RESUMO
Genome-wide association studies linked expression of the human neutrophil antigen 3b (HNA-3b) epitope on the Slc44a2 protein with a 30% decreased risk of venous thrombosis (VT) in humans. Slc44a2 is a ubiquitous transmembrane protein identified as a receptor for von Willebrand factor (VWF). To explain the link between Slc44a2 and VT, we wanted to determine how Slc44a2 expressing either HNA-3a or HNA-3b on neutrophils could modulate their adhesion and activation on VWF under flow. Transfected HEK293T cells or neutrophils homozygous for the HNA-3a- or HNA-3b-coding allele were purified from healthy donors and perfused in flow chambers coated with VWF at venous shear rates (100 s-1). HNA-3a expression was required for Slc44a2-mediated neutrophil adhesion to VWF at 100 s-1. This adhesion could occur independently of ß2 integrin and was enhanced when neutrophils were preactivated with lipopolysaccharide. Moreover, specific shear conditions with high neutrophil concentration could act as a "second hit," inducing the formation of neutrophil extracellular traps. Neutrophil mobilization was also measured by intravital microscopy in venules from SLC44A2-knockout and wild-type mice after histamine-induced endothelial degranulation. Mice lacking Slc44a2 showed a massive reduction in neutrophil recruitment in inflamed mesenteric venules. Our results show that Slc44a2/HNA-3a is important for the adhesion and activation of neutrophils in veins under inflammation and when submitted to specific shears. The fact that neutrophils expressing Slc44a2/HNA-3b have a different response on VWF in the conditions tested could thus explain the association between HNA-3b and a reduced risk for VT in humans.
Assuntos
Isoantígenos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Neutrófilos/citologia , Fator de von Willebrand/metabolismo , Animais , Circulação Sanguínea , Adesão Celular , Células Cultivadas , Armadilhas Extracelulares/genética , Armadilhas Extracelulares/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Isoantígenos/genética , Masculino , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Trombose Venosa/genética , Trombose Venosa/metabolismoRESUMO
Von Willebrand factor (VWF) modulates factor VIII (FVIII) clearance and the anti-FVIII immune response. Despite the high affinity that defines the FVIII/VWF interaction, association/dissociation kinetics dictates 2% to 5% FVIII being present as free protein. To avoid free FVIII when studying the FVIII-VWF complex in vivo, we designed a FVIII-nanobody fusion protein, with the nanobody part being directed against VWF. This fusion protein, designated FVIII-KB013bv, had a 25-fold higher affinity compared with B-domainless FVIII (BDD-FVIII) for VWF. In vitro analysis revealed full cofactor activity in 1-stage clotting and chromogenic assays (activity/antigen ratio 1.0 ± 0.3 and 1.1 ± 0.3, respectively). In vivo, FVIII-013bv displayed a twofold increased mean residence time compared with BDD-FVIII (3.0 hours vs 1.6 hours). In a tail clip-bleeding assay performed 24 hours after FVIII infusion, blood loss was significantly reduced in mice receiving FVIII-KB013bv vs BDD-FVIII (15 ± 7 µL vs 194 ± 146 µL; P = .0043). Unexpectedly, when examining anti-FVIII antibody formation in FVIII-deficient mice, the immune-response toward FVIII-KB013bv was significantly reduced compared with BDD-FVIII (1/8 vs 14/16 mice produced anti-FVIII antibodies after treatment with FVIII-KB013bv and BDD-FVIII, respectively). Our data show that a stabilized interaction between FVIII and VWF is associated with a prolonged survival of FVIII and a reduced immune response against FVIII.
Assuntos
Formação de Anticorpos/efeitos dos fármacos , Autoanticorpos , Fator VIII , Proteínas Recombinantes de Fusão , Anticorpos de Domínio Único/farmacologia , Fator de von Willebrand , Animais , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Fator VIII/imunologia , Fator VIII/farmacocinética , Fator VIII/farmacologia , Camundongos , Camundongos Mutantes , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Fator de von Willebrand/imunologia , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Postprocedural aortic regurgitation occurs in 10 to 20% of patients undergoing transcatheter aortic-valve replacement (TAVR) for aortic stenosis. We hypothesized that assessment of defects in high-molecular-weight (HMW) multimers of von Willebrand factor or point-of-care assessment of hemostasis could be used to monitor aortic regurgitation during TAVR. METHODS: We enrolled 183 patients undergoing TAVR. Patients with aortic regurgitation after the initial implantation, as identified by means of transesophageal echocardiography, underwent additional balloon dilation to correct aortic regurgitation. HMW multimers and the closure time with adenosine diphosphate (CT-ADP), a point-of-care measure of hemostasis, were assessed at baseline and 5 minutes after each step of the procedure. Mortality was evaluated at 1 year. A second cohort (201 patients) was studied to validate the use of CT-ADP in order to identify patients with aortic regurgitation. RESULTS: After the initial implantation, HMW multimers normalized in patients without aortic regurgitation (137 patients). Among the 46 patients with aortic regurgitation, normalization occurred in 20 patients in whom additional balloon dilation was successful but did not occur in the 26 patients with persistent aortic regurgitation. A similar sequence of changes was observed with CT-ADP. A CT-ADP value of more than 180 seconds had sensitivity, specificity, and negative predictive value of 92.3%, 92.4%, and 98.6%, respectively, for aortic regurgitation, with similar results in the validation cohort. Multivariable analyses showed that the values for HMW multimers and CT-ADP at the end of TAVR were each associated with mortality at 1 year. CONCLUSIONS: The presence of HMW-multimer defects and a high value for a point-of-care hemostatic test, the CT-ADP, were each predictive of the presence of aortic regurgitation after TAVR and were associated with higher mortality 1 year after the procedure. (Funded by Lille 2 University and others; ClinicalTrials.gov number, NCT02628509.).
