RESUMO
Congenital factor XIII (FXIII) deficiency is a rare, autosomal recessive bleeding disorder with potentially life-threatening consequences. FXIII is composed of two subunits (A and B), and a deficiency or dysfunction of either can result in FXIII deficiency. Traditionally, FXIII deficiency has been managed by infusing plasma-derived products containing FXIII (fresh frozen plasma, cryoprecipitate, and plasma-derived FXIII concentrates), all of which contain both subunits. Despite the increased safety of plasma-derived products, concern remains regarding potential viral safety issues. This review describes the development, from concept to clinical use, of a recombinant FXIII molecule (containing subunit A only; rFXIII-A2) for congenital FXIII-A subunit deficiency. Unmet needs and ongoing challenges in congenital FXIII deficiency are also discussed. Despite the challenges in developing a product for a very rare bleeding disorder, the information gathered on efficacy, safety, and pharmacokinetics of FXIII replacement therapy represents the largest dataset on congenital FXIII-A subunit deficiency in the world. It also provides evidence for the safety and efficacy of monthly prophylaxis with 35 IU/kg of rFXIII-A2 in patients with FXIII-A subunit deficiency. The issues encountered and overcome, along with lessons learned, may be applied to and encourage the development of new recombinant products for other rare bleeding disorders.
Assuntos
Deficiência do Fator XIII/tratamento farmacológico , Fator XIII/uso terapêutico , Proteínas Recombinantes/uso terapêutico , HumanosRESUMO
BACKGROUND: Refractoriness to platelet transfusion, prevalent among 15-20% of hemato-oncological patients, is associated with multitransfusions and inferior outcomes. We evaluated the effectiveness of extended slow-dose transfusion (ESDT) in increasing platelet increments in multitransfused patients. METHODS: Patients treated after the implementation of ESDT were compared with historical controls treated with standard single-donor platelet (SDP) transfusions. Cohorts of early and late recipients were assembled for comparison, i.e. the 8th or 9th and 11th platelet unit per patient, respectively. Patients in the ESDT group received transfusions equal to half an SDP unit, administered over 4 h. Effectiveness was defined as a higher corrected count increment (CCI) at 1, 12, and 24 h after transfusion. RESULTS: In the early-recipients cohort, 24-h-posttransfusion increments were available for 29 ESDT patients and 6 standard patients, and did not differ significantly between the groups (p = 0.078). The 24-h-posttransfusion increment was available for 20 ESDT patients and 7 standard patients in the late-recipients cohort. The CCI was significantly higher in the ESDT group (p = 0.042). ABO compatibility improved the CCI (p = 0.01). CONCLUSIONS: ESDT demonstrated slightly higher increments at 24 h after transfusion in late recipients, suggesting this could be a cost-effective approach for the treatment of thrombocytopenic multitransfused hemato-oncological patients.
Assuntos
Neoplasias Hematológicas/terapia , Transfusão de Plaquetas/métodos , Adulto , Idoso , Estudos de Coortes , Feminino , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/complicações , Humanos , Leucemia/sangue , Leucemia/complicações , Leucemia/terapia , Linfoma/sangue , Linfoma/complicações , Linfoma/terapia , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Transfusão de Plaquetas/efeitos adversos , Fatores de Risco , Trombocitopenia/sangue , Trombocitopenia/etiologia , Trombocitopenia/terapia , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Congenital factor XIII (FXIII) deficiency is a rare, autosomal-recessive disorder, with most patients having an A-subunit (FXIII-A) deficiency. Patients experience life-threatening bleeds, impaired wound healing, and spontaneous abortions. In many countries, only plasma or cryoprecipitate treatments are available, but these carry a risk for allergic reactions and infection with blood-borne pathogens. The present study was a multinational, open-label, single-arm, phase 3 prophylaxis trial evaluating the efficacy and safety of a novel recombinant FXIII (rFXIII) in congenital FXIII-A subunit deficiency. Forty-one patients ≥ 6 years of age (mean, 26.4; range, 7-60) with congenital FXIII-A subunit deficiency were enrolled. Throughout the rFXIII prophylaxis, only 5 bleeding episodes (all trauma induced) in 4 patients were treated with FXIII-containing products. The crude mean bleeding rate was significantly lower than the historic bleeding rate (0.138 vs 2.91 bleeds/patient/year, respectively) for on-demand treatment. Transient, non-neutralizing, low-titer anti-rFXIII Abs developed in 4 patients, none of whom experienced allergic reactions, any bleeds requiring treatment, or changes in FXIII pharmacokinetics during the trial or follow-up. These non-neutralizing Abs declined below detection limits in all 4 patients despite further exposure to rFXIII or other FXIII-containing products. We conclude that rFXIII is safe and effective in preventing bleeding episodes in patients with congenital FXIII-A subunit deficiency. This study is registered at http://www..clinicaltrials.gov as number NCT00713648.
