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.

Concurrent Cobalamin C and Plasminogen Deficiencies in a Patient with Chronic Thrombotic Microangiopathy.

BACKGROUND: Although most patients with atypical hemolytic uremic syndrome (aHUS) have variants in genes participating in alternative complement pathways, rare variants in non-complement pathway-related genes, including DGKE, INF2, MMACHC, PLG, and THBD, have also been described. CASE PRESENTATION: We report an 18-year-old male patient with renal biopsy-proven chronic thrombotic microangiopathy that raised suspicion of aHUS. Whole-exome sequencing revealed a novel pathogenic homozygous MMACHC c.484G>T (p.Gly162Trp) variant. Subsequently, clinical and laboratory findings confirmed cobalamin C (Cbl C) deficiency. Also, homozygous missense c.1112C>T PLG (p.Thr371Ile) variant was detected (it had been reported as a variant of unknown significance). However, the low serum plasminogen (PLG) activity proved the pathogenicity of c.1112C>T. Hence, the patient was diagnosed with concurrent Cbl C and PLG deficiencies. Segregation analysis revealed that the mother and father had the same heterozygous PLG and MMACHC variants. PLG variants have generally been described in aHUS patients concomitant with complement gene variants in the literature; therefore, the association between aHUS and PLG variants is controversial. The possible contribution of PLG deficiency to thrombotic microangiopathy was also discussed in this case. CONCLUSION: Non-complement-mediated aHUS is an exceptional disorder. A limited number of genes are involved in this entity. To our knowledge, this is the first aHUS patient diagnosed with both Cbl C and PLG deficiencies in the literature.

A clinical evaluation of patients with known mutations (plasminogen and factor XII) with a focus on prophylactic treatment.

BACKGROUND: Hereditary angioedema with normal C1-inhibitor (HAE-nC1-INH) is a rare genetic disease. The symptoms can resemble other forms of hereditary angioedema (HAE), but the specific laboratory values are inconspicuous. The knowledge about treatment strategies in HAE-nC1-INH remains insufficient; most of the drugs are only licensed and approved for other types of HAE. METHODS: An analysis of all patients with HAE-nC1-INH was carried out in a certified angioedema treatment center in southern Germany. Only patients with a confirmed HAE-nC1-INH mutation were included. The impact of disease was monitored with validated questionnaires. RESULTS: Eighteen patients were included: two families with a factor XII mutation and seven families with a plasminogen mutation. All individuals received icatibant for on-demand therapy-efficient treatment response was reported. Three patients were severely affected, and prophylaxis was initiated with lanadelumab. According to the questionnaires, the clinical course and symptoms improved significantly under this prophylactic regime. CONCLUSION: This is one of the first descriptions of the clinical outcomes as a response to prophylactic treatment with lanadelumab in HAE-nC1-INH patients with a known mutation. The therapeutic management of HAE-1 and HAE-2 should also be the basis of HAE-nC1-INH, including prophylaxis.

[Colonic pseudotumoral involvement as an expression of severe plasminogen deficiency]. / Compromiso pseudotumoral colónico como expresión de déficit grave de plasminogéno.

Plasminogen deficiency is a very rare multisystem entity that affects different tissues of the economy through the deposition of fibrin-rich pseudomembrane and determines a heterogeneous and diverse clinical presentation. It is transmitted in an autosomal recessive manner by mutations of the PLG gene on chromosome 6 and can be divided into hypoplasminogenemia or type I and dysplasminogenemia or type II, the latter not related to clinical pathology. Severe plasminogen deficiency has a prevalence of 1.6 individuals per million inhabitants and although it can be diagnosed in adulthood, the most severe symptoms are observed in infants and children. The most common form of onset is the so-called woody conjunctivitis, characterized by fibrin membranes that are deposited on the eyelids since childhood, causing exophytic lesions that affect vision. It can also affect other mucous membranes such as the gingival, respiratory, oropharyngeal, digestive and genital mucosa, among others. We present a rare case of severe plasminogen deficiency with conjunctivitis and woody cervicitis who was admitted with clinical acute abdominal symptoms, associated with a tumor mass due to pseudomembranous deposition in the ascending colon that simulated inflammatory bowel disease and resolved spontaneously.