Assuntos
Difosfato de Adenosina/sangue , Insuficiência da Valva Aórtica/diagnóstico , Estenose da Valva Aórtica/cirurgia , Complicações Pós-Operatórias/diagnóstico , Substituição da Valva Aórtica Transcateter , Fator de von Willebrand/análise , Idoso , Idoso de 80 Anos ou mais , Valva Aórtica/cirurgia , Insuficiência da Valva Aórtica/sangue , Estenose da Valva Aórtica/mortalidade , Biomarcadores/sangue , Feminino , Hemostasia/fisiologia , Humanos , Masculino , Análise Multivariada , Testes Imediatos , Complicações Pós-Operatórias/sangue , Curva ROC , Sensibilidade e Especificidade , Fator de von Willebrand/químicaRESUMO
Recently, we have identified scavenger receptor class A member I (SR-AI) as a receptor for coagulation factor X (FX), mediating the formation of an FX reservoir at the macrophage surface. Here, we demonstrate that the FX/SR-AI-complex comprises a third protein, pentraxin-2 (PTX2). The presence of PTX2 is essential to prevent internalization of FX by SR-AI, and the presence of FX is needed to interfere with internalization of PTX2. Binding studies showed that FX, SR-AI, and PTX2 independently bind to each other (KD,app: 0.2-0.7 µM). Surprisingly, immunoprecipitation experiments revealed that FX and PTX2 circulate as a complex in plasma, and complex formation involves the FX activation peptide. No binding of PTX2 to other vitamin K-dependent proteins was observed. Short hairpin RNA-mediated inhibition of PTX2 levels in mice resulted not only in reduced levels of PTX2, but also in similarly reduced FX levels. Moreover, PTX2 and FX levels were correspondingly reduced in SR-AI-deficient mice. Analysis of 71 human plasma samples uncovered a strong correlation between FX and PTX2 plasma levels. Furthermore, plasma samples of patients with reduced FX levels (congenital/acquired FX deficiency or after anti-vitamin K treatment) were characterized by concomitantly decreased PTX2 levels. In conclusion, we identified PTX2 as a novel partner for FX, and both proteins cooperate to prevent their SR-AI-mediated uptake by macrophages. Interestingly, their respective plasma levels are interdependent. These findings seem of relevance in perspective of ongoing clinical trials, in which plasma depletion of PTX2 is used as a therapeutical approach in the management of systemic amyloidosis.
Assuntos
Proteína C-Reativa/metabolismo , Deficiência do Fator X/sangue , Fator X/metabolismo , Macrófagos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Depuradores Classe A/metabolismo , Animais , Anticoagulantes/farmacologia , Proteína C-Reativa/genética , Linhagem Celular , Endocitose , Fator X/genética , Deficiência do Fator X/genética , Deficiência do Fator X/patologia , Expressão Gênica , Células HEK293 , Humanos , Cinética , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Depuradores Classe A/antagonistas & inibidores , Receptores Depuradores Classe A/deficiência , Receptores Depuradores Classe A/genética , Vitamina K/antagonistas & inibidores , Vitamina K/metabolismoRESUMO
Patients with type 2B von Willebrand disease (vWD) (caused by gain-of-function mutations in the gene coding for von Willebrand factor) display bleeding to a variable extent and, in some cases, thrombocytopenia. There are several underlying causes of thrombocytopenia in type 2B vWD. It was recently suggested that desialylation-mediated platelet clearance leads to thrombocytopenia in this disease. However, this hypothesis has not been tested in vivo The relationship between platelet desialylation and the platelet count was probed in 36 patients with type 2B von Willebrand disease (p.R1306Q, p.R1341Q, and p.V1316M mutations) and in a mouse model carrying the severe p.V1316M mutation (the 2B mouse). We observed abnormally high elevated levels of platelet desialylation in both patients with the p.V1316M mutation and the 2B mice. In vitro, we demonstrated that 2B p.V1316M/von Willebrand factor induced more desialylation of normal platelets than wild-type von Willebrand factor did. Furthermore, we found that N-glycans were desialylated and we identified αIIb and ß3 as desialylation targets. Treatment of 2B mice with sialidase inhibitors (which correct platelet desialylation) was not associated with the recovery of a normal platelet count. Lastly, we demonstrated that a critical platelet desialylation threshold (not achieved in either 2B patients or 2B mice) was required to induce thrombocytopenia in vivo In conclusion, in type 2B vWD, platelet desialylation has a minor role and is not sufficient to mediate thrombocytopenia.