Assuntos
Fator VIII/administração & dosagem , Hemofilia A/tratamento farmacológico , Hemorragia/tratamento farmacológico , Adolescente , Adulto , Anticorpos/sangue , Criança , Hipersensibilidade a Drogas/etiologia , Fator VIII/efeitos adversos , Feminino , Hemofilia A/sangue , Hemorragia/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Fatores de Risco , SegurançaRESUMO
BACKGROUND: A 75 year old patient presenting with mucocutaneous bleeding was diagnosed with acquired thrombasthenia. The diagnosis was based on lack of platelet aggregation with adenosine diphosphate (ADP), arachidonic acid and collagen, and normal aggregation induced by ristocetin. OBJECTIVE: To study the mechanism of platelet function inhibition in a patient with acquired thrombasthenia. METHODS: Aggregation assays of platelets from the patient and healthy controls were performed. In addition, anti-glycoprotein (GP) IIbIIIa antibodies bindingto normal in the presence or absence of the patient's serum was by flow cytometry. RESULTS: Aggregation of normal platelets in the presence of patient's plasma was inhibited four- and 2.5-fold in the presence of ADP and arachidonic acid respectively, while collagen-induced aggregation was completely abolished. Ristocetin-induced aggregation was normal. The patient's serum inhibited binding of commercial anti-glycoprotein IIbIIIa antibodies to normal platelets twofold by flow cytometry. Treatment with anti-CD20 monoclonal antibody (rituximab) normalized the patient's platelet aggregation. CONCLUSIONS: These results suggest that the patient developed inhibitory anti-GPIIbIIIa autoantibodies that caused acquired thrombasthenia.
Assuntos
Autoanticorpos/análise , Transtornos Plaquetários , Agregação Plaquetária , Testes de Função Plaquetária/métodos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Difosfato de Adenosina , Idoso , Anticorpos Monoclonais Murinos/administração & dosagem , Ácido Araquidônico , Transtornos Plaquetários/diagnóstico , Transtornos Plaquetários/etiologia , Transtornos Plaquetários/imunologia , Colágeno , Monitoramento de Medicamentos/métodos , Feminino , Humanos , Fatores Imunológicos/administração & dosagem , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/imunologia , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/imunologia , Indução de Remissão , Ristocetina , Rituximab , Resultado do TratamentoRESUMO
The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) has been shown to synergize doxorubicin (Dox) anticancer activity while attenuating its cardiotoxicity. In this study we further explored the selectivity of AN-7's action in several cancer and normal cells treated with anticancer agents. The cells studied were murine mammary 4T1, human breast T47D and glioblastoma U251 cancer cell lines, neonatal rat cardiomyocytes, cardiofibroblasts and astrocytes, and immortalized cardiomyocyte H9C2 cells. Cell death, ROS production and changes in protein expression were measured and in vivo effects were evaluated in Balb-c mice. AN-7 synergized Dox and anti-HER2 cytotoxicity against mammary carcinoma cells with combination indices of 0.74 and 0.79, respectively, while it protected cardiomyocytes against their toxicity. Additionally AN-7 protected astrocytes from Dox-cytoxicity. Cell-type specific changes in the expression of proteins controlling survival, angiogenesis and inflammation by AN-7 or AN-7+Dox were observed. In mice, the protective effect of AN-7 against Dox cardiotoxicity was associated with a reduction in inflammatory factors. In summary, AN-7 augmented the anticancer activity of Dox and anti-HER2 and attenuated their toxicity against normal cells. AN-7 modulation of c-Myc, thrombospondin-1, lo-FGF-2 and other proteins were cell type specific. The effects of AN-7, Dox and their combination were preserved in vivo indicating the potential benefit of combining AN-7 and Dox for clinical use.