RESUMO
BACKGROUND: The nuclear transcription factor c-Jun is preferentially expressed in basal-cell carcinoma. Dz13 is a deoxyribozyme that targets JUN messenger RNA and has inhibited the growth of a range of tumours in mice. We did a phase 1 study to assess safety and tolerability in human beings. METHODS: Adults with nodular basal-cell carcinoma were recruited from Royal Prince Alfred Hospital, Sydney, Australia, between September, 2010, and October, 2011. Patients were assigned to receive one intratumoral injected dose of 10, 30, or 100 µg Dz13, in a 50 µL volume of lipid carrier, and were assessed for adverse effects in the first 24 h then at 7, 14, and 28 days after injection. Treated tumours were surgically excised 14 days after injection and compared with the baseline biopsy samples for expression of c-Jun and tumorigenesis markers. FINDINGS: Nine patients were recruited, of whom three received each dose of Dz13. All patients completed the study with no drug-related serious adverse events. No systemic Dz13 exposure was detected. c-Jun expression was reduced in the excised tumours of all nine (100%) patients, compared with baseline, and histological tumour depth had decreased in five (56%) of nine. Proportions of cells positive for caspases 3, 8, and 9 and P53 were increased, but those of cells positive for Bcl-2 and MMP-9 were decreased. Infiltration by inflammatory and immune cells was stimulated. INTERPRETATION: Dz13 was safe and well tolerated after single intratumoral injections at all doses. FUNDING: Cancer Institute NSW, Cancer Council Australia, and National Health and Medical Research Council.
Assuntos
Antineoplásicos/administração & dosagem , Carcinoma Basocelular/tratamento farmacológico , DNA Catalítico/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patologia , DNA Catalítico/efeitos adversos , DNA Catalítico/farmacocinética , Feminino , Humanos , Injeções Intralesionais , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Resultado do TratamentoRESUMO
Current understanding of key transcription factors regulating angiogenesis is limited. Here we show that RNA-cleaving phosphodiester-linked DNA-based enzymes (DNAzymes), targeting a specific motif in the 5' untranslated region of early growth response (Egr-1) mRNA, inhibit Egr-1 protein expression, microvascular endothelial cell replication and migration, and microtubule network formation on basement membrane matrices. Egr-1 DNAzymes blocked angiogenesis in subcutaneous Matrigel plugs in mice, an observation that was independently confirmed by plug analysis in Egr-1-deficient animals, and inhibited MCF-7 human breast carcinoma growth in nude mice. Egr-1 DNAzymes suppressed tumor growth without influencing body weight, wound healing, blood coagulation or other hematological parameters. These agents inhibited endothelial expression of fibroblast growth factor (FGF)-2, a proangiogenic factor downstream of Egr-1, but not that of vascular endothelial growth factor (VEGF). Egr-1 DNAzymes also repressed neovascularization of rat cornea. Thus, microvascular endothelial cell growth, neovascularization, tumor angiogenesis and tumor growth are processes that are critically dependent on Egr-1.
Assuntos
DNA Catalítico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas Imediatamente Precoces , Transplante de Neoplasias/patologia , Neoplasias Experimentais/patologia , Neovascularização Patológica , Neovascularização Fisiológica , Fatores de Transcrição/metabolismo , Animais , Neoplasias da Mama , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/citologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfocinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Microtúbulos/metabolismo , Neoplasias Experimentais/irrigação sanguínea , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/genética , Transplante Heterólogo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
OBJECTIVE: Coronary reperfusion has been the mainstay therapy for reduced infarct size after a heart attack. However, this intervention also results in myocardial injury by initiating a marked inflammatory reaction, and new treatments are keenly sought. METHODS AND RESULTS: The basic-region leucine zipper protein, c-Jun is poorly expressed in the normal myocardium and is induced within 24 hours after myocardial ischemia-reperfusion injury. Synthetic catalytic DNA molecules (DNAzymes) targeting c-Jun (Dz13) reduce infarct size in the area-at-risk (AAR) regardless of whether it is delivered intramyocardially at the initiation of ischemia or at the time of reperfusion. Dz13 attenuates neutrophil infiltration, c-Jun and ICAM-1 expression in vascular endothelium, cardiomyocyte apoptosis, and the generation of reactive oxygen species in the reperfused myocardium. It inhibits infiltration into the AAR of complement 3 (C3), C3a receptor (C3aR), membrane attack complex-1 (Mac-1), or matrix metalloproteinase-2 (MMP-2) positive inflammatory cells. Dz13 also improves cardiac function without influencing myocardial vascularity or fibrosis. CONCLUSIONS: These findings demonstrate the regulatory role of c-Jun in the pathogenesis of myocardial inflammation and infarction following ischemia-reperfusion injury, and inhibition of this process using catalytic DNA.