La deficiencia de plasminógeno es una entidad multisistémica, muy infrecuente, que afecta diferentes tejidos de la economía mediante el depósito de pseudo membranas ricas en fibrina y que determina una presentación clínica heterogénea y diversa. Se transmite en forma autosómica recesiva por mutaciones del gen PLG del cromosoma 6 y se puede dividir en hipoplasminogenemia o tipo I y displasminogenemia o tipo II, esta última no relacionada con patología clínica. El déficit grave de plasminógeno tiene una prevalencia de 1.6 individuos por millón de habitantes y si bien puede diagnosticarse en edad adulta, los síntomas más graves se observan en lactantes y niños. La forma de inicio más común es la denominada conjuntivitis leñosa, caracterizada por membranas de fibrina que se depositan en los parpados desde la infancia, provocando lesiones exofíticas que afectan la visión. También puede afectar otras mucosas como la gingival, respiratoria, orofaríngea, digestiva y genital entre otros. Presentamos un raro caso de deficiencia grave de plasminógeno con conjuntivitis y cervicitis leñosa que ingresó con un cuadro de abdomen agudo clínico, asociado a una masa tumoral por depósito de pseudomembranas en el colon ascendente que simuló una enfermedad inflamatoria intestinal y que se resolvió espontáneamente.

Ligneous periodontitis exacerbated by Behçet's disease in a patient with plasminogen deficiency and a stop-gained variant PLG c.1468C > T: a case report.

BACKGROUND: Plasminogen serves as the precursor to plasmin, an essential element in the fibrinolytic process, and is synthesized primarily in the liver. Plasminogen activation occurs through the action of plasminogen activator, converting it into plasmin. This conversion greatly enhances the fibrinolytic system within tissues and blood vessels, facilitating the dissolution of fibrin clots. Consequently, congenital deficiency of plasminogen results in impaired fibrin degradation. Patients with plasminogen deficiency typically exhibit fibrin deposits in various mucosal sites throughout the body, including the oral cavity, eyes, vagina, and digestive organs. Behcet's disease is a chronic recurrent systemic inflammatory disease with four main symptoms: aphthous ulcers of the oral mucosa, vulvar ulcers, skin symptoms, and eye symptoms, and has been reported worldwide. This disease is highly prevalent around the Silk Road from the Mediterranean to East Asia. We report a case of periodontitis in a patient with these two rare diseases that worsened quickly, leading to alveolar bone destruction. Genetic testing revealed a novel variant characterized by a stop-gain mutation, which may be a previously unidentified etiologic gene associated with decreased plasminogen activity. CASE PRESENTATION: This case report depicts a patient diagnosed with ligneous gingivitis during childhood, originating from plasminogen deficiency and progressing to periodontitis. Genetic testing revealed a suspected association with the PLG c.1468C > T (p.Arg490*) stop-gain mutation. The patient's periodontal condition remained stable with brief intervals of supportive periodontal therapy. However, the emergence of Behçet's disease induced acute systemic inflammation, necessitating hospitalization and treatment with steroids. During hospitalization, the dental approach focused on maintaining oral hygiene and alleviating contact-related pain. The patient's overall health improved with inpatient care and the periodontal tissues deteriorated. CONCLUSIONS: Collaborative efforts between medical and dental professionals are paramount in comprehensively evaluating and treating patients with intricate complications from rare diseases. Furthermore, the PLG c.1468C > T (p.Arg490*) stop-gain mutation could contribute to the association between plasminogen deficiency and related conditions.

Diagnosis of Immunoglobulin G4-related disease in a child with ligneous conjunctivitis: a novel mutation in plasminogen gene and plasminogen activator inhibitor-1 polymorphism.