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Astrócitos/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Neoplasias da Mama/patologia , Fibroblastos/efeitos dos fármacos , Glioblastoma/patologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Angiogênicas/metabolismo , Animais , Anticorpos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Astrócitos/patologia , Neoplasias Encefálicas/enzimologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/imunologia , Butiratos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citoproteção , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Feminino , Fibroblastos/patologia , Glioblastoma/enzimologia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Mediadores da Inflamação/metabolismo , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Miócitos Cardíacos/patologia , Compostos Organofosforados/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptor ErbB-2/imunologia , Fatores de TempoAssuntos
Biomarcadores Tumorais/sangue , Detecção Precoce de Câncer/métodos , Fibrinogênio/análise , Leucócitos Mononucleares/química , Micose Fungoide/sangue , Neoplasias Cutâneas/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Feminino , Fibrinogênio/genética , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Micose Fungoide/genética , Micose Fungoide/patologia , Estadiamento de Neoplasias , Valor Preditivo dos Testes , RNA Mensageiro/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Regulação para Cima , Adulto JovemRESUMO
Acquired thrombotic thrombocytopenic purpura (TTP) is an uncommon disease in adults, characterized by fever, neurological manifestations, microangiopathic hemolytic anemia, thrombocytopenia, renal dysfunction, and the presence of antibodies against the enzyme ADAMTS13. Treatment with plasmapheresis has increased the survival from 10% to more than 90%. Still, there is a subset of patients with resistant TTP who fail to respond to plasmapheresis or remain dependent on this procedure. There is mounting evidence that rituximab may play an important role in remission induction of resistant/relapsing TTP, but the extent of the remission is unknown. We present here four patients with chronic-relapsing TTP who responded favorably to rituximab. All four patients achieved prolonged remission of 23 to 82 months after the treatment. One patient relapsed 6 years afterthe initial treatment with rituximab and re-entered remission following retreatment.
Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Fatores Imunológicos/uso terapêutico , Adulto , Antígenos CD20 , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Púrpura Trombocitopênica Trombótica/tratamento farmacológico , Recidiva , Indução de Remissão , Estudos Retrospectivos , Rituximab , Fatores de Tempo , Adulto JovemRESUMO
Von Willebrand disease (VWD) is the most common hereditary bleeding disorder, characterized by skin and mucosaL bleeding in most of the cases. In the Western world, the approximate number of bleeding VWD patients is 6.9 to 17 million. The disease is represented by several types with specific treatment for each type. Diagnosis can be troublesome and, therefore, n order to prevent bleeding complications, a professional clinical/laboratory team is required.