Assuntos
DNA Catalítico/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Infarto do Miocárdio/enzimologia , Miocardite/patologia , Miocardite/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/complicações , Animais , Apoptose/fisiologia , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Modelos Animais de Doenças , Testes de Função Cardíaca , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Proteínas Quinases JNK Ativadas por Mitógeno/farmacologia , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/patologia , Miocardite/complicações , Miocardite/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Probabilidade , Distribuição Aleatória , Valores de Referência , Traumatismo por Reperfusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Vascular injury initiates a cascade of phenotype-altering molecular events. Transcription factor function in this process, particularly that of negative regulators, is poorly understood. We demonstrate here that the forced expression of the injury-inducible GLI-Krüppel zinc finger protein Yin Yang-1 (YY1) inhibits neointima formation in human, rabbit and rat blood vessels. YY1 inhibits p21(WAF1/Cip1) transcription, prevents assembly of a p21(WAF1/Cip1)-cdk4-cyclin D1 complex, and blocks downstream pRb(Ser249/Thr252) phosphorylation and expression of PCNA and TK-1. Conversely, suppression of endogenous YY1 elevates levels of p21(WAF1/Cip1), PCNA, pRb(Ser249/Thr252) and TK-1, and increases intimal thickening. YY1 binds Sp1 and prevents its occupancy of a distinct element in the p21(WAF1/Cip1) promoter without YY1 itself binding the promoter. Additionally, YY1 induces ubiquitination and proteasome-dependent degradation of p53, decreasing p53 immunoreactivity in the artery wall. These findings define a new role for YY1 as both an inducer of p53 instability in smooth muscle cells, and an indirect repressor of p21(WAF1/Cip1) transcription, p21(WAF1/Cip1)-cdk4-cyclin D1 assembly and intimal thickening.
Assuntos
Quinase 4 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ciclinas/metabolismo , Complexos Multiproteicos/metabolismo , Miócitos de Músculo Liso/metabolismo , Túnica Íntima/crescimento & desenvolvimento , Fator de Transcrição YY1/metabolismo , Animais , Artérias/citologia , Artérias/crescimento & desenvolvimento , Linhagem Celular , Ciclina D , Quinase 4 Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Ciclinas/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Complexos Multiproteicos/genética , Miócitos de Músculo Liso/citologia , Antígeno Nuclear de Célula em Proliferação/biossíntese , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica/fisiologia , Coelhos , Ratos , Elementos de Resposta/fisiologia , Proteína do Retinoblastoma/biossíntese , Proteína do Retinoblastoma/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Timidina Quinase/biossíntese , Timidina Quinase/genética , Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Túnica Íntima/citologia , Fator de Transcrição YY1/genéticaRESUMO
Conventional anti-inflammatory strategies induce multiple side effects, highlighting the need for novel targeted therapies. Here we show that knockdown of the basic-region leucine zipper protein, c-Jun, by a catalytic DNA molecule, Dz13, suppresses vascular permeability and transendothelial emigration of leukocytes in murine models of vascular permeability, inflammation, acute inflammation and rheumatoid arthritis. Treatment with Dz13 reduced vascular permeability due to cutaneous anaphylactic challenge or VEGF administration in mice. Dz13 also abrogated monocyte-endothelial cell adhesion in vitro and abolished leukocyte rolling, adhesion and extravasation in a rat model of inflammation. Dz13 suppressed neutrophil infiltration in the lungs of mice challenged with endotoxin, a model of acute inflammation. Finally, Dz13 reduced joint swelling, inflammatory cell infiltration and bone erosion in a mouse model of rheumatoid arthritis. Mechanistic studies showed that Dz13 blocks cytokine-inducible endothelial c-Jun, E-selectin, ICAM-1, VCAM-1 and VE-cadherin expression but has no effect on JAM-1, PECAM-1, p-JNK-1 or c-Fos. These findings implicate c-Jun as a useful target for anti-inflammatory therapies.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , DNA Catalítico/farmacologia , Proteínas Proto-Oncogênicas c-jun/efeitos dos fármacos , Animais , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Linhagem Celular , Técnicas de Cocultura/métodos , Células Endoteliais/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Microscopia de Fluorescência , Monócitos , Proteínas Proto-Oncogênicas c-jun/genética , RatosRESUMO
