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.

[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.

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.

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.

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.

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.

Plasminogen gene polymorphisms [c.924C>T and IVS 8+14 G>A] in periodontitis and familial Mediterranean fever: A case-control study.

BACKGROUND AND OBJECTIVE: The plasminogen (PLG) activation system plays an essential role in severe inflammation based diseases such as periodontitis, destructive membranous periodontal disease (ligneous periodontitis), familial Mediterranean fever (FMF), and amyloidosis. We have aimed to evaluate variations in PLG and the associations between PLG and MEFV genotypes in patients with FMF/ FMF-related secondary amyloidosis and periodontitis. MATERIAL AND METHODS: A total of 247 individuals who were either diagnosed with FMF or systemically healthy were recruited to this human observational study with a cross-sectional design. All individuals were also diagnosed with periodontitis or periodontally healthy. Blood samples were obtained from patients with FMF and systemically healthy controls. Clinical periodontal indicators were recorded. All polymorphisms located in exons 6 and 8 of PLG and mutations located on exons 2 and 10 of the MEFV gene were analyzed by DNA Sanger Sequencing. Genotypes and allele frequencies of PLG and MEFV were detected and tested by the Hardy-Weinberg equilibrium. Serum levels of amyloid A (SAA), high-sensitive C-reactive protein (hs-CRP), PLG, and salivary PLG levels were determined by using enzyme-linked immunosorbent assay (ELISA). RESULTS: Two polymorphisms were identified in PLG: G to A polymorphism on the 14th nucleotide of intron 8 and C to T polymorphism on the 924th nucleotide of the coding region (IVS 8+14 G>A and c.924C>T, respectively). In IVS 8+14 G>A polymorphisms, wild-type genotype: GG, heterozygote genotype: GA and homozygote genotype: AA. In c.924C>T polymorphism, wild-type genotype: CC, heterozygote genotype: CT and homozygote genotype: TT. The frequency of the heterozygous polymorphisms of PLG was significantly increased (17.6%) in FMF patients with periodontitis (p = .027). A large proportion of the test group that was heterozygous for MEFV-R202Q also had heterozygous PLG polymorphisms. Remarkable exacerbation in periodontal parameters was observed in patients with FMF and amyloidosis. SAA and hs-CRP levels were significantly correlated with salivary PLG levels in patients with periodontitis and heterozygous PLG. CONCLUSIONS: The current study describes IVS 8+14 G>A (rs2295368) and c.924C>T (rs1380916375) polymorphisms for the first time in the periodontal literature, which might play an important role in the pathogenesis of periodontitis, FMF, or amyloidosis. The elucidation of PLG polymorphisms is beneficial from a public health perspective by increasing the quality of life in these patients and reducing the mortality and morbidity associated with inflammatory diseases such as periodontal disease, FMF, and FMF-related amyloidosis.

Pseudogene PLGLA exerts anti-tumor effects on hepatocellular carcinoma through modulating miR-324-3p/GLYATL1 axis.

BACKGROUND: Pseudogenes are dysfunctional copies of protein-coding genes that showed critical regulatory roles during carcinogenesis. Plasminogen like A (PLGLA) is a transcribed unprocessed pseudogene and biasedly expressed in liver. But its function has not been studied in hepatocellular carcinoma (HCC). AIMS: We aimed to explore the role of PLGLA in HCC. METHODS: The expression of PLGLA and its association with pathological features in HCC patients was analyzed using The Cancer Genome Atlas (TCGA) datasets. Quantitative reverse transcription PCR (qRT-PCR) was used to validate PLGLA level in HCC tissue samples and cell lines. Gain-of-function experiments in vitro and in vivo were employed to assess the impact of PLGLA on HCC cell proliferation, migration and invasion. Luciferase reporter assay and RNA pull-down assay were conducted to confirm the interaction among PLGLA, miR-324-3p and GLYATL1. RESULTS: PLGLA expression was significantly downregulated in HCC tissues and cell lines. Furthermore, low PLGLA expression was positively associated with tumor progression and poor prognosis. PLGLA restoration markedly suppressed cell proliferation, migration and invasion. Mechanistically, PLGLA could competitively bind to miR-324-3p and acted as a competitive endogenous RNA (ceRNA) to enhance GLYATL1 expression. CONCLUSIONS: Our results established a novel tumor suppressive role of PLGLA in HCC pathogenesis and highlighted its potential as a therapeutic target for HCC treatment.

Analysis of cold activation of the contact system in hereditary angioedema with normal C1 inhibitor.