Assuntos
Doenças de von Willebrand/diagnóstico , Doenças de von Willebrand/terapia , Hemorragia/etiologia , Hemorragia/prevenção & controle , Humanos , Incidência , Equipe de Assistência ao Paciente , Doenças de von Willebrand/classificação , Doenças de von Willebrand/epidemiologiaRESUMO
Tissue transglutaminase was reported to act as protein disulfide isomerase (PDI). We studied whether plasma transglutaminase - coagulation factor XIII (FXIII) - has PDI activity as well. PDI activity was measured by determining the ability to renature reduced-denatured RNase (rdRNase). We found that FXIII can renature rdRNase, with efficiency comparable to commercial PDI. This PDI activity was inhibited by bacitracin. Like tissue transglutaminase, FXIII-mediated PDI activity is independent of its transglutaminase activity and is located on the A subunit. Surface-associated PDI has been previously shown to catalyse two distinct functions: transnitrosation with subsequent release of intracellular nitric oxide and disulfide bond rearrangement during platelet integrin ligation. Our results imply that FXIII-PDI activity may have a role in platelet function.
Assuntos
Fator XIII/metabolismo , Fator XIIIa/metabolismo , Isomerases de Dissulfetos de Proteínas/sangue , Ribonuclease Pancreático/metabolismo , Animais , Anticorpos , Bacitracina/farmacologia , Bovinos , Inibidores Enzimáticos/farmacologia , Fator XIII/antagonistas & inibidores , Fator XIII/química , Fator XIII/imunologia , Fator XIIIa/antagonistas & inibidores , Fator XIIIa/química , Fator XIIIa/imunologia , Humanos , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/imunologia , Renaturação Proteica , Subunidades ProteicasAssuntos
Anticoagulantes/uso terapêutico , Síndrome Antifosfolipídica/tratamento farmacológico , Transplante de Rim/métodos , Fragmentos de Peptídeos/uso terapêutico , Adulto , Anticoagulantes/efeitos adversos , Antitrombinas/efeitos adversos , Antitrombinas/uso terapêutico , Doença Catastrófica , Feminino , Heparina/efeitos adversos , Heparina/uso terapêutico , Hirudinas/efeitos adversos , Humanos , Fragmentos de Peptídeos/efeitos adversos , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Trombocitopenia/induzido quimicamenteRESUMO
Histone deacetylase inhibitory prodrugs that are metabolized to butyric acid and formaldehyde possess antineoplastic properties and low toxicity. We sought to characterize the antiangiogenic and antimetastatic activities of two lead prodrugs, pivaloyloxymethyl butyrate (AN-9) and butyroyloxymethyl-diethyl phosphate (AN-7) in murine cancer models. In the sc implanted human colon carcinoma HT-29 xenograft model AN-7, exhibited superior anticancer activity compared to AN-9, as was evident by the significantly greater inhibition of tumor growth and reduction of serum CEA. AN-7 was also more effective in reducing mean vessel density (MVD) by 7-fold, bFGF, Ki-67 (7-fold) and HIF-1alpha in immunohistochemically stained tumor sections. Semi-quantitative evaluation of the levels of bFGF, HDAC1 and HIF-1alpha by Western blot analysis showed a decrease in expression only in the tumors of mice treated with AN-7. The level of bFGF was reduced 3-fold in the tumor and that of TIMP1 was elevated (by 3-fold) in the serum of AN-7 treated mice. In a 4T1 metastatic breast carcinoma model, AN-7 inhibited the formation of lung lesions by 76% and AN-9 by 47%, further demonstrating the greater efficacy of AN-7 compared to AN-9 (P<0.02). Both AN-7 and AN-9 exhibited antimetastatic and antiangiogenic activities by reducing vascularization, bFGF expression and HIF-1alpha. Yet, AN-7 was more potent than AN-9.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Butiratos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Metástase Neoplásica/prevenção & controle , Compostos Organofosforados/farmacologia , Animais , Antígenos CD34/análise , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Camundongos , PTEN Fosfo-Hidrolase/análise , Inibidor Tecidual de Metaloproteinase-1/análiseRESUMO