Platelet and leucocyte activity are important in the acute development of thrombosis and in the pathogenesis of ischaemic vascular disease. Dan Shen Di Wan (DS, Cardiotonic Pill or Dantonic(R) Pill) is one of the most commonly used Chinese herbal formulations for treating patients with atherosclerotic disease in China and several Asian countries. We studied the effect of DS on platelet and leucocyte function and compared the effects with conventional antiplatelet agents, cangrelor (ADP P2Y(12) receptor antagonist) and aspirin (acetyl salicylic acid, ASA). Measurements were made by platelet aggregation (%) and activation (CD62P %), platelet-monocyte conjugate formation (P/M, CD42a median fluorescence, mf), platelet-neutrophil conjugate formation (P/N, mf), and leucocyte activation (CD11b median fluorescence on monocytes and neutrophils, mf) in response to 3.3 micromol/L adenosine diphosphate (ADP), 1.0 micromol/L platelet activating factor (PAF), 5.0 micromol/L adrenaline and 0.5 microg/mL collagen. We also evaluated the effect of its main component, water soluble extract of salvia miltiorrhiza (SME) on intracellular calcium mobilization in platelets triggered by 10 micromol/L ADP, 10 micromol/L PAF, 2 microg/mL collagen and 15 micromol/L thrombin receptor activating peptide (TRAP). Overall DS showed inhibition of platelet aggregation, platelet activation, platelet-leucocyte conjugate formation and leucocyte activation in response to all the agonists apart from adrenaline (all p < 0.01). DS showed inhibition of platelet aggregation and leucocyte activation equivalent to cangrelor 100 nmol/L and ASA 100 micromol/L. SME dose-dependently inhibited intracellular calcium mobilization in platelets following stimulation with all the platelet agonists with maximum effective at 0.36 mg/mL (all p < 0.01). When used at 0.18 mg/mL its inhibitory effect was equivalent to cangrelor and ASA. We conclude that DS is a potential inhibitor of both platelet and leucocyte activation.
Assuntos
Antioxidantes/farmacologia , Plaquetas/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Leucócitos/metabolismo , Extratos Vegetais/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Antioxidantes/uso terapêutico , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/uso terapêutico , Feminino , Humanos , Isquemia/tratamento farmacológico , Isquemia/metabolismo , Masculino , Extratos Vegetais/uso terapêutico , Inibidores da Agregação Plaquetária/uso terapêutico , Salvia miltiorrhiza , Trombose/tratamento farmacológico , Trombose/metabolismoRESUMO
Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (Ki 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.
Assuntos
Angioplastia com Balão/efeitos adversos , Músculo Liso Vascular/efeitos dos fármacos , Oligossacarídeos/farmacologia , Túnica Íntima/efeitos dos fármacos , Animais , Ligação Competitiva , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/etiologia , Lesões das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/prevenção & controle , Divisão Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Oligossacarídeos/metabolismo , Coelhos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Túnica Média/efeitos dos fármacos , Túnica Média/metabolismo , Túnica Média/patologia , Tempo de Coagulação do Sangue TotalRESUMO
Animal models of neointima (NI) formation have proven useful in gaining insights into the mechanisms of restenosis after coronary angioplasty and stenting, but the events at a molecular level remain incompletely understood. Here, we describe a technically straightforward, rat model of NI formation, involving complete ligation of the common carotid artery and demonstrate the importance of the immediate-early gene and zinc finger transcription factor Egr-1 in this process. Acute cessation of common carotid blood flow by vessel ligation, was followed by the expression of Egr-1 in the arterial media within 3 h and NI formation proximal to the point of ligation at 18 days. Local delivery of catalytic oligodeoxynucleotides (ODN) targeting rat Egr-1 mRNA at the time of ligation reduced both Egr-1 expression and NI formation in this model. In contrast, a scrambled version of this ODN had no inhibitory effect. These studies demonstrate for the first time that arterial intimal thickening following artery ligation is critically-dependent on the activation of Egr-1.