OBJECTIVES: Ligneous conjunctivitis (LC) is a chronic conjunctivitis characterized by recurrent, firm, fibrin-rich, woody pseudomembranes on the palpebral conjunctiva. It is an ultrarare autosomal recessive disease associated with congenital plasminogen (PLG) deficiency due to mutations in the PLG gene (6q26). Immunoglobulin G4-related disease (IgG4-RD) is an idiopathic, systemic fibroinflammatory disease characterized by elevated serum IgG4 concentration and tissue infiltration of IgG4-positive plasma cells leading to organ enlargement, fibrosis and damage. CASE REPORT: A 7-year-old girl with LC was hospitalized for recurrent pancreatitis and diagnosed as IgG4-RD. PLG activity level was 15% (normal range 55-145%). Co-segregation analysis indicated that the patient was homozygous for the c. NG_016200.1(NM_000301.5):c.1465 T>C mutation in PLG gene. c. NG_016200.1(NM_000301.5):c.1465 T>C PLG variant was found to be heterozygous by NGS analysis in both parents. She also had plasminogen activator inhibitor - 1 (PAI-1) NG_013213.1(NM_000602.5):c.-816A>G (4G/4G) homozygous polymorphism and a heterozygote NG_001333.2 (NM_002769.5):c.292_293insC mutation in the serine protease 1 (PRSS-1) gene. However, heterozygous PRSS-1NG_001333.2 (NM_002769.5):c.292_293insC variant was found in the mother of the patient. All detected variants are currently considered as a variant of uncertain (or unknown) significance (VUS) according to the American College of Medical Genetics and Genomics (ACMG) classification. Oral steroid, oral azathioprine, topical fresh frozen plasma, topical heparin, topical steroid and topical cyclosporine were given. After 3 years of follow-up, IgG4-RD is under partial remission and no pseudomembranes. CONCLUSION: She is the second case had both LC and IgG4-RD. We identified a NG_016200.1(NM_000301.5):c.1465 T>C novel homozygous mutation in PLG gene and a PAI-1 NG_016200.1(NM_000301.5):c.1465 T>C (4G/4G) homozygous polymorphism, which has been reported as a risk factor for thrombotic events.

Plasmin-Mediated Fibrinolysis in Periodontitis Pathogenesis.

The hemostatic and inflammatory systems work hand in hand to maintain homeostasis at mucosal barrier sites. Among the factors of the hemostatic system, fibrin is well recognized for its role in mucosal homeostasis, wound healing, and inflammation. Here, we present a basic overview of the fibrinolytic system, discuss fibrin as an innate immune regulator, and provide recent work uncovering the role of fibrin-neutrophil activation as a regulator of mucosal/periodontal homeostasis. We reason that the role of fibrin in periodontitis becomes most evident in individuals with the Mendelian genetic defect, congenital plasminogen (PLG) deficiency, who are predisposed to severe periodontitis in childhood due to a defect in fibrinolysis. Consistent with plasminogen deficiency being a risk factor for periodontitis, recent genomics studies uncover genetic polymorphisms in PLG, encoding plasminogen, being significantly associated with periodontal disease, and suggesting PLG variants as candidate risk indicators for common forms of periodontitis.

Increased glutamate in type 2 diabetes in the Korean population is associated with increased plasminogen levels.

BACKGROUND: Glutamate is a major neurotransmitter, although it causes cytotoxicity and inflammation in nonneuronal organs. This study aimed to investigate the metabolic disorders in which glutamate, associated with type 2 diabetes onset, is induced in the liver. METHODS: An analysis of Korean community-based Ansan-Ansung cohort study data as well as functional research using in vitro and mouse models were performed. RESULTS: Groups with high plasma glutamate levels (T2, T3) had a significantly increased risk of diabetes incidence after 8 years, compared to the group with relatively low glutamate levels (T1). Analysis of the effect of glutamate on diabetes onset in vitro showed that glutamate induces insulin resistance by increasing glucose-related protein 78 (GRP78) and phosphoenolpyruvate carboxykinase (PEPCK) expression in SK-Hep-1 human liver cells. In addition, three different genes, FRMB4B, PLG, and PARD3, were significantly associated with glutamate and were identified via genome-wide association studies. Among glutamate-related genes, plasminogen (PLG) levels were most significantly increased in several environments in which insulin resistance was induced, and was also upregulated by glutamate. Glutamate-induced increase in PLG in liver cells was caused by metabotropic glutamate receptor 5 activation, and PLG levels were also upregulated after extracellular secretion. Moreover, glutamate increased the expression of plasminogen activator inhibitor-1 (PAI-1). Thus, extracellular secreted PLG cannot be converted to plasmin (fibrinolytic enzyme) by increased PAI-1. CONCLUSIONS: Increased glutamate is closely associated with the development of diabetes, and it may cause metabolic disorders by inhibiting the fibrinolytic system, which plays an important role in determining blood clots, a hallmark of diabetes.