Assuntos
Plaquetas/patologia , Mutação , Ácido N-Acetilneuramínico/química , Trombocitopenia/patologia , Doença de von Willebrand Tipo 2/complicações , Fator de von Willebrand/genética , Animais , Plaquetas/metabolismo , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Integrina alfa2beta1/metabolismo , Integrina beta3/metabolismo , Masculino , Camundongos , Ácido N-Acetilneuramínico/metabolismo , Contagem de Plaquetas , Polissacarídeos/metabolismo , Prognóstico , Processamento de Proteína Pós-Traducional , Trombocitopenia/etiologia , Trombocitopenia/metabolismo , Doença de von Willebrand Tipo 2/genética , Doença de von Willebrand Tipo 2/patologiaRESUMO
Previously, we found that LDL-receptor related protein-1 on macrophages mediated shear stress-dependent clearance of von Willebrand factor. In control experiments, however, we observed that von Willebrand factor also binds to macrophages independently of this receptor under static conditions, suggesting the existence of additional clearance-receptors. In search for such receptors, we focused on the macrophage-specific scavenger-receptor SR-AI. von Willebrand factor displays efficient binding to SR-AI (half-maximum binding 14±5 nM). Binding is calcium-dependent and is inhibited by 72±4% in the combined presence of antibodies against the A1- and D4-domains. Association with SR-AI was confirmed in cell-binding experiments. In addition, binding to bone marrow-derived murine SR-AI-deficient macrophages was strongly reduced compared to binding to wild-type murine macrophages. Following expression via hydrodynamic gene transfer, we determined ratios for von Willebrand factor-propeptide over von Willebrand factor-antigen, a marker of von Willebrand factor clearance. Propeptide/antigen ratios were significantly reduced in SR-AI-deficient mice compared to wild-type mice (0.6±0.2 versus 1.3±0.3; P<0.0001), compatible with a slower clearance of von Willebrand factor in SR-AI-deficient mice. Interestingly, mutants associated with increased clearance (von Willebrand factor/p.R1205H and von Willebrand factor/p.S2179F) had significantly increased binding to purified SR-AI and SR-AI expressed on macrophages. Accordingly, propeptide/antigen ratios for these mutants were reduced in SR-AI-deficient mice. In conclusion, we have identified SR-AI as a novel macrophage-specific receptor for von Willebrand factor. Enhanced binding of von Willebrand factor mutants to SR-AI may contribute to the increased clearance of these mutants.
Assuntos
Receptores Depuradores Classe A/fisiologia , Fator de von Willebrand/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Macrófagos , Camundongos , Proteínas Mutantes/metabolismo , Ligação Proteica , Receptores Depuradores/fisiologia , Fator de von Willebrand/genéticaRESUMO
OBJECTIVE: von Willebrand factor (VWF) is crucial to hemostasis, but also plays a role in inflammatory processes. Unfortunately, no proper monoclonal antibodies to study VWF function in mice are currently available. We therefore aimed to generate single-domain antibodies (sdAbs) recognizing murine VWF and blocking its function in vivo. APPROACH AND RESULTS: Llama-derived sdAbs recognizing both human and murine VWF were isolated via phage display technology. One of them (designated KB-VWF-006) recognized the VWF A1 domain with picomolar affinity. This sdAb avidity was strongly enhanced via dimerization using a triple Ala linker (KB-VWF-006bi). When administered in vivo to wild-type mice, KB-VWF-006bi dose dependently induced bleeding in a tail clip model. In 2 distinct models of inflammation, KB-VWF-006bi efficiently interfered with leukocyte recruitment and vascular leakage. CONCLUSIONS: KB-VWF-006bi is an sdAb recognizing the A1 domain of human VWF and murine VWF that interferes with VWF-platelet interactions in vivo. By using this sdAb, we now also show that the A1 domain is pertinent to the participation of VWF in the inflammatory response.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Quimiotaxia de Leucócito/efeitos dos fármacos , Inflamação/tratamento farmacológico , Leucócitos/efeitos dos fármacos , Anticorpos de Cadeia Única/farmacologia , Fator de von Willebrand/antagonistas & inibidores , Animais , Especificidade de Anticorpos , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Reações Cruzadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hemorragia/induzido quimicamente , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Domínios Proteicos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/toxicidade , Fator de von Willebrand/genética , Fator de von Willebrand/imunologia , Fator de von Willebrand/metabolismoRESUMO
RATIONALE: Percutaneous aortic valve procedures are a major breakthrough in the management of patients with aortic stenosis. Residual gradient and residual aortic regurgitation are major predictors of midterm and long-term outcome after percutaneous aortic valve procedures. We hypothesized that (1) induction/recovery of high molecular weight (HMW) multimers of von Willebrand factor defect could be instantaneous after acute changes in blood flow, (2) a bedside point-of-care assay (platelet function analyzer-closure time adenine DI-phosphate [PFA-CADP]), reflecting HMW multimers changes, could be used to monitor in real-time percutaneous aortic valve procedures. OBJECTIVE: To investigate the time course of HMW multimers changes in models and patients with instantaneous induction/reversal of pathological high shear and its related bedside assessment. METHODS AND RESULTS: We investigated the time course of the induction/recovery of HMW multimers defects under instantaneous changes in shear stress in an aortic stenosis rabbit model and in patients undergoing implantation of a continuous flow left ventricular assist device. We further investigated the recovery of HMW multimers and monitored these changes with PFA-CADP in aortic stenosis patients undergoing transcatheter aortic valve implantation or balloon valvuloplasty. Experiments in the aortic stenosis rabbit model and in left ventricular assist device patients demonstrated that induction/recovery of HMW multimers occurs within 5 minutes. Transcatheter aortic valve implantation patients experienced an acute decrease in shear stress and a recovery of HMW multimers within minutes of implantation which was sustained overtime. In patients with residual high shear or with residual aortic regurgitation, no recovery of HMW multimers was observed. PFA-CADP profiles mimicked HMW multimers recovery both in transcatheter aortic valve implantation patients without aortic regurgitation (correction) and transcatheter aortic valve implantation patients with aortic regurgitation or balloon valvuloplasty patients (no correction). CONCLUSIONS: These results demonstrate that variations in von Willebrand factor multimeric pattern are highly dynamic, occurring within minutes after changes in blood flow. It also demonstrates that PFA-CADP can evaluate in real time the results of transcatheter aortic valve procedures.