Assuntos
Estenose das Carótidas/metabolismo , DNA Antissenso/farmacologia , DNA Catalítico/farmacologia , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Imediatamente Precoces , Fatores de Transcrição/fisiologia , Túnica Íntima/patologia , Animais , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Estenose das Carótidas/etiologia , Estenose das Carótidas/genética , Estenose das Carótidas/patologia , DNA Antissenso/administração & dosagem , DNA Catalítico/administração & dosagem , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Genes Precoces/efeitos dos fármacos , Injeções Intra-Arteriais , Ligadura , Masculino , Modelos Animais , RNA Mensageiro/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Reviewing advances in cardiology and haematology together may appear at first sight to require some artificiality to make a satisfying fit. For two reasons, at least, this is not the case. Firstly, convergence in biology has become very clear over the past decade and this could not be better illustrated by the demonstration that the haemangioblast is the common progenitor of both haemapoietic stem cells and vascular endothelium. This opens the way to common (and differential) approaches to the manipulation of these cells, a field at present in its infancy. A second convergence is the common goal of understanding the processes resulting in haemostasis, thrombosis and vascular occlusion and the means for developing effective antithrombotics. This is exemplified by a number of agents either in use or in clinical trial as a result of haematological and cardiological collaboration. This collaboration is recognisable with the development, many years ago, of streptokinase and the use of aspirin in vascular disease and continues to this day with specific antiplatelet inhibitors and oral thrombin inhibitors as they become accepted into clinical use over the next few years. Here we review current advances in pharmacological treatments in cardiology and haematology, grouped primarily by disease process, focusing on novel and emerging therapies likely to be of importance in the future.
Assuntos
Fármacos Cardiovasculares/uso terapêutico , Cardiopatias/tratamento farmacológico , Cardiopatias/terapia , Doenças Hematológicas/tratamento farmacológico , Angioplastia Coronária com Balão/efeitos adversos , Animais , Doença das Coronárias/tratamento farmacológico , Cardiopatias/prevenção & controle , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Infarto do Miocárdio/tratamento farmacológico , Transplante de Células-Tronco , Trombose/prevenção & controleRESUMO
Worldwide, one in three cancers is skin-related, with increasing incidence in many populations. Here, we demonstrate the capacity of a DNAzyme-targeting c-jun mRNA, Dz13, to inhibit growth of two common skin cancer types-basal cell and squamous cell carcinomas-in a therapeutic setting with established tumors. Dz13 inhibited tumor growth in both immunodeficient and immunocompetent syngeneic mice and reduced lung nodule formation in a model of metastasis. In addition, Dz13 suppressed neovascularization in tumor-bearing mice and zebrafish and increased apoptosis of tumor cells. Dz13 inhibition of tumor growth, which required an intact catalytic domain, was due in part to the induction of tumor immunity. In a series of good laboratory practice-compliant toxicology studies in cynomolgus monkeys, minipigs, and rodents, the DNAzyme was found to be safe and well tolerated. It also did not interfere in more than 70 physiologically relevant in vitro bioassays, suggesting a reduced propensity for off-target effects. If these findings hold true in clinical trials, Dz13 may provide a safe, effective therapy for human skin cancer.