Single-cell RNA seq identifies Plg-RKT-PLG as signals inducing phenotypic transformation of scar-associated macrophage in liver fibrosis.

Hepatic macrophages play a central role in liver fibrosis. Scar-associated macrophages (SAMs), a recently identified subgroup of macrophages, play an important role in this process. However, the mechanism by which SAMs transform during liver fibrosis is still unclear. In this study, we aimed to characterize SAMs and elucidate the underlying mechanism of SAM transformation. Bile duct ligation (BDL) and carbon tetrachloride (CCl4) were used to induce mouse liver fibrosis. Non-parenchymal cells were isolated from normal/fibrotic livers and were analyzed using single cell RNA sequencing (scRNA-seq) or mass cytometry (CyTOF). The glucan-encapsulated siRNA particles (siRNA-GeRPs) was employed to perform macrophage selective gene knockdown. The results of scRNA-seq and CyTOF revealed that SAMs, which derived from bone marrow-derived macrophages (BMMs), accumulated in mouse fibrotic livers. Further analysis showed that SAMs highly expressed genes related to fibrosis, indicating the pro-fibrotic functions of SAMs. Moreover, plasminogen receptor Plg-RKT was highly expressed by SAMs, suggesting the role of Plg-RKT and plasminogen (PLG) in SAM transformation. In vitro, PLG-treated BMMs transformed into SAMs and expressed SAM functional genes. Knockdown of Plg-RKT blocked the effects of PLG. In vivo, selective knockdown of Plg-RKT in intrahepatic macrophages of BDL- and CCl4-treated mice reduced the number of SAMs and alleviated BDL- and CCl4-induced liver fibrosis, suggesting that Plg-RKT-PLG played an important role in liver fibrosis by mediating SAM transformation. Our findings reveal that SAMs are crucial participants in liver fibrosis. Inhibition of SAM transformation by blocking Plg-RKT might be a potential therapeutic target for liver fibrosis.

Anti-fibrinolytic agent tranexamic acid suppresses the endotoxin-induced expression of Tnfα and Il1α genes in a plasmin-independent manner.

INTRODUCTION: Tranexamic acid (TXA) is widely used as an antifibrinolytic agent in hemorrhagic trauma patients. The beneficial effects of TXA exceed the suppression of blood loss and include the ability to decrease inflammation and edema. We found that TXA suppresses the release of mitochondrial DNA and enhances mitochondrial respiration. These results allude that TXA could operate through plasmin-independent mechanisms. To address this hypothesis, we compared the effects of TXA on lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines in plasminogen (Plg) null and Plg heterozygous mice. METHODS: Plg null and Plg heterozygous mice were injected with LPS and TXA or LPS only. Four hours later, mice were sacrificed and total RNA was prepared from livers and hearts. Real time quantitative polymerase chain reaction with specific primers was used to assess the effects of LPS and TXA on the expression of pro-inflammatory cytokines. RESULTS: LPS enhanced the expression of Tnfα in the livers and hearts of recipient mice. The co-injection of TXA significantly decreased the effect of LPS both in Plg null and heterozygous mice. A similar trend was observed with LPS-induced Il1α expression in hearts and livers. CONCLUSIONS: The effects of TXA on the endotoxin-stimulated expression of Tnfα and Il1α in mice do not depend on the inhibition of plasmin generation. These results indicate that TXA has other biologically important target(s) besides plasminogen/plasmin. Fully understanding the molecular mechanisms behind the extensive beneficial effects of TXA and future identification of its targets may lead to improvement in the use of TXA in trauma, cardiac, and orthopedic surgical patients.

Gene Mutations Linked to Hereditary Angioedema in Solitary Angioedema Patients With Normal C1 Inhibitor.

