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1.
Blood ; 128(1): 110-9, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27207796

RESUMO

Infection or inflammation may precede and trigger formation of microvascular thrombosis in patients with acquired thrombotic thrombocytopenic purpura (TTP). However, the mechanism underlying this clinical observation is not fully understood. Here, we show that human neutrophil peptides (HNPs) released from activated and degranulated neutrophils inhibit proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 in a concentration-dependent manner. Half-maximal inhibitory concentrations of native HNPs toward ADAMTS13-mediated proteolysis of peptidyl VWF73 and multimeric VWF are 3.5 µM and 45 µM, respectively. Inhibitory activity of HNPs depends on the RRY motif that is shared by the spacer domain of ADAMTS13. Native HNPs bind to VWF73 (KD = 0.72 µM), soluble VWF (KD = 0.58 µM), and ultra-large VWF on endothelial cells. Enzyme-linked immunosorbent assay (ELISA) demonstrates markedly increased plasma HNPs1-3 in most patients with acquired autoimmune TTP at presentation (median, ∼170 ng/mL; range, 58-3570; n = 19) compared with healthy controls (median, ∼23 ng/mL; range, 6-44; n = 18) (P < .0001). Liquid chromatography plus tandem mass spectrometry (LC-MS/MS) reveals statistically significant increases of HNP1, HNP2, and HNP3 in patient samples (all P values <.001). There is a good correlation between measurement of HNPs1-3 by ELISA and by LC-MS/MS (Spearman ρ = 0.7932, P < .0001). Together, these results demonstrate that HNPs1-3 may be potent inhibitors of ADAMTS13 activity, likely by binding to the central A2 domain of VWF and physically blocking ADAMTS13 binding. Our findings may provide a novel link between inflammation/infection and the onset of microvascular thrombosis in acquired TTP and potentially other immune thrombotic disorders.


Assuntos
Proteína ADAMTS13/metabolismo , Defensinas/metabolismo , Neutrófilos/metabolismo , Proteólise , Púrpura Trombocitopênica Trombótica/metabolismo , Fator de von Willebrand/metabolismo , Motivos de Aminoácidos , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Neutrófilos/patologia , Púrpura Trombocitopênica Trombótica/patologia
2.
J Thromb Haemost ; 17(4): 596-606, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30741476

RESUMO

Essentials Biological activity of human neutrophil peptide (HNP)-1 in hemostasis under physiological conditions is not fully understood. HNP-1 inhibits the adhesion/aggregation of murine platelets on a fibrillar collagen surface or an activated endothelial cell surface under flow. The anti-adhesion activity appears to depend on the terminal free thiols of HNP-1, which may inhibit VWF-VWF lateral associations. Our results suggest a protective role and potential novel therapeutic use of HNP-1 for arterial thrombosis. SUMMARY: Background Human neutrophil peptides (HNPs), also known as α-defensins, are released from degranulated neutrophils and play an important role in innate immunity. However, their biological roles in hemostasis under flow are not fully explored. Objective This study aims to determine the role of HNP-1 on platelet adhesion and aggregation on a collagen surface or ultra large von Willebrand factor (ULVWF) on endothelium under flow and elucidate the structural elements required for its activity. Methods Anticoagulated whole blood from wild-type or Adamts13-/- mice was incubated with a fluorescein-conjugated anti-human CD41 in the presence of increasing concentrations of a synthetic HNP-1 and perfused over a collagen surface or a tumor necrosis factor (TNF)-α activated murine endothelial cell surface under arterial flow. The rate of accumulation and the final surface coverage of fluoresceinated murine platelets or the rate of forming platelet-decorated ULVWF strings were determined using the BioFlux microfluidic system. Results HNP-1 inhibited the rate and final coverage of fluorescein-labeled murine platelets on a fibrillar collagen surface under flow (100 dyne/cm2 ) in a concentration-dependent manner and the anti-adhesive activity of HNP-1 depended on its terminal free cysteine thiols. HNP-1 (20 µM) also dramatically inhibited the formation of platelets-decorated ULVWF strings on TNF-α activated murine endothelial surface under arterial flow. Conclusions Our results demonstrate for the first time an antiplatelet adhesion or antithrombotic activity of HNP-1; this activity depends on its terminal free thiols, likely affecting VWF-VWF lateral associations. These findings may suggest a potential novel therapeutic strategy for arterial thrombosis.