Assuntos
Valva Aórtica/cirurgia , Coração Auxiliar , Hemorreologia , Multimerização Proteica , Substituição da Valva Aórtica Transcateter , Fator de von Willebrand/química , Idoso , Idoso de 80 Anos ou mais , Angioplastia com Balão , Animais , Insuficiência da Valva Aórtica/sangue , Insuficiência da Valva Aórtica/fisiopatologia , Insuficiência da Valva Aórtica/cirurgia , Estenose da Valva Aórtica/sangue , Estenose da Valva Aórtica/fisiopatologia , Estenose da Valva Aórtica/cirurgia , Biomarcadores , Velocidade do Fluxo Sanguíneo , Sistemas Computacionais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Testes de Função Plaquetária/métodos , Estudos Prospectivos , CoelhosRESUMO
Two unrelated families were recruited in the French Reference Center for von Willebrand Disease with moderate bleeding symptoms associated with low von Willebrand factor (VWF) antigen levels, decreased collagen binding assay, and no or partial response to desmopressin. Genetic analysis showed the presence of heterozygous mutations in the A3 domain away from the collagen-binding surface: 1 never reported previously (p.L1696R) and another (p.P1824H) described in a Spanish family. The mutations were reproduced by site-directed mutagenesis and mutant VWF was expressed in different expression systems, COS-7 cells, baby hamster kidney cells, and in VWF-deficient mice through hydrodynamic injection. p.L1696R and p.P1824H were associated with very low expression levels both in vitro and in vivo, with intracellular retention for p.P1824H. Both homozygous mutants displayed decreased binding to collagen types I and III but also decreased binding to platelet glycoproteins Ib and IIbIIIa. Co-transfections with wild-type VWF partially corrected these defects, except that collagen binding remained abnormal. The in vivo thrombosis response was severely reduced for both heterozygous mutants. In conclusion, we report 2 VWF A3 domain mutations that induce a combined qualitative and quantitative defect.
Assuntos
Mutação de Sentido Incorreto/fisiologia , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo , Fator de von Willebrand/fisiologia , Adulto , Animais , Animais Recém-Nascidos , Células COS , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Família , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Transfecção , Doenças de von Willebrand/genética , Doenças de von Willebrand/metabolismoRESUMO
von Willebrand factor (VWF) is a promising target for developing antithrombotic drugs. The absence of accessible animal models impedes the study of specific human VWF (huVWF) targeting molecules in thrombosis. huVWF is not functional in the mouse because of a lack of interaction between huVWF and murine glycoprotein Ib. Using site-directed mutagenesis, we have replaced single or multiple amino acids in huVWF with their murine counterparts to eliminate species incompatibility. Using hydrodynamic injection, we have expressed the different chimeric VWF constructs into VWF(-/-) mice. Only huVWF with a complete murine A1 domain insertion was able to correct bleeding in vivo and form occlusive thrombi in mesenteric vessels after FeCl(3) treatment. Using this model, we tested the antithrombotic effect of monoclonal antibodies against huVWF, blocking its interaction with collagens (mAbs 203 and 505) or with glycoprotein IIbIIIa (mAb 9). The 3 mAbs inhibited the thrombotic process in arterioles of VWF(-/-) mice expressing huVWFmuA1. Inhibiting VWF-interaction with collagens was more potent, emphasizing the potential of such a target as an antithrombotic tool. Our results validate our murine model as a simple in vivo tool to evaluate anti-huVWF agents.