Assuntos
DNA Catalítico/uso terapêutico , Proteínas Proto-Oncogênicas c-jun/genética , Neoplasias Cutâneas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , DNA Catalítico/farmacologia , Relação Dose-Resposta a Droga , Humanos , Imunidade Celular/efeitos dos fármacos , Camundongos , Peixe-ZebraRESUMO
The past decade has seen the rapid evolution of small-molecule gene-silencing strategies, driven largely by enhanced understanding of gene function in the pathogenesis of disease. Over this time, many genes have been targeted by specifically engineered agents from different classes of nucleic acid-based drugs in experimental models of disease to probe, dissect, and characterize further the complex processes that underpin molecular signaling. Arising from this, a number of molecules have been examined in the setting of clinical trials, and several have recently made the successful transition from the bench to the clinic, heralding an exciting era of gene-specific treatments. This is particularly important because clear inadequacies in present therapies account for significant morbidity, mortality, and cost. The broad umbrella of gene-silencing therapeutics encompasses a range of agents that include DNA enzymes, short interfering RNA, antisense oligonucleotides, decoys, ribozymes, and aptamers. This review tracks current movements in these technologies, focusing mainly on DNA enzymes and short interfering RNA, because these are poised to play an integral role in antigene therapies in the future.
Assuntos
DNA Catalítico/química , DNA Catalítico/uso terapêutico , Inativação Gênica , Terapia Genética/métodos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/uso terapêutico , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/uso terapêutico , DNA Catalítico/metabolismo , Marcação de Genes , Humanos , Camundongos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/uso terapêutico , RNA Catalítico/química , RNA Catalítico/uso terapêutico , RNA Interferente Pequeno/metabolismo , Coelhos , RatosRESUMO
The amino acid sequence of the subunit of human platelet factor 4 has been determined. Human platelet factor 4 consists of identical subunits containing 70 amino acids, each with a molecular weight of 7,756. The molecule contains no methionine, phenylalanine or tryptophan. The proposed amino acid sequence of PF4 is: Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-Ser- Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gin- Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Lys- Ile-Ile-Lys-Lys-Leu-Leu-Glu-Ser. From consideration of the homology with p-thromboglobulin, disulphide bonds between residues 10 and 36 and between residues 12 and 52 can be inferred.
RESUMO
The recent discovery of the von Willebrand Factor (vWF) cleaving protease (ADAMTS13) and the association of its deficiency with thrombotic thrombocytopaenic purpura (TTP) has generated both enormous interest and considerable confusion. Ultra large von Willebrand Factor (UL vWF) multimers are present in the plasmas of patients with chronic relapsing TTP in remission but disappear during an attack. This observation led to the recognition that UL vWF multimers precipitate the thrombotic occlusion of arterioles, a feature that characterizes TTP. Multiple mutations in ADAMTS13 are associated with congenital TTP and neutralizing autoantibodies have been demonstrated in the acquired TTP syndrome. Although a number of functional assays for this enzyme have been described, the more rigorously evaluated assays are difficult to perform outside a research laboratory. There is also an enduring uncertainty about the specificity of ADAMTS13 deficiency for the diagnosis of acquired TTP and a perception that the result does not alter patient management. The cloning of the ADAMTS13 gene has also raised the prospect of recombinant enzyme therapy for the treatment of TTP, and this has heightened the need for a simple assay. In this review, we evaluate the value of measuring this enzyme in the management of TTP.
Assuntos
Eletroforese das Proteínas Sanguíneas , Ensaios Enzimáticos Clínicos/métodos , Metaloendopeptidases/sangue , Agregação Plaquetária , Púrpura Trombocitopênica Trombótica/diagnóstico , Fator de von Willebrand/análise , Proteínas ADAM , Proteína ADAMTS13 , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Compostos de Bário/farmacologia , Biopolímeros , Cloreto de Cálcio/farmacologia , Administração de Caso , Cloretos/farmacologia , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Hemorreologia , Humanos , Metaloendopeptidases/química , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Metaloendopeptidases/imunologia , Microscopia de Vídeo , Mutação de Sentido Incorreto , Fragmentos de Peptídeos/sangue , Púrpura Trombocitopênica Trombótica/genética , Púrpura Trombocitopênica Trombótica/imunologia , Púrpura Trombocitopênica Trombótica/terapia , Recidiva , Fator de von Willebrand/metabolismoRESUMO
Plasma von Willebrand factor (VWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury; however, only the very large VWF multimers are effective in promoting platelet adhesion in flowing blood. The multimeric size of VWF can be controlled by the glycoprotein, thrombospondin-1 (TSP-1), which facilitates reduction of the disulfide bonds that hold VWF multimers together. The TSP family of extracellular glycoproteins consists of 5 members in vertebrates, TSP-1 through TSP-4 and TSP-5/COMP. TSP-1 and TSP-2 are structurally similar trimeric proteins composed of disulfide-linked 150-kDa monomers. Recombinant pieces of TSP-1 and TSP-2 incorporating combinations of domains that span the entire subunit were produced in insect cells and examined for VWF reductase activity. VWF reductase activity was present in the Ca(++)-binding repeats and C-terminal sequence of TSP-1, but not of TSP-2. Alkylation of Cys974 in the C-terminal TSP-1 construct, which is a serine in TSP-2, ablated VWF reductase activity. These results imply that the reductase function of TSP-1 centers around Cys974 in the C-terminal sequence.