Assuntos
Coagulação Sanguínea , Plaquetas/metabolismo , Cisteína/metabolismo , Compostos de Sulfidrila/metabolismo , Trombose/metabolismo , alfa-Defensinas/metabolismo , Proteína ADAMTS13/deficiência , Proteína ADAMTS13/genética , Animais , Linhagem Celular , Colágeno/metabolismo , Células Endoteliais/metabolismo , Humanos , Camundongos Knockout , Adesividade Plaquetária , Agregação Plaquetária , Fluxo Sanguíneo Regional , Trombose/sangue , Fator de von Willebrand/metabolismo
3.
Thromb Haemost ; 118(4): 676-687, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29618154

RESUMO

Decrease of plasma activity of ADAMTS13, a metalloenzyme that cleaves von Willebrand factor (VWF) and prevents adhesion and aggregation of platelets, has been reported early after onset of systemic inflammation resulting from infections and after severe trauma. Here, we determined whether trauma-induced systemic (sterile) inflammation would be associated with a reduction of plasma ADAMTS13 activity in paediatric patients and its association with disease severity and outcome. Paediatric patients (n = 106) with severe trauma at a level 1 paediatric trauma centre between 2014 and 2016 were prospectively enrolled. Blood samples were collected upon arrival and at 24 hours and analysed for plasma levels of ADAMTS13 activity, VWF antigen, collagen binding activity, human neutrophil peptides (HNP) 1-3, coagulation abnormalities, endothelial glycocalyx damage and clinical outcome. Plasma samples were also collected for similar measurements from 52 healthy paediatric controls who underwent elective minor surgery. The median age of patients was 9 years with 81% sustaining blunt trauma. The median injury severity score was 22 and the mortality rate was 11%. Plasma levels of ADAMTS13 activity were significantly lower and plasma levels of VWF antigen and HNP 1-3 proteins were significantly higher for paediatric trauma patients on admission and at 24 hours when compared with controls. Finally, the lowest plasma ADAMTS13 activity was found in patients who died from their injuries. We conclude that relative plasma deficiency of ADAMTS13 activity may be associated with more severe traumatic injury, significant endothelial glycocalyx damage, coagulation abnormalities and mortality after severe trauma in paediatric patients.


Assuntos
Proteína ADAMTS13/sangue , Transtornos da Coagulação Sanguínea/complicações , Coagulação Sanguínea , Células Endoteliais/metabolismo , Ferimentos e Lesões/complicações , Adolescente , Testes de Coagulação Sanguínea , Plaquetas/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Colágeno/química , Feminino , Glicocálix/química , Humanos , Lactente , Inflamação , Masculino , Plasma/metabolismo , Estudos Prospectivos , Índice de Gravidade de Doença , Sindecana-1/sangue , Resultado do Tratamento , alfa-Defensinas/sangue , Fator de von Willebrand/análise
4.
Thromb Haemost ; 117(4): 691-699, 2017 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-28102428

RESUMO

Increased von Willebrand factor (VWF) and reduced ADAMTS13 activity are associated with arterial thrombosis. This may also be the culprit mechanism implicated in delayed cerebral ischaemia after aneurysmal subarachnoid haemorrhage (SAH). It was our objective to determine plasma VWF and ADAMTS13 in patients with SAH and healthy subjects; and to explore the levels of those markers and outcome after SAH. Forty consecutive patients were enrolled between September 2007 and April 2014 in a pilot study. Plasma samples were collected from SAH patients on post-bleed day (PBD) 0, 1, 3, 5, 7 and 10 and healthy controls. VWF antigen (VWFAg) and VWF activity (VWFAc) were determined by enzyme-linked immunoassay and collagen binding assay, respectively. ADAMTS13 activity was determined by the cleavage of a fluorescent substrate. Univariate descriptive statistics and cluster analyses were performed based on outcomes in the group with SAH only. Mean age of SAH patients was 52.4 years (26-84 years) and 30 (75 %) were women. 12/40 (30 %) had a high Hunt and Hess grade (IV-V) and 25 (62.5 %) were treated with coil embolisation. Plasma VWFAg and VWFAc were significantly higher in SAH patients than those in healthy subjects on each PBD (p<0.0001). Concurrently, plasma ADAMTS13 activity in SAH patients was significantly lower than that in healthy subjects (p<0.0001). Among those with SAH, cluster analysis demonstrated that patients with higher VWFAg and VWFAc and/or lower ADAMTS13 activity might be at risk of increased mortality. In conclusion, the relative deficiency of plasma ADAMTS13 activity in SAH patients may associate with worse outcome.


Assuntos
Proteína ADAMTS13/sangue , Hemorragia Subaracnóidea/sangue , Fator de von Willebrand/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Coagulação Sanguínea , Estudos de Casos e Controles , Análise por Conglomerados , Embolização Terapêutica/instrumentação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos Prospectivos , Hemorragia Subaracnóidea/enzimologia , Hemorragia Subaracnóidea/mortalidade , Hemorragia Subaracnóidea/terapia , Fatores de Tempo , Resultado do Tratamento
5.
Pharmaceuticals (Basel) ; 9(2)2016 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-27213405

RESUMO

Pediatric Non-Hodgkin Lymphomas (NHL) are a diverse group of malignancies and as such treatment can vary based on the different biological characteristics of each malignancy. Significant advancements are being made in the treatment and outcomes of this group of malignancies. This is in large part due to novel targeted drug therapies that are being used in combination with traditional chemotherapy. Here, we discuss several new lines of therapy that are being developed or are in current use for pediatric patients with NHL.

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