Assuntos
Anticorpos Monoclonais/farmacologia , Modelos Animais de Doenças , Hemorragia/prevenção & controle , Mutação/genética , Adesividade Plaquetária/efeitos dos fármacos , Trombose/prevenção & controle , Fator de von Willebrand/fisiologia , Proteína ADAMTS13 , Animais , Cloretos/toxicidade , Colágeno/antagonistas & inibidores , Colágeno/imunologia , Colágeno/metabolismo , Ensaio de Imunoadsorção Enzimática , Compostos Férricos/toxicidade , Fibrinolíticos/toxicidade , Hemorragia/genética , Humanos , Metaloendopeptidases/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Adesividade Plaquetária/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trombose/induzido quimicamente , Trombose/genética , Fator de von Willebrand/antagonistas & inibidoresRESUMO
The relationship between low-density lipoprotein receptor-related protein-1 (LRP1) and von Willebrand factor (VWF) has remained elusive for years. Indeed, despite a reported absence of interaction between both proteins, liver-specific deletion of LRP1 results in increased VWF levels. To investigate this discrepancy, we used mice with a macrophage-specific deficiency of LRP1 (macLRP1(-)) because we previously found that macrophages dominate VWF clearance. Basal VWF levels were increased in macLRP1(-) mice compared with control mice (1.6 ± 0.4 vs 1.0 ± 0.4 U/mL). Clearance experiments revealed that half-life of human VWF was significantly increased in macLRP1(-) mice. Ubiquitous blocking of LRP1 or additional lipoprotein receptors by overexpressing receptor-associated protein in macLRP1(-) mice did not result in further rise of VWF levels (0.1 ± 0.2 U/mL), in contrast to macLRP1(+) mice (rise in VWF, 0.8 ± 0.4 U/mL). This points to macLRP1 being the only lipoprotein receptor regulating VWF levels. When testing the mechanism(s) involved, we observed that VWF-coated beads adhered efficiently to LRP1 but only when exposed to shear forces exceeding 2.5 dyne/cm(2), implying the existence of shear stress-dependent interactions. Furthermore, a mechanism involving ß2-integrins that binds both VWF and LRP1 also is implicated because inhibition of ß2-integrins led to increased VWF levels in control (rise, 0.19 ± 0.16 U/mL) but not in macLRP1(-) mice (0.08 ± 0.15 U/mL).
Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Macrófagos/metabolismo , Fator de von Willebrand/metabolismo , Animais , Fator VIII/metabolismo , Humanos , Cadeias beta de Integrinas/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Receptores de LDL/antagonistas & inibidores , Resistência ao CisalhamentoRESUMO
INTRODUCTION: ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) deficiency has been reported in patients with sepsis but its clinical relevance and pathophysiology remain unclear. Our objectives were to assess the clinical significance, prognostic value and pathophysiology of ADAMTS13 deficiency in patients with septic shock with and without disseminated intravascular coagulation (DIC). METHODS: This was a prospective monocenter cohort study of patients with septic shock. Von Willebrand Factor, ADAMTS13-related parameters and plasma IL-6 concentration were measured at inclusion to the study. Patients were categorized into three groups according to the presence of ADAMT13 deficiency (<30%) or DIC. RESULTS: This study included 72 patients with a median age of 59 years (interquartile range (IQR) 50 to 71). Each of the included patients received vasopressors; 55 (76%) were under mechanical ventilation and 22 (33%) underwent renal replacement therapy. Overall, 19 patients (26%) had DIC, and 36 patients had ADMTS13 deficiency (50%). Patients with DIC, ADAMTS13 deficiency or both were more severe at ICU admission. Mortality was higher in septic shock patients from group one. By multivariate analysis, Simplified Acute Physiology Score 2 (SAPS2) score (odds ratio (OR) 1.11/point; 95% CI 1.01 to 1.24) and ADAMTS13 activity <30% (OR 11.86; 95% CI 1.36 to 103.52) were independently associated with hospital mortality. There was no correlation between ADAMTS13 activity and the International Society for Thrombosis and Haemostasis (ISTH) score (rs = -0.97, P = 0.41) suggesting that ADAMTS13 functional deficiency and DIC were independent parameters. IL-6 level was higher in patients with ADAMTS13 activity <30% [895 (IQR 330 to 1843) pg/mL versus 83 (IQR 43 to 118), P = 0.0003). CONCLUSIONS: Septic shock was associated with a functional deficiency of ADAMTS13, independently of DIC. ADAMTS13 functional deficiency is then a prognostic factor for mortality in septic shock patients, independently of DIC.