Assuntos
Trombospondina 1/metabolismo , Trombospondinas/metabolismo , Fator de von Willebrand/metabolismo , Alquilação , Sítios de Ligação , Humanos , Maleimidas/farmacologia , Modelos Moleculares , Oxirredução , Fragmentos de Peptídeos/química , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/metabolismo , Compostos de Sulfidrila/metabolismo , Trombospondina 1/químicaRESUMO
Severe deficiency of the von Willebrand Factor (VWF)-cleaving proteinase, ADAMTS13, is associated with the development of thrombotic thrombocytopenic purpura (TTP). Several mutations spread across the ADAMTS13 gene have been identified in association with a deficiency of VWF-cleaving proteinase activity in patients with congenital TTP. The spread of these dysfunctional mutations and the domain structure of ADAMTS13 are suggestive of a complex interaction between the enzyme and its substrate. We have studied a patient with congenital TTP who is a compound heterozygote for the Thr196Ile mutation in the metalloproteinase domain and a frameshift mutation (4143-4144insA) in the second CUB domain that results in loss of the last 49 amino acids of the protein. The VWF-cleaving proteinase activity of the truncated enzyme was comparable to that of the wild-type enzyme but its secretion from transfected COS-7 cells was about 14% of the wild type.
Assuntos
Mutação da Fase de Leitura , Metaloendopeptidases/genética , Púrpura Trombocitopênica Trombótica/congênito , Púrpura Trombocitopênica Trombótica/genética , Proteínas ADAM , Proteína ADAMTS13 , Animais , Sequência de Bases , Sítios de Ligação , Células COS , Chlorocebus aethiops , Humanos , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Modelos Moleculares , Polimorfismo de Nucleotídeo Único/genética , Conformação Proteica , Transfecção , Fator de von Willebrand/genéticaRESUMO
Plasma von Willebrand factor (VWF) is a multimeric glycoprotein from endothelial cells and platelets that mediates adhesion of platelets to sites of vascular injury. In the shear force of flowing blood, however, only the very large VWF multimers are effective in capturing platelets. The multimeric size of VWF can be controlled by proteolysis at the Tyr(842)-Met(843) peptide bond by ADAMTS13 or cleavage of the disulfide bonds that hold VWF multimers together by thrombospondin-1 (TSP-1). The average multimer size of plasma VWF in TSP-1 null mice was significantly smaller than in wild type mice. In addition, the multimer size of VWF released from endothelium in vivo was reduced more rapidly in TSP-1 null mice than in wild type mice. TSP-1, like ADAMTS13, bound to the VWF A3 domain. TSP-1 in the wild type mice, therefore, may compete with ADAMTS13 for interaction with the A3 domain and slow the rate of VWF proteolysis. TSP-1 is stored in platelet alpha-granules and is released upon platelet activation. Significantly, platelet VWF multimer size was reduced upon lysis or activation of wild type murine platelets but not TSP-1 null platelets. This difference had functional consequences in that there was an increase in collagen- and VWF-mediated aggregation of the TSP-1 null platelets under both static and shear conditions. These findings indicate that TSP-1 influences plasma and platelet VWF multimeric size differently and may be more relevant for control of the VWF released from platelets.