Hereditary angioedema (HAE) attacks are caused by excessive activation of the contact system. Understanding how the contact system is activated in HAE, especially in patients with normal C1 inhibitor (HAEnCI), is essential to effectively treat this disease. Contact system activation involves the cleavage of several proteins including Factor XII (FXII), high molecular weight kininogen (HK), prekallikrein, sgp120 (ITIH4) and C1 inhibitor (C1-INH) before the subsequent generation of bradykinin that mediates HAE. In this study, we evaluated the fragmentation and enzymatic activity of contact system proteins in HAEnCI plasma samples before and after contact system activation induced by incubation in the cold. Our results show that in contrast to normal plasma, cold activation induced contact system activation in the majority of the HAEnCI patient samples we tested, in which each contact system protein exhibited fragmentation, FXII and kallikrein enzymatic activity increased, and C1-INH functional activity decreased. HAEnCI samples with low FXII concentrations or functional activity were not affected by cold activation. One HAEnCI sample with a plasminogen gene mutation activated the fibrinolytic system, as shown by an increase in concentration of plasma D dimers. Our results suggest that cold activation seems to be initiated by the cleavage of prekallikrein, and that it needs FXII in order to occur. Reported to be susceptible to excessive contact system activation after incubation in the cold, we further applied this system of study to the evaluation of plasma from women undergoing estrogen treatment. Similar to plasma from HAEnCI patients, excessive contact system activation was demonstrated.

Screening for Plasminogen Mutations in Hereditary Angioedema Patients.

Hereditary angioedema (HAE) is a rare disease belonging to the group of bradykinin-mediated angioedemas, characterized by recurring edematous episodes involving the subcutaneous and/or submucosal tissues. Most cases of HAE are caused by mutations in the SERPING1 gene encoding C1-inhibitor (C1-INH-HAE); however, mutation analysis identified seven further types of HAE: HAE with Factor XII mutation (FXII-HAE), with plasminogen gene mutation (PLG-HAE), with angiopoietin-1 gene mutation (ANGPT1-HAE), with kininogen-1 gene mutation (KNG1-HAE), with a myoferlin gene mutation (MYOF-HAE), with a heparan sulfate-glucosamine 3-sulfotransferase 6 (HS3ST6) mutation, and hereditary angioedema of unknown origin (U-HAE). We sequenced DNA samples stored from 124 U-HAE patients in the biorepository for exon 9 of the PLG gene. One of the 124 subjects carried the mutation causing a lysine to glutamic acid amino acid exchange at position 330 (K330E). Later, the same PLG mutation was identified in the patient's son. The introduction of new techniques into genetic testing has increased the number of genes identified. As shown by this study, a biorepository creates the means for the ex-post analysis of recently identified genes in stored DNA samples of the patients. This makes the diagnosis more accurate with the possibility of subsequent family screening and the introduction of appropriate therapy.

Type 1 Plasminogen Deficiency With Pulmonary Involvement: Novel Treatment and Novel Mutation.

Type 1 plasminogen deficiency is a rare genetic disorder. Type 1 plasminogen deficiency is characterized by fibrin-rich pseudomembrane formation on mucosal surfaces, particularly the conjunctiva. Tracheobronchial tree involvement is a less common reported manifestation of type 1 plasminogen deficiency. Pseudomembranes in the tracheobronchial tree may result in respiratory compromise and ultimately fail if not recognized and treated. Currently, there is no specific replacement therapy approved for the treatment of congenital plasminogen deficiency. In the present paper, we report that type 1 plasminogen deficiency with novel frameshift mutation and pulmonary involvement was treated initially with systemic fresh frozen plasma followed by pulmonary lavage with fresh frozen plasma and tissue plasminogen activator.

Plasmin-Induced Activation of Human Platelets Is Modulated by Thrombospondin-1, Bona Fide Misfolded Proteins and Thiol Isomerases.