BACKGROUND: Chronic recurrent angioedema without wheals (CRA) with normal C1 inhibitor (C1-INH) that is unresponsive to antihistamines may involve patients with recurrent angioedema of unknown cause (ie, so-called non-histaminergic idiopathic angioedema) as well as patients with hereditary angioedema with normal C1-INH (HAEnCI) when HAEnCI occurs in only one family member. OBJECTIVE: To identify patients with one of type of HAEnCI in a group of patients with CRA with normal C1-INH that was unresponsive to antihistamines. METHODS: A total of 132 patients with CRA and normal C1-INH that was unresponsive to antihistamines underwent mutational and clinical analysis. The presence of hereditary angioedema-specific mutations in Factor XII, plasminogen, ANGPT1, KNG1, MYOF, and HS3ST6 genes was tested by Sanger sequencing. When an HAEnCI-causing mutation was identified, available asymptomatic relatives were genetically tested. RESULTS: In 116 of 132 solitary patients with CRA (87.9%), none of the six HAEnCI-linked mutations could be found. Ten patients (7.6%) had the Factor XII mutation c.983C>A (p.T328K) and six (4.5%) the plasminogen mutation c.988A>G (p.K330E). Other mutations linked to HAEnCI were not found in this patient series. In the 16 families with HAEnCI, 11 asymptomatic carriers of one of the HAEnCI-linked mutations were identified. CONCLUSIONS: A search for HAEnCI-linked mutations in patients with solitary CRA may lead to the detection of patients and families with HAEnCI. This is important because family members can be identified who are at risk for developing potentially life-threatening angioedema, although they were previously asymptomatic. Without genetic investigation, the risk for an HAEnCI would have remained undetected in these patients and asymptomatic relatives.

Posttranscriptional Regulation of the Plasminogen Activation System by Non-Coding RNA in Cancer.

Various species of non-coding RNAs (ncRNAs) may act as functional molecules regulating diverse biological processes. In cancer cell biology, ncRNAs include RNAs that regulate the expression of oncogenes and tumor suppressor genes through various mechanisms. The urokinase (uPA)-mediated plasminogen activation system (PAS) includes uPA, its inhibitors PAI-1 and PAI-2 and its specific cellular receptor uPAR; their increased expression represents a negative prognostic factor in several cancers. Here, we will briefly describe the main uPA-mediated PAS components and ncRNA species; then, we will review more recent evidence of the roles that ncRNAs may play in regulating the expression and functions of uPA-mediated PAS components in cancer.

Ligneous conjunctivitis mimicking preseptal cellulitis in a 3-month-old infant.

BACKGROUND: Ligneous conjunctivitis is a very rare form of pseudomembranous conjunctivitis with few published cases in literature. We aim to describe the ocular findings and treatment in an infant with ligneous conjunctivitis resembling preseptal cellulitis on presentation. MATERIALS AND METHODS: Case report of a 3-month-old girl who was referred to a tertiary centre for ophthalmic assessment due to progressive eyelid oedema with no response to initiated topical and systemic antibiotics. Ethical approval has been achieved from the local ethics committee of the Ghent University Hospital and informed consent has been obtained from the parents of the child. RESULTS: Examination under general anaesthesia showed multiple, wood-like fibrinous pseudomembranes, originating from the conjunctiva, consistent with ligneous conjunctivitis. After careful removal of the coagulated exudate covering the cornea, a central corneal epithelial defect was evident without stromal infiltration. Histopathologic examination confirmed the predominance of fibrin within the pseudomembranes. Plasminogen activity was below the normal range. Genetic analysis did not identify a pathogenic variant in the PLG gene. The corneal epithelium re-epithelialised during the following days and the conjunctival lesions gradually subsided over the ensuing weeks whilst continuing heparin-containing artificial tears. CONCLUSION: A high level of suspicion is warranted in atypical cases of preseptal cellulitis which show no response to antibiotic treatment. Particularly in young children, examination under general anesthesia is warranted to allow diagnosis of rare causes of secondary eyelid oedema. We report an infant with unilateral ligneous conjunctivitis who responded well to topical, commercially-available heparin-containing artificial tears treatment. This approach is an effective and easy first-line treatment option in this condition, particularly in milder phenotypes.