Recombinant factor XIII-A2 (rFXIII-A2) was developed for prophylaxis and treatment of bleeds in patients with congenital FXIII A-subunit deficiency. mentor™2 (NCT00978380), a multinational, open-label, single-arm, multiple-dosing extension to the pivotal mentor™1 trial, assessed long-term safety and efficacy of rFXIII-A2 prophylaxis in eligible patients (patients with severe [<0.05 IU/mL] congenital FXIII subunit A deficiency) aged ≥6 years. Patients received 35 IU/kg rFXIII-A2 (exact dosing) every 28 ± 2 days for ≥52 weeks. Primary endpoint was safety (adverse events including immunogenicity); secondary endpoints were rate of bleeds requiring FXIII treatment, haemostatic response after one 35 IU/kg rFXIII-A2 dose for breakthrough bleeds and withdrawals due to lack of rFXIII-A2 efficacy. Steady-state pharmacokinetic variables were also summarized. Elective surgery was permitted during the treatment period. Sixty patients were exposed to rFXIII-A2; their median age was 26.0 years (range: 7.0-77.0). rFXIII-A2 was well tolerated without any safety concerns. No non-neutralizing or neutralizing antibodies (inhibitors) against FXIII were detected. Mean annualized bleeding rate (ABR) was 0.043/patient-year. Mean spontaneous ABR was 0.011/patient-year. No patients withdrew due to lack of efficacy. Geometric mean FXIII trough level was 0.17 IU/mL. Geometric terminal half-life was 13.7 days. rFXIII-A2 prophylaxis provided sufficient haemostatic coverage for 12 minor surgeries without the need for additional FXIII therapy; eight procedures were performed within 7 days of the patient's last scheduled rFXIII-A2 dose, and four were performed 10 to 21 days after the last dose.
Assuntos
Fator VIIIa/uso terapêutico , Deficiência do Fator XIII/terapia , Hemorragia/prevenção & controle , Procedimentos Cirúrgicos Operatórios , Adolescente , Adulto , Idoso , Criança , Deficiência do Fator XIII/congênito , Deficiência do Fator XIII/cirurgia , Feminino , Hemostasia , Humanos , Masculino , Pessoa de Meia-Idade , Segurança do Paciente , Proteínas Recombinantes/uso terapêutico , Adulto JovemRESUMO
Tyrosine kinase inhibitors (TKIs) have revolutionized the prognosis of chronic myeloid leukemia. With the advent of highly efficacious therapy, the focus has shifted toward managing TKI adverse effects, such as vascular adverse events (VAEs). We used an in vitro angiogenesis model to investigate the TKI-associated VAEs. Our data show that imatinib, nilotinib, and ponatinib reduce human umbilical vein endothelial cells (HUVECs) viability. Pharmacological concentrations of ponatinib induced apoptosis, reduced migration, inhibited tube formation of HUVECs, and had a negative effect on endothelial progenitor cell (EPC) function. Furthermore, in HUVECs transfected with VEGF receptor 2 (VEGFR2), the effect of ponatinib on tube formation and on all parameters representing normal endothelial cell function was less prominent than in control cells. This is the first report regarding the pathogenesis of ponatinib-associated VAEs. The antiangiogenic effect of ponatinib, possibly mediated by VEGFR2 inhibition, as shown in our study, is another piece in the intricate puzzle of TKI-associated VAEs.
Assuntos
Antineoplásicos/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/farmacologia , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Biomarcadores , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Imidazóis/efeitos adversos , Imidazóis/uso terapêutico , Imunofenotipagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Fenótipo , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/uso terapêutico , Piridazinas/efeitos adversos , Piridazinas/uso terapêutico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Thrombin-activated Factor XIII (FXIIIa), a plasma transglutaminase, stabilizes fibrin clots by crosslinking fibrin chains. FXIIIa was previously shown by us to exhibit proangiogenic activity associated with downregulation of thrombospondin-1, phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2), and upregulation of c-Jun. In the current study, we evaluated the proangiogenic effect of FXIIIa in two murine models: a neonatal heterotopic cardiac allograft model in normal mice, and a Matrigel plug model in FXIII-deficient mice. In the neonatal cardiac allograft model, the number of new vessels as well as graft viability (contractile performance) was significantly higher in FXIIIa-injected animals than in controls. A significant increase in the level of c-Jun mRNA and a significant decrease in the level of TSP-1 mRNA were observed in heart allografts treated with FXIIIa. A marked decrease in TSP-1 protein expression was observed within the endothelial cells of hearts treated with FXIIIa. In the Matrigel plug model, FXIII-deficient mice showed a significantly decreased number of new vessels compared to that of the control mice, and the number of vessels almost reached normal levels following addition of FXIIIa. The results of this study provide substantial in vivo evidence for the proangiogenic activity of FXIIIa.