Assuntos
Proteínas ADAM/deficiência , Coagulação Intravascular Disseminada/sangue , Interleucina-6/sangue , Choque Séptico/sangue , Fator de von Willebrand/análise , Proteínas ADAM/sangue , Proteína ADAMTS13 , APACHE , Idoso , Biomarcadores/sangue , Feminino , França , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Escores de Disfunção Orgânica , Prognóstico , Estudos Prospectivos , Terapia de Substituição Renal , Respiração Artificial , Choque Séptico/mortalidade , Choque Séptico/terapia , Análise de Sobrevida , Vasoconstritores/uso terapêutico , Vasopressinas/uso terapêuticoRESUMO
Von Willebrand disease (VWD)-type 2B originates from a gain-of-function mutation in von Willebrand factor (VWF), resulting in enhanced platelet binding. Clinical manifestations include increased bleeding tendency, loss of large multimers, thrombocytopenia, and circulating platelet aggregates. We developed a mouse model to study phenotypic consequences of VWD-type 2B mutations in murine VWF: mVWF/R1306Q and mVWF/V1316M. Both mutations allow normal multimerization but are associated with enhanced ristocetin-induced platelet aggregation, typical for VWD-type 2B. In vivo expression resulted in thrombocytopenia and circulating aggregates, both of which were more pronounced for mVWF/V1316M. Furthermore, both mutants did not support correction of bleeding time or arterial vessel occlusion in a thrombosis model. They further displayed a 2- to 3-fold reduced half-life and induced a 3- to 6-fold increase in number of giant platelets compared with wild-type VWF. Loss of large multimers was observed in 50% of the mice. The role of ADAMTS13 was investigated by expressing both mutants in VWF/ADAMTS13 double-deficient mice. ADAMTS13 deficiency resulted in more and larger circulating platelet aggregates for both mutants, whereas the full multimer range remained present in all mice. Thus, we established a mouse model for VWD-type 2B and found that phenotype depends on mutation and ADAMTS13.
Assuntos
Plaquetas/metabolismo , Metaloendopeptidases , Mutação de Sentido Incorreto , Multimerização Proteica , Doença de von Willebrand Tipo 2 , Fator de von Willebrand , Proteína ADAMTS13 , Substituição de Aminoácidos , Animais , Antibacterianos/efeitos adversos , Antibacterianos/farmacologia , Tempo de Sangramento , Modelos Animais de Doenças , Meia-Vida , Humanos , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Mutantes , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/genética , Ristocetina/efeitos adversos , Ristocetina/farmacologia , Índice de Gravidade de Doença , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombose/induzido quimicamente , Trombose/genética , Trombose/metabolismo , Doença de von Willebrand Tipo 2/genética , Doença de von Willebrand Tipo 2/metabolismo , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
OBJECTIVE: The goal of this study was to search for an association between a desintegrin-like and metalloprotease thrombospondin type 1 motif, member 13 (ADAMTS13) levels and the occurrence of preeclampsia, its characteristics (time-onset and severity), and its consequences (occurrence of fetal growth restriction or preterm delivery). METHODS AND RESULTS: We studied 140 pairs of women in a case-control study with 3 matching criteria: maternal age, gestational age, and ethnic origin. We measured ADAMTS13 activity using a fluorescence resonance energy transfer assay with the fluorescence resonance energy transfer-VWF73 peptide. ELISA was used to assess protein antigen levels: ADAMTS13, von Willebrand Factor (VWF), interleukin-6, C-reactive protein, P-selectin, and thrombospondin-1. The lowest levels of ADAMTS13 (activity ≤ 70% or antigen ≤ 592 ng/mL) were significantly associated with preeclampsia (odds ratios [OR] [95% confidence interval] of 4.2 [1.1 to 15] and 14.3 [1.7 to 123], respectively). This association was independent of VWF levels and preeclampsia risk factors but dependent on interleukin-6 and C-reactive protein levels for ADAMTS13 activity. Levels of ADAMTS13 activity (≤ 57%) were significantly associated with early-onset preeclampsia (OR = 2.5 [1.1 to 5.8]). Severe preeclampsia was associated with the highest levels of P-selectin (>57 ng/mL) (OR = 3.4 [1.2 to 9.7]). CONCLUSIONS: Preeclampsia is associated with decreased levels of ADAMTS13, independently of VWF. This decrease is quantitative, occurs early, and seems to be dependent on inflammation. Our results suggest that ADAMTS13 could participate in the pathophysiology of preeclampsia.