A Missense Mutation of the Plasminogen Gene in a Japanese Family with Hereditary Angioedema with Normal C1 Inhibitor: Third Family Survey in Asia.

Hereditary angioedema (HAE) is a life-threatening disease associated with recurrent episodes of subcutaneous and mucosal swelling, painful abdominal cramping, and asphyxiation. HAE has long been thought to be caused by genetic defects of C1 inhibitors (C1-INH). Recently, HAE with a normal C1 inhibitor expression (HAEnCI) was reported, and the missense mutation p.Lys330Glu (K330E) in exon 9 of the plasminogen (PLG) gene was shown to be responsible for a subset of HAEnCI. HAE with the K330E mutation in the PLG gene-PLG (HAE-PLG) has been reported in only two Japanese families in Asia. We herein report a third family with HAE-PLG in Japan.

Multiple Mutations on α, ß and γ Domains of Streptokinase Lead to the Generation of Highly Efficient Cysteine Analogues with Promising Features.

BACKGROUND: Streptokinase, one of the most widely used thrombolytic medicines, is a favorable protein for site-specific PEGylation as it lacks any cysteine residues in its amino acid sequence; however, any changes in the protein's structure should be carefully planned to avoid undesired changes in its function. OBJECTIVES: This study aimed to design and produce novel di/tri-cysteine variants of streptokinase from previously developed cysteine analogues, Arg45, Glu263, and Arg319, as candidates for multiple site-specific PEGylation. METHODS: Using bioinformatics tools and site-directed mutagenesis, we incorporated concurrent mutations at Arg45, Glu263, and Arg319 (carried out in our previous study) to create di/tri-cysteine variants of streptokinase proteins (SK45-319cys, SK263-319cys, and SK45-263-319cys) and evaluated their kinetic activity parameters by a colorimetric method, using H-D-Val-Leu-Lys-pNA.2HCl (S2251) as substrate. RESULTS: Based on the kinetic results, SK263-319cys with 44% enzyme efficiency increment compared to wild-type SK was the superior protein in terms of activity; as well, SK45-319cys and SK45-263-319cys showed 17 and 22% activity enhancement, respectively. Docking of the mutant streptokinase proteins with µ-plasmin demonstrated that changes in intermolecular interactions caused by amino acid substitution could be the reason for activity difference. CONCLUSION: The novel mutant proteins created in this study exhibit remarkable biological activity and may be uniquely suitable for simultaneous PEGylation on two/three domains. As well, PEGylated derivates of these variants might prove to be more proficient proteins, compared to the singlecysteine analogs of streptokinase; because of their more surface coverage and increased molecular weight.

Case-only design identifies interactions of genetic risk variants at SIGLEC5 and PLG with the lncRNA CTD-2353F22.1 implying the importance of periodontal wound healing for disease aetiology.

AIM: The basis of phenotypic variation of periodontitis is genetic variability. Disease relevant effects of individual risk alleles are considered to result from genetic interactions. We investigated gene × gene (G×G) interactions of suggestive periodontitis susceptibility alleles. MATERIALS AND METHODS: We used the case-only design and investigated single-nucleotide polymorphism (SNPs) that showed associations in our recent genome-wide association study (GWAS) and GWAS meta-analysis with p < 5 × 10-6 . CRISPR-dCas9 gene activation followed by RNA-sequencing and gene-set enrichment analyses elucidated differentially expressed genes and gene networks. With the databases of SNPInspector and Transfac professional, luciferase reporter gene assays and antibody electrophoretic mobility shift experiments, we analysed allele-specific effects on transcription factor binding. RESULTS: SNPs at the genes sialic acid binding Ig-like lectin 5 (SIGLEC5) and plasminogen (PLG) showed G×G interactions with rs1122900 at the long non-coding RNA (lncRNA) CTD-2353F22. Associated chromatin cis-activated CTD-2353F22.1 6.5-fold (p = .003), indicating CTD-2353F22.1 as target gene of this interaction. CTD-2353F22.1 regulated GADD45A (padj < 4.9 × 10-11 , log2 fold change (FC) = -0.55), THBS1, SERPINE1 and Tissue Factor F3 (padj < 5 × 10-7 , log2 FC ≥ -0.35) and the gene set "angiogenesis" (area under the curve = 0.71, padj  = 8.2 × 10-5 ). rs1122900 effect C-allele decreased reporter gene activity (5.5-fold, p = .0003) and PRDM14 binding (76%). CONCLUSIONS: CTD-2353F22.1 mediates interaction of SIGLEC5 and PLG, together with genes that function in periodontal wound healing.