Inflammatory processes are triggered by the fibrinolytic enzyme plasmin. Tissue-type plasminogen activator, which cleaves plasminogen to plasmin, can be activated by the cross-ß-structure of misfolded proteins. Misfolded protein aggregates also represent substrates for plasmin, promoting their degradation, and are potent platelet agonists. However, the regulation of plasmin-mediated platelet activation by misfolded proteins and vice versa is incompletely understood. In this study, we hypothesize that plasmin acts as potent agonist of human platelets in vitro after short-term incubation at room temperature, and that the response to thrombospondin-1 and the bona fide misfolded proteins Eap and SCN--denatured IgG interfere with plasmin, thereby modulating platelet activation. Plasmin dose-dependently induced CD62P surface expression on, and binding of fibrinogen to, human platelets in the absence/presence of plasma and in citrated whole blood, as analyzed by flow cytometry. Thrombospondin-1 pre-incubated with plasmin enhanced these plasmin-induced platelet responses at low concentration and diminished them at higher dose. Platelet fibrinogen binding was dose-dependently induced by the C-terminal thrombospondin-1 peptide RFYVVMWK, Eap or NaSCN-treated IgG, but diminished in the presence of plasmin. Blocking enzymatically catalyzed thiol-isomerization decreased plasmin-induced platelet responses, suggesting that plasmin activates platelets in a thiol-dependent manner. Thrombospondin-1, depending on the concentration, may act as cofactor or inhibitor of plasmin-induced platelet activation, and plasmin blocks platelet activation induced by misfolded proteins and vice versa, which might be of clinical relevance.

Functional prediction and comparative population analysis of variants in genes for proteases and innate immunity related to SARS-CoV-2 infection.

New coronavirus SARS-CoV-2 is capable to infect humans and cause a novel disease COVID-19. Aiming to understand a host genetic component of COVID-19, we focused on variants in genes encoding proteases and genes involved in innate immunity that could be important for susceptibility and resistance to SARS-CoV-2 infection. Analysis of sequence data of coding regions of FURIN, PLG, PRSS1, TMPRSS11a, MBL2 and OAS1 genes in 143 unrelated individuals from Serbian population identified 22 variants with potential functional effect. In silico analyses (PolyPhen-2, SIFT, MutPred2 and Swiss-Pdb Viewer) predicted that 10 variants could impact the structure and/or function of proteins. These protein-altering variants (p.Gly146Ser in FURIN; p.Arg261His and p.Ala494Val in PLG; p.Asn54Lys in PRSS1; p.Arg52Cys, p.Gly54Asp and p.Gly57Glu in MBL2; p.Arg47Gln, p.Ile99Val and p.Arg130His in OAS1) may have predictive value for inter-individual differences in the response to the SARS-CoV-2 infection. Next, we performed comparative population analysis for the same variants using extracted data from the 1000 Genomes project. Population genetic variability was assessed using delta MAF and Fst statistics. Our study pointed to 7 variants in PLG, TMPRSS11a, MBL2 and OAS1 genes with noticeable divergence in allelic frequencies between populations worldwide. Three of them, all in MBL2 gene, were predicted to be damaging, making them the most promising population-specific markers related to SARS-CoV-2 infection. Comparing allelic frequencies between Serbian and other populations, we found that the highest level of genetic divergence related to selected loci was observed with African, followed by East Asian, Central and South American and South Asian populations. When compared with European populations, the highest divergence was observed with Italian population. In conclusion, we identified 4 variants in genes encoding proteases (FURIN, PLG and PRSS1) and 6 in genes involved in the innate immunity (MBL2 and OAS1) that might be relevant for the host response to SARS-CoV-2 infection.

Angioedema without urticaria: novel findings which must be measured in clinical setting.

PURPOSE OF REVIEW: Angioedema without urticaria is composed of an increasing subtype's variety and presents a challenging diagnosis. This review summarizes the subtypes recently described and subsequent new findings helpful within their classification. RECENT FINDINGS: New methods to measure cleaved high molecular weight kininogen and activated plasma kallikrein have emerged as potential biochemical tests to identify bradykinin-mediated angioedema. Three new subtypes of hereditary angioedema (HAE) with normal C1 inhibitor were described in the past two years: HAE due to mutation in plasminogen gene, in kininogen gene, and in angiopoietin-1 gene; implicating the fibrinolytic and contact systems, and the regulation of vasculature, respectively. The understanding of some mechanisms in angioedema has been improved, compatible to the dominant-negative for some C1 inhibitor variants; furthermore, the increased activation of truncated F12 mutants by plasma kallikrein; and the diminished binding of angiopoietin-1 to its receptor. SUMMARY: The validation of biomarkers for the contact system activation could be beneficial in differentiating bradykinin - from histaminergic-mediated angioedema. Currently, the available laboratorial tests are still somewhat restricted to the evaluation of the complement activation and the mediators of nonhistaminergic and nonbradykinin-mediated angioedema remain to be identified.

Nonsynonymous SNPs in LPA homologous to plasminogen deficiency mutants represent novel null apo(a) alleles.

Plasma lipoprotein (a) [Lp(a)] levels are largely determined by variation in the LPA gene, which codes for apo(a). Genome-wide association studies (GWASs) have identified nonsynonymous variants in LPA that associate with low Lp(a) levels, although their effect on apo(a) function is unknown. We investigated two such variants, R990Q and R1771C, which were present in four null Lp(a) individuals, for structural and functional effects. Sequence alignments showed the R990 and R1771 residues to be highly conserved and homologous to each other and to residues associated with plasminogen deficiency. Structural modeling showed both residues to make several polar contacts with neighboring residues that would be ablated on substitution. Recombinant expression of the WT and R1771C apo(a) in liver and kidney cells showed an abundance of an immature form for both apo(a) proteins. A mature form of apo(a) was only seen with the WT protein. Imaging of the recombinant apo(a) proteins in conjunction with markers of the secretory pathway indicated a poor transit of R1771C into the Golgi. Furthermore, the R1771C mutant displayed a glycosylation pattern consistent with ER, but not Golgi, glycosylation. We conclude that R1771 and the equivalent R990 residue facilitate correct folding of the apo(a) kringle structure and mutations at these positions prevent the proper folding required for full maturation and secretion. To our knowledge, this is the first example of nonsynonymous variants in LPA being causative of a null Lp(a) phenotype.

Study of angiopoietin and plasminogen genes in hereditary angioedema

SUMMARY OBJECTIVE To investigate the presence of the Angiopoietin 1 (ANGPT1) and Plasminogen (PLG) mutations in patients with Hereditary Angioedema (HAE) and normal C1 esterase inhibitor (C1-INH) levels, who do not harbor the F12 gene mutation. METHODS Patients clinically diagnosed with HAE but without C1-INH deficiency or dysfunction and F12 gene mutation were evaluated. DNA extraction, quantification, and dilution were performed at a concentration of 100 ng/µL, followed by a DNA amplification (PCR) for molecular evaluation of exon 2 of the ANGPT1 gene and exon 9 of the PLG gene for identification of mutations c.807G>T / p.A119S and c.988A>G / p.K330E, respectively. The PCR product was evaluated in 1% agarose gel electrophoresis. Sequencing was performed using the Sanger method. The electropherograms were analyzed using the FASTA® program. RESULTS DNA samples from 15 women were sequenced. Their ages ranged from 10 to 60 years and the normal C1 esterase and C4 inhibitor serum levels ranged from 22 to 39 mg/dL and from 10 to 40 mg/dL, respectively. No mutations were detected in the analyzed exons of ANGPT1 and PLG. However, a single-nucleotide polymorphism (SNP) was detected in two homozygotic and five heterozygotic patients. CONCLUSION Further studies are needed to evaluate these SNPs and scrutinize their potential for use as molecular markers of HAE and as novel therapeutic targets.

RESUMO OBJETIVO Investigar a presença das mutações no gene Angiopoietina (ANGPT1) e gene Plasminogênio (PLG) em pacientes com Angioedema Hereditário (AEH) com inibidor C1 esterase (C1-INH) normal e negativos para mutação do gene F12. MÉTODOS Foram avaliados pacientes com diagnóstico clínico de AEH sem deficiência ou disfunção de C1-INH e negativos para mutação do gene F12. Realizou-se extração, quantificação e diluição do DNA a uma concentração de 100 ng/uL, em seguida amplificação do DNA (PCR) para avaliação molecular do exon 2 do gene ANGPT1 e do exon 9 do gene PLG para identificação das mutações c.807G>T.p.A119S e c.988A>G p.K330E, respectivamente. O produto da PCR foi avaliado em eletroforese em gel de agarose 1%. Foi realizado o sequenciamento pelo método de Sanger. As análises dos eletroferogramas foram realizadas pelo programa FASTA®. RESULTADOS Foram sequenciadas amostras de 15 mulheres, idade entre 10 e 60 anos, com níveis séricos de inibidor de C1 esterase e C4 normais variando de 22 a 39mg/dL e 10 a 40mg/dL, respectivamente. Não foram identificadas mutações nos éxons analisados dos genes ANGPT1 e PLG. Entretanto no gene PLG foram encontrados polimorfismo de nucleotídeo único (SNP), em duas pacientes homozigotas e cinco heterozigotas. CONCLUSÃO Mais estudos sobre SNP são necessários para esclarecer estes achados pois eles podem ser utilizados como marcadores moleculares do AEH e alvo para novos tratamentos.