Assuntos
Indutores da Angiogênese/farmacologia , Deficiência do Fator XIII/genética , Fator XIIIa/farmacologia , Transplante de Coração , Neovascularização Fisiológica/efeitos dos fármacos , Transplante Heterotópico , Animais , Animais Recém-Nascidos , Colágeno , Vasos Coronários/efeitos dos fármacos , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Orelha Externa , Fator XIII/genética , Sobrevivência de Enxerto/efeitos dos fármacos , Laminina , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Modelos Animais , Miocárdio/metabolismo , Proteoglicanas , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/metabolismo , Trombospondina 1/genética , Trombospondina 1/metabolismoRESUMO
OBJECTIVE: Coagulation Factor XIII (FXIII) was previously shown by us to induce angiogenesis. The aim of this study was to elucidate the molecular events underlying the proangiogenic effects of activated FXIII (FXIIIa) on human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: As shown by coimmunoprecipitation studies, FXIIIa crosslinked alpha(v)beta3 with vascular endothelial growth factor receptor 2 (VEGFR-2) and enhanced the noncovalent interaction between the 2 receptors. In addition, FXIIIa induced tyrosine phosphorylation of VEGFR-2 in both the crosslinked high-molecular-weight and the noncovalent VEGFR-2/alpha(v)beta3 complexes. These effects as well as FXIIIa-induced proliferation and migration of HUVECs were abolished by iodoacetamide treatment of FXIIIa (I-FXIII) or by PTKI, an inhibitor of VEGFR-2. FXIIIa induced upregulation of c-Jun and Egr-1 as revealed by quantitative RT-PCR. Electrophoretic mobility-shift assay experiments showed that FXIIIa treatment of HUVECs enhanced binding of Wilm's tumor-1 (WT-1) but not of early growth response (Egr)-1 to the thrombospondin-1 (TSP-1) promoter sequence, suggesting that WT-1 but not Egr-1 is involved in downregulation of TSP-1 expression. CONCLUSIONS: The proangiogenic effect of FXIIIa is mediated by (1) enhancement of crosslinked and noncovalent alpha(v)beta3/VEGFR-2 complex formation; (2) tyrosine phosphorylation and activation of VEGFR-2; (3) upregulation of c-Jun and Egr-1; and (4) downregulation of TSP-1 induced indirectly by c-Jun through WT-1. These processes may clarify FXIII role in vascular remodeling and tissue repair.
Assuntos
Endotélio Vascular/fisiologia , Fator XIII/farmacologia , Fator XIII/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Células Cultivadas , Reagentes de Ligações Cruzadas/farmacologia , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Proteínas Imediatamente Precoces/genética , Integrina alfaVbeta3/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Proteínas Proto-Oncogênicas c-jun/genética , Transdução de Sinais/efeitos dos fármacos , Trombospondina 1/genética , Fatores de Transcrição/genética , Tirosina/metabolismo , Veias Umbilicais/citologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas WT1/metabolismoRESUMO
Four missense mutations in the factor XIII A-subunit gene, Arg260Leu, Ala318Val, Thr398Asn and Gly210Arg, were previously reported by us in patients with severe factor XIII deficiency. The objective of our study was to discern the effect of all four mutations on the stability and intracellular localization of the factor XIII A-subunit by their expression in COS cells. In-vitro mutagenesis, transient expression of the mutants in COS cells and subsequent pulse-chase analyses were carried out. Intracellular localization of wild-type and mutant proteins was analyzed by immunohistochemistry using a monoclonal antibody against factor XIII A-subunit. Pulse-chase analyses of metabolically labeled proteins demonstrated rapid intracellular degradation of each mutant protein as compared with wild type. Immunocytochemical and immunofluorescence analyses disclosed that wild-type and all four mutant factor XIII A-subunit proteins were diffusely distributed within the cytoplasm but not in the endoplasmic reticulum of the COS-7 cells. The Arg260Leu, Ala318Val, Thr398Asn and Gly210Arg mutations in FXIII A-subunit cause rapid intracellular degradation of the corresponding mutated protein.