Assuntos
Proteínas ADAM/sangue , Pré-Eclâmpsia/sangue , Fator de von Willebrand/análise , Proteína ADAMTS13 , Adulto , Biomarcadores/sangue , Proteína C-Reativa/análise , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Retardo do Crescimento Fetal/sangue , Retardo do Crescimento Fetal/etiologia , Transferência Ressonante de Energia de Fluorescência , França , Humanos , Interleucina-6/sangue , Modelos Logísticos , Razão de Chances , Selectina-P/sangue , Pré-Eclâmpsia/etiologia , Pré-Eclâmpsia/fisiopatologia , Gravidez , Nascimento Prematuro/sangue , Nascimento Prematuro/etiologia , Medição de Risco , Fatores de Risco , Índice de Gravidade de Doença , Trombospondina 1/sangueRESUMO
This report describes the first case of splenic injury in a patient with p.V1316M-associated von Willebrand disease type 2B (VWD2B) with chronic thrombocytopenia, successfully treated with nonoperative management including von Willebrand factor (VWF) replacement therapy, and platelet transfusions relayed by a thrombopoietin receptor agonist (TPO-RA, Eltrombopag). Eltrombopag was initially introduced to rescue an unusual post-platelet-transfusion reaction exacerbating the thrombocytopenia. In-depth analysis of the dramatic platelet count drop and VWF measurements timeline ruled out an allo-immune reaction and supported an alternative hypothesis of a sudden platelet clearance as a consequence of stress-induced release of abnormal VWF. One year later, a second life-threatening bleeding episode required urgent surgery successfully managed with VWF replacement therapy and platelet transfusions. Eltrombopag was further introduced in the post-surgery period to allow bleeding-free and platelet-transfusion-free successful recovery. Treatment decisions are particularly challenging in patients with VWD2B, and this case highlights how such decisions can benefit from understanding the molecular origin of platelet count fluctuations observed in these patients. Here, we successfully used a new therapeutic approach combining VWF-replacement therapy and initial platelet-transfusion relayed by TPO-RA to optimize patient management. PLAIN LANGUAGE SUMMARY: A combination of von Willebrand factor replacement and thrombopoietin receptor agonist in thrombocytopenic patients with von Willebrand disease type 2B: a new therapy approach to optimize patient management?Therapeutic management of patients with von Willebrand disease type 2B are particularly challenging in case of severe thrombocytopenia.Treatment includes von Willebrands factor replacement therapy and iterative platelet transfusions.We describe the first case of splenic injury in a patient with p.V1316M-associated von Willebrand disease type 2B successfully treated with nonoperative management including von Willebrand factor replacement therapy and platelet transfusions relayed by a thrombopoietin receptor agonist.We showed that the unusual post-platelet-transfusion reaction associated with a dramatic platelet count drop was a consequence of stress-induced release of abnormal von Willebrand factor.The combination of von Willebrand factor replacement therapy and thrombopoietin receptor agonist may offer a new therapeutic approach to optimize patient management.
RESUMO
AIMS: Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary haemostasis, while also having additional roles beyond haemostasis namely in cancer, inflammation, angiogenesis, and potentially in vascular smooth muscle cell (VSMC) proliferation. Here, we addressed how VWF modulates VSMC proliferation and investigated the underlying molecular pathways and the in vivo pathophysiological relevance. METHODS AND RESULTS: VWF induced proliferation of human aortic VSMCs and also promoted VSMC migration. Treatment of cells with a siRNA against αv integrin or the RGT-peptide blocking αvß3 signalling abolished proliferation. However, VWF did not bind to αvß3 on VSMCs through its RGD-motif. Rather, we identified the VWF A2 domain as the region mediating binding to the cells. We hypothesized the involvement of a member of the LDL-related receptor protein (LRP) family due to their known ability to act as co-receptors. Using the universal LRP-inhibitor receptor-associated protein, we confirmed LRP-mediated VSMC proliferation. siRNA experiments and confocal fluorescence microscopy identified LRP4 as the VWF-counterreceptor on VSMCs. Also co-localization between αvß3 and LRP4 was observed via proximity ligation analysis and immuno-precipitation experiments. The pathophysiological relevance of our data was supported by VWF-deficient mice having significantly reduced hyperplasia in carotid artery ligation and artery femoral denudation models. In wild-type mice, infiltration of VWF in intimal regions enriched in proliferating VSMCs was found. Interestingly, also analysis of human atherosclerotic lesions showed abundant VWF accumulation in VSMC-proliferating rich intimal areas. CONCLUSION: VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvß3 signalling. Our findings provide new insights into the mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall. In addition, the VWF/LRP4-axis may represent a novel therapeutic target to modulate VSMC proliferation.
Assuntos
Aterosclerose/metabolismo , Proliferação de Células , Integrina alfaVbeta3/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fator de von Willebrand/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Movimento Celular , Células Cultivadas , Hiperplasia , Integrina alfaVbeta3/genética , Proteínas Relacionadas a Receptor de LDL/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Neointima , Placa Aterosclerótica , Transdução de Sinais , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/patologia , Fator de von Willebrand/genéticaRESUMO
Essentials Treatment options for von Willebrand disease (VWD) patients are limited. The p.P1127_C1948delinsR deletion/variant is a useful model to study VWD in vitro and in vivo. Counteracting dominant-negative effects restores von Willebrand factor multimerization in mice. This is the first siRNA-based treatment applied to a mouse model of VWD-type 2A. ABSTRACT: Background Treatment options for patients suffering from von Willebrand disease (VWD) are limited. Von Willebrand factor (VWF) is a polymeric protein that undergoes regulated dimerization and subsequent multimerization during its biosynthesis. Numerous heterozygous variants within the VWF gene display a dominant-negative effect and result in severe VWD. Previous studies have suggested that preventing the assembly of wild-type and mutant heteropolymers using siRNAs may have beneficial effects on VWF phenotypes in vitro. Objectives To study heterozygous dominant-negative variants in vivo, we developed a mouse model of VWD-type 2A and tested two independent strategies to modulate its detrimental effect. Methods The p.P1127_C1948delinsR deletion/variant, causing defective VWF multimerization, was expressed in mice as a model of VWD-type 2A variant. Two corrective strategies were applied. For the first time in a mouse model of VWD, we applied siRNAs selectively inhibiting translation of the mutant transcripts and we combined the VWD-type 2A deletion with the Cys to Arg substitution at position 2773, which is known to prevent dimerization. Results The RNA silencing approach induced a modest but consistent improvement of the VWF multimer profile. However, due to incomplete efficiency, the dominant-negative effect of the original variant could not be completely prevented. In contrast, the DNA approach resulted in increased antigen levels and restoration of a normal multimer profile. Conclusions Our data showed that preventing the detrimental impact of dominant-negative VWF variants by independent molecular mechanisms has beneficial consequences in vivo, in mouse models of dominant VWD.
