore, a reduced TGF-ß1 degradation was also involved in the pathological phenotype of DD, because caveolin-1 expression was significantly downregulated, and if rescued, myofibroblastogenesis was also inhibited. Therefore, our study demonstrates that a deficient release and degradation of TGF-ß1 are important players in the pathological phenotype of DD and should be addressed in future research studies to improve DD therapy or other related fibrotic conditions.