Insufficiency of hepatocyte growth factor activator inhibitor-1 confers lymphatic invasion of tongue carcinoma cells.

Hepatocyte growth factor (HGF) activator inhibitor type-1 (HAI-1), encoded by the SPINT1 gene, is a transmembrane protease inhibitor that regulates membrane-anchored serine proteases, particularly matriptase. Here, we explored the role of HAI-1 in tongue squamous cell carcinoma (TSCC) cells. An immunohistochemical study of HAI-1 in surgically resected TSCC revealed the cell surface immunoreactivity of HAI-1 in the main portion of the tumor. The immunoreactivity decreased in the infiltrative front, and this decrease correlated with enhanced lymphatic invasion as judged by podoplanin immunostaining. In vitro homozygous deletion of SPINT1 (HAI-1KO) in TSCC cell lines (HSC3 and SAS) suppressed the cell growth rate but significantly enhanced invasion in vitro. The loss of HAI-1 resulted in enhanced pericellular activities of proteases, such as matriptase and urokinase-type plasminogen activator, which induced activation of HGF/MET signaling in the co-culture with pro-HGF-expressing fibroblasts and plasminogen-dependent plasmin generation, respectively. The enhanced plasminogen-dependent plasmin generation was abrogated partly by matriptase silencing. Culture supernatants of HAI-1KO cells had enhanced potency for converting the proform of vascular endothelial growth factor-C (VEGF-C), a lymphangiogenesis factor, into the mature form in a plasminogen-dependent manner. Furthermore, HGF significantly stimulated VEGF-C expression in TSCC cells. Orthotopic xenotransplantation into nude mouse tongue revealed enhanced lymphatic invasion of HAI-1KO TSCC cells compared to control cells. Our results suggest that HAI-1 insufficiency leads to dysregulated pericellular protease activity, which eventually orchestrates robust activation of protease-dependent growth factors, such as HGF and VEGF-C, in a tumor microenvironment to contribute to TSCC progression.

Severe dysplasminogenemia due to homozygous PLG Ala620Thr variant in a Korean woman without a history of venous thromboembolism: A case report and literature review.

RATIONALE: Plasminogen plays an important role in fibrinolysis and is encoded by the PLG gene. The missense variant PLG Ala620Thr is the major cause of dysplasminogenemia in East Asian countries, including Korea. Although dysplasminogenemia was first reported in a Japanese patient with recurrent venous thromboembolism (VTE), subsequent studies have not demonstrated any clear association between the PLG Ala620Thr variant and the risk of VTE. To the best of our knowledge, this is the first report of a homozygous PLG Ala620Thr variant case from Korea. PATIENT CONCERNS: Here, we report a Korean family with PLG Ala620Thr mutation. The proband was a 34-year-old man who presented with multiple thrombotic arterial embolism and cardiac myxoma. INTERVENTIONS: Laboratory workup, including coagulation profile and PLG gene sequencing, was carried out for the affected family. DIAGNOSIS AND OUTCOME: The proband carried a heterozygous PLG Ala620Thr variant with decreased plasminogen activity of 65%. His 53-year-old mother, who had no reported history of VTE, was homozygous for the PLG Ala620Thr variant with decreased plasminogen activity of just 25%. Decreased plasminogen activity indicates dysplasminogenemia. LESSONS: We believe that this clinically silent homozygous case supports the previous findings that isolated PLG Ala620Thr variant does not confer a significant risk of VTE.

A mechanism for hereditary angioedema caused by a lysine 311-to-glutamic acid substitution in plasminogen.

Patients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates.