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1.
Zh Nevrol Psikhiatr Im S S Korsakova ; 122(11): 137-142, 2022.
Artigo em Russo | MEDLINE | ID: mdl-36440791

RESUMO

OBJECTIVE: To determine the serum concentrations of some proteins identified in the blood serum of patients with bipolar affective disorder (BD) and to evaluate the relationship of their concentrations with clinical characteristics of BD. MATERIAL AND METHODS: Protein concentrations of cadherin 5, coagulation factor XIII and ANKRD12 gene product in the blood serum of patients with BD and healthy individuals were determined using commercial ELISA kits. The severity of current depression and the main clinical manifestations of BD were assessed by SIGH-SAD and CGI upon admission to the hospital before the prescription of therapy. Data analysis was carried out using the Kruskal-Wallis and Mann-Whitney tests. RESULTS: Comparison between subgroups of BD patients, selected in accordance with the current affective episode (hypomanic, manic, depressive and mixed), and control group by the Kruskal-Wallis test has shown the significant difference in ANKRD12 protein product concentration (p=0.0448). When comparing pairwise each subgroup with the control group, the significant difference in ANKRD12 product concentration was found only in the case of a subgroup of BD patients with current depressive episode (Mann-Whitney test, p=0.006). Pairwise comparison of the ANKRD12 product concentrations in subgroups of BD patients with different disease duration (<5 years, 6-10 years, and >11 years) and in the control group by the Mann-Whitney test has revealed significant differences (p=0.04, p=0.01, p=0.02, respectively), the maximum protein concentration was found in patients with disease duration >11 years. Significant negative correlation of cadherin 5 concentration with the severity of atypical depressive symptoms assessed by SIGH-SAD (r=-0.69, p=0.038) and the decrease in cadherin 5 concentration in BD patients with greater severity of the current depressive episode were revealed. Significant negative correlation was found between factor XIII concentration and CGI scores (r=-0.62, p=0.032). CONCLUSION: The results indicate a possible link of the studied proteins with BD pathogenesis. Further study of these proteins may contribute to the development of new therapeutic and diagnostic techniques.


Assuntos
Transtorno Bipolar , Humanos , Transtorno Bipolar/diagnóstico , Transtorno Bipolar/psicologia , Fator XIII , Caderinas , Transtornos do Humor , Proteínas Nucleares
2.
BMJ Case Rep ; 15(10)2022 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-36207057

RESUMO

A term Caucasian neonate with an uncomplicated birth history presented with persistent umbilical stump bleeding unresponsive to extensive topical haemostatic measures initially. He subsequently developed hypovolaemic shock. Routine full blood count and basic coagulation screen were unremarkable. He received packed red cell and cryoprecipitate transfusions. Further specialist coagulation studies performed revealed factor XIII deficiency. Genetic investigations demonstrated a compound heterozygosity for the disorder. He was later started on monthly prophylactic treatment of plasma-derived factor XIII. Clinicians should have a high index of suspicion for factor XIII deficiency for newborns with abnormal umbilical stump bleeding in the presence of no bleeding risk factors and normal routine blood investigations.


Assuntos
Deficiência do Fator XIII , Transtornos Hemorrágicos , Hemostáticos , Testes de Coagulação Sanguínea , Fator XIII/uso terapêutico , Deficiência do Fator XIII/complicações , Deficiência do Fator XIII/diagnóstico , Deficiência do Fator XIII/genética , Hemorragia/complicações , Transtornos Hemorrágicos/complicações , Humanos , Recém-Nascido , Masculino
3.
Sci Rep ; 12(1): 17269, 2022 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-36241854

RESUMO

Viscoelastic coagulation tests have been increasingly used for hemostasis management in cardiac surgery. The ClotPro system is a novel viscoelastic device based on principles of rotational thromboelastometry. We aimed to compare ClotPro with ROTEM and plasma coagulation assays in cardiopulmonary bypass (CPB) patients. Blood samples were collected from 25 CPB patients at (1) baseline, (2) start of CPB, (3) end of CPB, and (4) end of surgery. The EX-test, IN-test, HI-test, FIB-test parameters on ClotPro were compared with corresponding ROTEM assay (EXTEM, INTEM, HEPTEM, and FIBTEM). Standard plasma coagulation assays and endogenous thrombin generation (TG) were simultaneously evaluated. Pearson correlation analyses showed moderate correlations between clotting times (CTs) (r = 0.63-0.67; p < 0.001, respectively), and strong correlations with maximal clot firmness (MCF) (r = 0.93-0.98; p < 0.001, respectively) between ClotPro and ROTEM. EX-test and IN-test MCF parameters were interchangeable with acceptable percentage errors (EX-test MCF: 7.3%, IN-test MCF: 8.3%), but FIB-test MCF (27.0%) and CT results were not (EX-test CT: 44.7%, IN-test CT: 31.4%). The correlations of PT/INR or peak TG with EX-test CTs were higher than with EXTEM CTs (PT/INR: r = 0.80 and 0.41, peak TG: 0.43 and 0.18, respectively). FIB-test MCF has strong correlation with plasma fibrinogen and factor XIII level (r = 0.84 and 0.66, respectively). ROC analyses showed that ClotPro was capable of emulating well-established ROTEM thresholds (area under curves: 0.83-1.00). ClotPro demonstrated strong correlations in MCF parameters of ROTEM in CPB patients. It may be reasonable to modify ROTEM-based transfusion algorithm pertaining to MCF parameters to establish cut-off values for ClotPro device.


Assuntos
Procedimentos Cirúrgicos Cardíacos , Tromboelastografia , Coagulação Sanguínea , Testes de Coagulação Sanguínea/métodos , Procedimentos Cirúrgicos Cardíacos/métodos , Fator XIII , Fibrinogênio , Humanos , Tromboelastografia/métodos , Trombina
4.
J Thromb Haemost ; 20(12): 2873-2886, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36111375

RESUMO

BACKGROUND: Obesity predisposes individuals to metabolic syndrome, which increases the risk of cardiovascular diseases, non-alcoholic fatty liver disease (NAFLD), and type 2 diabetes. A pathological manifestation of obesity is the activation of the coagulation system. In turn, extravascular fibrin(ogen) deposits accumulate in adipose tissues and liver. These deposits promote adiposity and downstream sequelae by driving pro-inflammatory macrophage function through binding the leukocyte integrin receptor αM ß2 . OBJECTIVES: An unresolved question is whether conversion of soluble fibrinogen to a crosslinked fibrin matrix is required to exacerbate obesity-driven diseases. METHODS: Here, fibrinogen-deficient/depleted mice (Fib- or treated with siRNA against fibrinogen [siFga]), mice expressing fibrinogen that cannot polymerize to fibrin (FibAEK ), and mice deficient in the fibrin crosslinking transglutaminase factor XIII (FXIII-) were challenged with a high-fat diet (HFD) and compared to mice expressing a mutant form of fibrinogen lacking the αM ß2 -binding domain (Fib𝛾390-396A ). RESULTS AND CONCLUSIONS: Consistent with prior studies, Fib𝛾390-396A mice were significantly protected from increased adiposity, NAFLD, hypercholesterolemia, and diabetes while Fib- and siFga-treated mice gained as much weight and developed obesity-associated pathologies identical to wildtype mice. FibAEK and FXIII- mice displayed an intermediate phenotype with partial protection from some obesity-associated pathologies. Results here indicate that fibrin(ogen) lacking αM ß2 binding function offers substantial protection from obesity and associated disease that is partially recapitulated by preventing fibrin polymer formation or crosslinking of the wildtype molecule, but not by reduction or complete elimination of fibrinogen. Finally, these findings support the concept that fibrin polymerization and crosslinking are required for the full implementation of fibrin-driven inflammation in obesity.


Assuntos
Afibrinogenemia , Diabetes Mellitus Tipo 2 , Hemostáticos , Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Fibrina/metabolismo , Polímeros , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Fibrinogênio/genética , Fibrinogênio/metabolismo , Fator XIII/metabolismo , Obesidade , Dieta
5.
Crit Care ; 26(1): 290, 2022 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-36163263

RESUMO

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


Assuntos
Transtornos da Coagulação Sanguínea , Hemostáticos , Trombose , Fator XIII/farmacologia , Fibrina/química , Fibrina/farmacologia , Fibrinogênio/uso terapêutico , Fibrinolisina/farmacologia , Fibrinólise , Hemostáticos/farmacologia , Humanos , Inibidor 1 de Ativador de Plasminogênio , Trombose/terapia
7.
J Thromb Thrombolysis ; 54(4): 593-596, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36094687

RESUMO

BACKGROUND: FXIII deficiency is a very rare coagulation disorder that can affect equally males and females with an estimated incidence of 1 in 2 million persons worldwide. Due to this rarity, there are only few clinical and pharmacokinetic (PK) data deriving from the real-world. AIM: The aim of this report is to compare head-to-head the pharmacokinetic data of catridecacog derived from the MENTORTM2 trial with our real-world (RW) study. METHODS: The PK-profiles of all patients with FXIII deficiency treated with catridecacog at eleven Italian Hemophilia Centers were compared with PK data obtained by Kerlin et al. in the MENTORTM2. RESULTS: Overall 18 real-world PK were compared with 23 PK derived from the pivotal study. In the RW 55.6% of patients were females, 26.2% in the MENTORTM2 (p < 0.05). The mean dosage of drug used for the PK assessment was 35 IU/kg in the MENTORTM2, and 33.9 IU/kg in the RW study.


Assuntos
Deficiência do Fator XIII , Hemofilia A , Feminino , Humanos , Masculino , Fator XIII , Hemofilia A/tratamento farmacológico , Mentores , Proteínas Recombinantes/uso terapêutico , Ensaios Clínicos como Assunto
8.
J Biochem ; 172(5): 293-302, 2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-35997167

RESUMO

At the final stages of blood coagulation, fibrinogen is processed into insoluble fibrin by thrombin resulting in fibril-like structure formation. Via further cross-linking reactions between the fibrin gamma subunit by the catalytic action of blood transglutaminase (Factor XIII), this molecule gains further physical stability. Meanwhile, since fibrinogen is expressed in various cells and tissues, this molecule can exhibit other functions apart from its role in blood coagulation. To create a system studying on aberrant coagulation and investigate the physiological functions, using a model fish medaka (Oryzias latipes), we established gene-deficient mutants of fibrinogen gamma subunit protein in parallel with its biochemical analysis, such as tissue distribution pattern and substrate properties. By genetic deletion via genome editing, two distinct mutants displayed retardation of blood coagulation. The mutants showed lower hematocrit with aberrant erythrocyte maturation, which indicates that fibrin deficiency caused severe anemia, and also appeared as a model for investigation of the fibrin function.


Assuntos
Anemia , Oryzias , Animais , Oryzias/genética , Oryzias/metabolismo , Fator XIII/química , Fator XIII/genética , Fator XIII/metabolismo , Trombina/metabolismo , Transglutaminases/metabolismo , Fibrina/metabolismo , Fibrinogênio/genética , Fibrinogênio/química , Fibrinogênio/metabolismo , Anemia/genética
9.
Blood Coagul Fibrinolysis ; 33(6): 337-341, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35981255

RESUMO

The objective of the study was to analyse a novel F13A1 gene mutation in a Chinese patient with factor XIII (FXIII) deficiency and explore the molecular mechanism. Pedigree investigation, clinical diagnosis, phenotypic and genetic analysis were conducted. The F13A1 gene was amplified by PCR and directly sequenced. Online bioinformatics software was needed to analyse the mutation. A novel mutation c.515G>C (p.Arg208Pro) in exon 4 was found in the proband. Protein Arg208 is conserved highly among homologous species. Bioinformatics software showed that Arg208Pro mutation might affect the protein function. We preliminarily believed the mutation Arg208Pro was responsible for the decrease FXIII level. We reported a novel mutation in the F13A1 gene, which can flesh out the mutant library.


Assuntos
Deficiência do Fator XIII , China , Fator XIII/genética , Deficiência do Fator XIII/diagnóstico , Deficiência do Fator XIII/genética , Humanos , Mutação , Linhagem
10.
Clin Lab ; 68(8)2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35975501

RESUMO

BACKGROUND: Congenital factor XIII (FXIII) deficiency is an extremely rare bleeding disorder with defects in the F13A1 or F13B genes. Here, we report a case of congenital FXIII deficiency patient who presented with trauma-induced intramuscular hemorrhage accompanied with transient platelet dysfunction with increased endogenous thrombin potential (ETP). METHODS: FXIII antigen and activity, F13A1 gene sequencing, and thrombin generation assay were measured. RESULTS: The diagnosis of FXIII deficiency was confirmed by a double heterozygous mutation of the F13A1 gene and decreased levels of FXIII antigen and activity. Platelet dysfunction caused by an antiplatelet drug was revealed in both platelet aggregation test and PFA-100. After a bleeding event, the PFA-100 results returned to normal and the thrombin generation assay in patient's plasma showed a higher ETP than normal. CONCLUSIONS: This increase in ETP may protect against bleeding and may explain why some patients show only a mild bleeding tendency despite undetectable FXIII activity.


Assuntos
Deficiência do Fator XIII , Fator XIII/genética , Deficiência do Fator XIII/complicações , Deficiência do Fator XIII/diagnóstico , Deficiência do Fator XIII/genética , Hemorragia/genética , Humanos , Mutação , Trombina
11.
Arterioscler Thromb Vasc Biol ; 42(8): 931-941, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35652333

RESUMO

As the third most common vascular disease, venous thromboembolism is associated with significant mortality and morbidity. Pathogenesis underlying venous thrombosis is still not fully understood. Accumulating data suggest fibrin network structure and factor XIII-mediated crosslinking are major determinants of venous thrombus mass, composition, and stability. Understanding the cellular and molecular mechanisms mediating fibrin(ogen) and factor XIII production and function and their ability to influence venous thrombosis and resolution may inspire new anticoagulant strategies that target these proteins to reduce or prevent venous thrombosis in certain at-risk patients. This article summarizes fibrinogen and factor XIII biology and current knowledge of their function during venous thromboembolism.


Assuntos
Hemostáticos , Trombose , Tromboembolia Venosa , Trombose Venosa , Coagulação Sanguínea , Fator XIII/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Humanos , Trombose Venosa/metabolismo
12.
Gene ; 834: 146637, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35671883

RESUMO

Factor XIII, a transglutaminase that plays a crucial role in clot formation, consists of subunits A and B. Single nucleotide polymorphisms in Factor XIII-A have been linked to thrombotic risk. In Type 2 Diabetes mellitus (T2DM), a hypercoagulable state is thought to contribute to the high mortality rate associated with thrombotic diseases. Due to the lack of prevalence data of FXIII-A single nucleotide polymorphisms (SNPs) in T2DM in a South African cohort, this study assessed the prevalence FXIII-A Val34Leu (rs5985) and Tyr204Phe (rs3024477) SNP's and the effect on clot kinetics in T2DM. MATERIALS AND METHODS: A cohort of T2DM patients (n = 100) and race, age and gender matched healthy controls (n = 101) were recruited following ethical approval. Thromboelastography® (TEG®) was used to assess the viscoelastic properties in platelet poor plasma (PPP) in controls (n = 91) and T2DM patients (n = 91) younger than 50 years old. Genomic DNA was isolated from whole blood using the Quick-DNA™ Miniprep Plus Kit and PCR-RFLP was used to genotype each sample for FXIII-A rs5985 and rs3024477 SNPs. RESULTS: TEG® analyses indicated a longer R-time (p < 0.0001) and higher TMRTG (p < 0.0001) in PPP of T2DM patients. Control and T2DM genotype distribution conformed to Hardy-Weinberg equilibrium (p > 0.05). There was a higher prevalence of the wildtype genotype of FXIII-A Tyr204Phe (rs3024477) SNP in T2DM (OR = 0.23, 95% CI = 0.12-0.42, p < 0.0001). The 204Phe variant was more frequent in the Caucasians (OR = 0.39, 95% CI = 0.05-0.33, p < 0.0001). The presence of the 204Phe variant in T2DM affected TMRTG (p = 0.0207). The variant affected R time (p = 0.0432) and TMRTG (p = 0.0209 and p = 0.0207) in controls and T2DM, respectively. CONCLUSION: An inverse association with T2DM and FXIII-A Tyr204Phe was found. A hypo coagulable PPP clot profile was observed in T2DM. A shorter reaction time was observed and but faster rate at which the clot reached maximum strength in both controls and T2DM in the presence of the 204Phe variant.


Assuntos
Diabetes Mellitus Tipo 2 , Fator VIII/genética , Trombose , Diabetes Mellitus Tipo 2/genética , Fator XIII/genética , Fator XIIIa/genética , Humanos , Cinética , Pessoa de Meia-Idade , África do Sul , Trombose/genética
15.
Int J Mol Sci ; 23(9)2022 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-35563115

RESUMO

Coagulation factor XIII (FXIII) circulates in plasma as a pro-transglutaminase heterotetrameric complex (FXIIIA2B2), which upon activation by thrombin and calcium covalently crosslinks preformed fibrin polymers. The heterotetrameric complex is composed of a catalytic FXIIIA2 subunit and a protective/regulatory FXIII-B2 subunit coded by F13A1 and F13B genes, respectively. The catalytic FXIIIA2 subunit is encoded by the F13A1 gene, expressed primarily in cells of mesenchymal origin, whereas the FXIIIB subunit encoded by the F13B gene is expressed and secreted from hepatocytes. The plasma FXIIIA2 subunit, which earlier was believed to be secreted from cells of megakaryocytic lineage, is now understood to result primarily from resident macrophages. The regulation of the FXIII subunits at the genetic level is still poorly understood. The current study adopts a purely bioinformatic approach to analyze the temporal, time-specific expression array-data corresponding to both the subunits in specific cell lineages, with respect to the gene promoters. We analyze the differentially expressed genes correlated with F13A1 and F13B expression levels in an array of cell types, utilizing publicly available microarray data. We attempt to understand the regulatory mechanism underlying the variable expression of FXIIIA2 subunit in macrophages (M0, M1, M2 and aortic resident macrophages). Similarly, the FXIIIB2 subunit expression data from adult, fetal hepatocytes and embryonic stem cells derived hepatoblasts (hESC-hepatoblast) was analyzed. The results suggest regulatory dependence between the two FXIII subunits at the transcript level. Our analysis also predicts the involvement of the FXIIIA2 subunit in macrophage polarization, plaque stability, and inflammation.


Assuntos
Biologia Computacional , Fator XIII , Testes de Coagulação Sanguínea , Fator XIII/genética , Fator XIII/metabolismo , Fibrina , Trombina/metabolismo
16.
Int J Mol Sci ; 23(6)2022 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-35328366

RESUMO

Loss of fibrinogen is a feature of trauma-induced coagulopathy (TIC), and restoring this clotting factor is protective against hemorrhages. We compared the efficacy of cryoprecipitate, and of the fibrinogen concentrates RiaSTAP® and FibCLOT® in restoring the clot integrity in models of TIC. Cryoprecipitate and FibCLOT® produced clots with higher maximal absorbance and enhanced resistance to lysis relative to RiaSTAP®. The fibrin structure of clots, comprising cryoprecipitate and FibCLOT®, mirrored those of normal plasma, whereas those with RiaSTAP® showed stunted fibers and reduced porosity. The hemodilution of whole blood reduced the maximum clot firmness (MCF) as assessed by thromboelastography. MCF could be restored with the inclusion of 1 mg/mL of fibrinogen, but only FibCLOT® was effective at stabilizing against lysis. The overall clot strength, measured using the Quantra® hemostasis analyzer, was restored with both fibrinogen concentrates but not cryoprecipitate. α2antiplasmin and plasminogen activator inhibitor-1 (PAI-1) were constituents of cryoprecipitate but were negligible in RiaSTAP® and FibCLOT®. Interestingly, cryoprecipitate and FibCLOT® contained significantly higher factor XIII (FXIII) levels, approximately three-fold higher than RiaSTAP®. Our data show that 1 mg/mL fibrinogen, a clinically achievable concentration, can restore adequate clot integrity. However, FibCLOT®, which contained more FXIII, was superior in normalizing the clot structure and in stabilizing hemodiluted clots against mechanical and fibrinolytic degradation.


Assuntos
Transtornos da Coagulação Sanguínea , Hemostáticos , Trombose , Fator XIII/farmacologia , Fator XIII/uso terapêutico , Fibrina/química , Fibrinogênio/metabolismo , Humanos , Tromboelastografia
17.
Crit Care ; 26(1): 69, 2022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-35331308

RESUMO

Factor XIII (FXIII) is a protein involved in blood clot stabilisation which also plays an important role in processes including trauma, wound healing, tissue repair, pregnancy, and even bone metabolism. Following surgery, low FXIII levels have been observed in patients with peri-operative blood loss and FXIII administration in those patients was associated with reduced blood transfusions. Furthermore, in patients with low FXIII levels, FXIII supplementation reduced the incidence of post-operative complications including disturbed wound healing. Increasing awareness of potentially low FXIII levels in specific patient populations could help identify patients with acquired FXIII deficiency; although opinions and protocols vary, a cut-off for FXIII activity of ~ 60-70% may be appropriate to diagnose acquired FXIII deficiency and guide supplementation. This narrative review discusses altered FXIII levels in trauma, surgery and wound healing, diagnostic approaches to detect FXIII deficiency and clinical guidance for the treatment of acquired FXIII deficiency.


Assuntos
Transtornos da Coagulação Sanguínea , Deficiência do Fator XIII , Transtornos da Coagulação Sanguínea/etiologia , Fator XIII/metabolismo , Fator XIII/uso terapêutico , Deficiência do Fator XIII/complicações , Deficiência do Fator XIII/diagnóstico , Deficiência do Fator XIII/tratamento farmacológico , Hemorragia/tratamento farmacológico , Humanos , Cicatrização
18.
J Thromb Haemost ; 20(5): 1223-1235, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35146910

RESUMO

BACKGROUND: Platelets contain a high amount of potentially active A subunit dimer of coagulation factor XIII (cellular FXIII; cFXIII). It is of cytoplasmic localization, not secreted, but becomes translocated to the surface of platelets activated by convulxin and thrombin (CVX+Thr). OBJECTIVE: To explore the difference in cFXIII translocation between receptor mediated and non-receptor mediated platelet activation and if translocation can also be detected on platelet-derived microparticles. Our aim was also to shed some light on the mechanism of cFXIII translocation. METHODS: Gel-filtered platelets were activated by CVX+Thr or Ca2+ -ionophore (calcimycin). The translocation of cFXIII and phosphatidylserine (PS) to the surface of activated platelets and platelet-derived microparticles was investigated by flow cytometry, immunofluorescence, and immune electron microscopy. Fluo-4-AM fluorescence was used for the measurement of intracellular Ca2+ concentration. RESULTS: Receptor mediated activation by CVX+Thr exposed cFXIII to the surface of more than 60% of platelets. Electron microscopy revealed microparticles with preserved membrane structure and microparticles devoid of labeling for membrane glycoprotein CD41a. cFXIII was observed on both types of microparticles but was more abundant in the absence of CD41a. Rhosin, a RhoA inhibitor, significantly decreased cFXIII translocation. Non-receptor mediated activation of platelets by calcimycin elevated intracellular Ca2+ concentration, induced the translocation of PS to the surface of platelets and microparticles, but failed to expose cFXIII. CONCLUSIONS: The elevation of intracellular Ca2+ concentration is sufficient for the translocation of PS from the internal layer of the membrane, while the translocation of cFXIII from the platelet cytoplasm requires additional receptor mediated mechanism(s).


Assuntos
Micropartículas Derivadas de Células , Fator XIII , Plaquetas , Calcimicina/farmacologia , Proteínas de Transporte , Humanos , Fosfatidilserinas , Ativação Plaquetária , Trombina/farmacologia
19.
Blood Coagul Fibrinolysis ; 33(3): 153-158, 2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-35221320

RESUMO

Factor XIII (FXIII) deficiency is one of the most severe congenital bleeding disorders, with an estimated incidence of one person per one million. Patients with severe FXIII deficiency present a wide range of clinical manifestations, including umbilical cord bleeding, intracranial haemorrhage and recurrent miscarriages. Due to the high rate of life-threatening bleeding, primary prophylaxis is mandatory from the time of diagnosis. Although replacement therapy is the most common therapeutic choice, gene therapy remains the only curative option. In the present study, we assessed the efficacy of the clustered regularly interspaced short palindromic repeats - CRISPR-associated protein 9 (CRISPR/Cas9) system in the correction of the most common FXIII disease-causing mutation (c.562 T > C). A dermal fibroblast was harvested from the human skin biopsy of a young patient with FXIII deficiency. Sanger sequencing was used to confirm the presence of c.562 T>C mutation in the patient and in the harvested fibroblasts. PX459 vector was digested with BbsI restriction enzyme, and after annealing and ligation of two 20-bp guide-RNAs (g-RNAs) close to the PAM (NGG) sequence, the constructed vectors were amplified in Escherichia coli Top 10. Transfection was performed by a nucleofector device, and DNA extraction was performed after puromycin selection and serial dilution from potentially transfected colonies. A 50-bp template oligonucleotide was used to aid homologous repair for correction of the underlying mutation and synonymous mutation as an internal control. The synonymous mutation (AAT to ACT) near the mutation site was used as internal control. Sanger sequencing was done in order to check the gene correction. The c.562 T > C mutation was detected in homozygote state in the primary fibroblasts of the patient and wild-type alleles were confirmed in the normal individual. Colony PCR and sequencing revealed successful cloning of the designed gRNAs. The detected mutation was corrected from a homozygote mutant state (c.562 T > C) to a homozygote wild type in transfected dermal fibroblasts of the patient. The control mutation, as an internal control, was also corrected in the same fibroblasts in the heterozygote manner. The result of the study shows that the CRISPR/CAS9 gene editing system is an effective tool for correction of point mutations in transfected fibroblasts of patients with congenital FXIII deficiency and represents a new, potentially curative, option.


Assuntos
Deficiência do Fator XIII , Edição de Genes , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Escherichia coli , Fator XIII/genética , Deficiência do Fator XIII/genética , Deficiência do Fator XIII/terapia , Humanos , Mutação
20.
Cancer Sci ; 113(5): 1587-1600, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35178836

RESUMO

Evolutionarily conserved DDB1-and CUL4-associated factor 13 (DCAF13) is a recently discovered substrate receptor for the cullin RING-finger ubiquitin ligase 4 (CRL4) E3 ubiquitin ligase that regulates cell cycle progression. DCAF13 is overexpressed in many cancers, although its role in breast cancer is currently elusive. In this study we demonstrate that DCAF13 is overexpressed in human breast cancer and that its overexpression closely correlates with poor prognosis, suggesting that DCAF13 may serve as a diagnostic marker and therapeutic target. We knocked down DCAF13 in breast cancer cell lines using CRISPR/Cas9 and found that DCAF13 deletion markedly reduced breast cancer cell proliferation, clone formation, and migration both in vitro and in vivo. In addition, DCAF13 deletion promoted breast cancer cell apoptosis and senescence, and induced cell cycle arrest in the G1/S phase. Genome-wide RNAseq analysis and western blotting revealed that loss of DCAF13 resulted in both mRNA and protein accumulation of p53 apoptosis effector related to PMP22 (PERP). Knockdown of PERP partially reversed the hampered cell proliferation induced by DCAF13 knockdown. Co-immunoprecipitation assays revealed that DCAF13 and DNA damage-binding protein 1 (DDB1) directly interact with PERP. Overexpression of DDB1 significantly increased PERP polyubiquitination, suggesting that CRL4DCAF13 E3 ligase targets PERP for ubiquitination and proteasomal degradation. In conclusion, DCAF13 and the downstream effector PERP occupy key roles in breast cancer proliferation and potentially serve as prognostics and therapeutic targets.


Assuntos
Neoplasias da Mama , Fator XIII , Neoplasias da Mama/genética , Proliferação de Células/genética , Proteínas Culina/genética , Fator XIII/genética , Fator XIII/metabolismo , Feminino , Genes Supressores de Tumor , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
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