Assuntos
Deficiência do Fator XIII/genética , Fator XIII/genética , Mutação de Sentido Incorreto , Animais , Células COS , Chlorocebus aethiops , Citoplasma/genética , Citoplasma/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Fator XIII/metabolismo , Deficiência do Fator XIII/metabolismo , Expressão Gênica , Humanos , Mutagênese , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Factor XIII that stabilizes fibrin clots in the final stages of blood coagulation also participates in wound healing, as can be inferred from a delay in wound repair in some patients with inherited FXIII deficiency. In this study we evaluated the effect of FXIII on wound healing in FXIII-deficient mice. Three groups of mice (n = 10) were employed: control group, FXIII-deficient group and FXIII-deficient group treated with FXIII concentrate. Excisional wounds were left unsutured and undressed, and mice were followed for eleven days. FXIII-deficient mice exhibited impaired wound healing as has been demonstrated by 15%, 27% and 27% decrease in percentage of wound closure on day 4, 8 and 11, respectively. On day 11 complete healing was observed in control (100% closure), 73.23% in FXIII-deficient and 90.06% in FXIII deficient/FXIII-treated groups (p = 0.007 by ANOVA and p = 0.001 by t-test between control and FXIII-deficient groups). Scoring system representing maturation rate of the wounds showed that the scores for the control, FXIII-deficient and FXIII deficient/FXIII treated groups were 94.9 +/- 4.7, 61.5 +/- 14.5 and 94.5 +/- 6.4, respectively (p < 0.001 by ANOVA). Histological analysis of the lesions performed at day 11 disclosed delayed reepithelization and necrotized fissure in FXIII-deficient mice and normal healing in FXIII-deficient/FXIII-treated mice. The findings of this study confirm that in FXIII-deficient mice wound healing is delayed and the cellular and tissue defects can be corrected by treatment with FXIII, providing evidence for the essential role of FXIII in wound repair and remodeling.
Assuntos
Fator XIII/genética , Fator XIII/fisiologia , Inflamação/metabolismo , Cicatrização , Análise de Variância , Animais , Éxons , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos CBA , Fatores de TempoRESUMO
OBJECTIVE: Factor XIII (FXIII), a plasma transglutaminase that stabilizes fibrin clots at the final stages of blood coagulation by crosslinking fibrin monomers, is essential for embryo implantation and participates in tissue remodeling and wound healing, processes that involve angiogenesis. The aim of our study was to analyze the effect of FXIII on angiogenesis using in vitro and in vivo models and to examine the role of FXIII in the basic steps of angiogenesis, ie, migration, proliferation, and apoptosis/cell survival. METHODS AND RESULTS: In the Matrigel tube formation model, only FXIIIa caused a dose-dependent enhancement of array formation. This proangiogenic effect was not associated with alterations in vascular endothelial growth factor (VEGF) protein levels nor VEGF or VEGFR2 mRNA levels. FXIIIa, but not nonactivated or transglutaminase-inactivated FXIII, significantly enhanced endothelial cell migration and proliferation and inhibited apoptosis. After treatment of HUVECs with FXIIIa, almost complete disappearance of mRNA of thrombospondin 1 (TSP-1) and a marked reduction in the secretion of TSP-1 protein were observed. A reduction in TSP-1 protein synthesis, although to a lesser extent, was observed on treatment of microvascular endothelial cells with FXIIIa. In a rabbit cornea model, injection of FXIIIa caused neovascularization associated with almost complete disappearance of TSP-1 in the cornea. CONCLUSIONS: These results show that FXIIIa exhibits a novel proangiogenic activity that is associated with downregulation of TSP-1 and also involves stimulation of endothelial cell proliferation and migration and inhibition of apoptosis. These findings might shed light on the mechanism by which FXIII mediates tissue repair and remodeling.
Assuntos
Endotélio Vascular/metabolismo , Fator XIII/metabolismo , Neovascularização Fisiológica/fisiologia , Trombospondina 1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Córnea/irrigação sanguínea , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Fator XIII/farmacologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Coelhos , Trombospondina 1/biossínteseRESUMO
The aim of the study was to further investigate the role of fibrinogen-like protein 2 (FGL-2), a transmembrane prothrombinase that directly cleaves prothrombin to thrombin, in angiogenesis and tumor development and the mechanism(s) underlying these processes. To study angiogenesis HUVEC clones with decreased fgl-2 mRNA were generated by specific siRNA. To study tumorigenesis SCID mice were implanted with intact (wild type) and fgl-2-silenced PC-3 clones. IFN-γ treated HUVEC expressing increased fgl-2 mRNA exhibited significant capillary sprouting that was not inhibited by hirudin, whereas fgl-2 silencing completely inhibited blood-vessel formation. Tumors (poorly differentiated carcinoma) developed in all 12 mice injected with wild type PC-3 compared with 8/12 mice injected with the fgl-2-silenced PC-3 clone. The tumors developed by fgl-2-silenced PC-3 clones were smaller and less aggressive and contained significantly fewer blood vessels (p<0.05). All tumors' sections were negative for thrombin staining, indicating that FGL-2-induced tumorigenesis was not mediated by thrombin. In fgl-2-silenced tumors there was a decrease in fgl-2 mRNA (p=0.02) and ERK1/2 phosphorylation (p<0.05) by 80% and a 20%, respectively. The mechanism underlying these processes, studied in PC-3 clones, revealed that fgl-2 silencing was associated with a 65% decrease in FGF-2 mRNA (p<0.01) and a 30% down regulation of ERK1/2 phosphorylation (p<0.05). Together, these results suggest that FGL-2 mediates angiogenesis and tumorigenesis not by thrombin-mediated mechanism but rather through FGF-2/ERK signaling pathway. FGL-2 may serve as a valuable therapeutic target in the future.
Assuntos
Carcinogênese/metabolismo , Fibrinogênio/metabolismo , Sistema de Sinalização das MAP Quinases , Neovascularização Patológica/metabolismo , Neoplasias da Próstata/metabolismo , Tromboplastina/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células , Fibrinogênio/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos SCID , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Interferência de RNA , RNA Interferente Pequeno/genética , Trombina/metabolismo , Tromboplastina/genéticaRESUMO
OBJECTIVE: To assesses the live birth rate without treatment in women with hereditary thrombophilia who have recurrent miscarriage and women without thrombophilia who have recurrent miscarriage. DESIGN: Prospective observational study. SETTING: Tertiary referral unit in university hospital. PATIENT(S): One hundred twenty women with thrombophilia and 65 women without thrombophilia. MAIN OUTCOME MEASURE(S): Number of live births or repeated miscarriages. RESULTS: Of the 185 patients, 44 with thrombophilia and 26 without thrombophilia have conceived. Nineteen of the 44 pregnancies (43.2%) in thrombophilia patients have terminated in live births, compared with 8 of 26 pregnancies (30.8%) in patients without thrombophilia. This difference is not statistically significant. CONCLUSIONS: Hereditary thrombophilia did not seem to affect the live birth rate in women with recurrent miscarriage.