Assuntos
Modelos Animais de Doenças , Doença de von Willebrand Tipo 2 , Doenças de von Willebrand , Animais , Heterozigoto , Humanos , Camundongos , Fenótipo , Doença de von Willebrand Tipo 2/genética , Doença de von Willebrand Tipo 2/terapia , Doenças de von Willebrand/genética , Doenças de von Willebrand/terapia , Fator de von Willebrand/genéticaRESUMO
ADAMTS13 mutations S203P, R268P, R507Q and A596V were previously identified in French patients with hereditary thrombotic thrombocytopenic purpura (TTP) (Upshaw-Schulman syndrome). Mutated recombinant (r) ADAMTS13 were transiently expressed in COS-7 cells and characterized in comparison with wild-type (WT) rADAMTS13. ADAMTS13 antigen was qualitatively and quantitatively estimated by electrophoretic analysis and ELISA. Enzymatic activity was qualitatively and quantitatively estimated using GST-VWF73, FRETS-VWF73 fragments and full-length rVWF-WT as substrates. The four mutants and rADAMTS13-WT were present within the cells. Secretion level of rADAMTS13-WT reached 1,200 ng/ml. The four mutations strongly altered the secretion and biological activity of rADAMTS13. The percentage secretion was 21, 38 and 17% for rADAMTS13-S203P, -R268P and -A596V compared with rADAMTS13-WT. rADAMTS13-R507Q concentration was under the detection limit of the assay. In the four cases, no enzymatic activity was detected. After concentration, we confirmed that mutations S203P and R268P totally abolished the proteolytic activity of ADAMTS13. Due to the very low protease concentration, activity of rADAMTS13-R507Q was below the threshold of the assays. rADAMTS13-A596V had no proteolytic activity towards the full-length rVWF-WT whereas it exhibited a decreased specific activity of about 30% of that of rADAMTS13-WT towards FRETS-VWF73 fragment. Binding study of mutated rADAMTS13-S203P, -R268P and -A596V showed that the three mutations strongly decreased the interaction of ADAMTS13 with VWF. In conclusion, the four mutations, which led to a secretion defect, a loss of enzymatic activity and a decreased binding to the substrate, are responsible for the hereditary TTP in patients.
Assuntos
Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Mutação de Sentido Incorreto , Púrpura Trombocitopênica Trombótica/genética , Púrpura Trombocitopênica Trombótica/metabolismo , Proteínas ADAM/química , Proteína ADAMTS13 , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Predisposição Genética para Doença , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Dobramento de Proteína , Proteínas Recombinantes/metabolismo , Fatores de Risco , Síndrome , Transfecção , Fator de von Willebrand/metabolismoRESUMO
von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Willebrand factor (VWF) A1-domain, leading to increased affinity for its platelet-receptor, glycoprotein Ibα. We engineered the first knock-in (KI) murine model for VWD-type 2B by introducing the p.V1316M mutation in murine VWF. Homozygous KI-mice replicated human VWD-type 2B with macrothrombocytopenia (platelet counts reduced by 55%, platelet volume increased by 44%), circulating platelet-aggregates and a severe bleeding tendency. Also, vessel occlusion was deficient in the FeCl3-induced thrombosis model. Platelet aggregation induced by thrombin or collagen was defective for KI-mice at all doses. KI-mice manifested a loss of high molecular weight multimers and increased multimer degradation. In a model of VWF-string formation, the number of platelets/string and string-lifetime were surprisingly enhanced in KI-mice, suggesting that proteolysis of VWF/p.V1316M is differentially regulated in the circulation versus the endothelial surface. Furthermore, we observed increased leukocyte recruitment during an inflammatory response induced by the reverse passive Arthus reaction. This points to an active role of VWF/p.V1316M in the exfiltration of leukocytes under inflammatory conditions. In conclusion, our genetically-engineered VWD-type 2B mice represent an original model to study the consequences of spontaneous VWF-platelet interactions and the physiopathology of this human disease.
Assuntos
Doença de von Willebrand Tipo 2/sangue , Doença de von Willebrand Tipo 2/patologia , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Feminino , Engenharia Genética , Hemostasia/genética , Humanos , Inflamação/genética , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/sangue , Proteínas Mutantes/química , Proteínas Mutantes/genética , Adesividade Plaquetária , Contagem de Plaquetas , Doença de von Willebrand Tipo 2/genética , Fator de von Willebrand/química , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
Thrombocytopenia and increased platelet clearance observed in von Willebrand disease-type 2B (VWD-2B) may be explained by platelet apoptosis triggered by the constitutive binding of VWF to its receptor, glycoprotein Ib (GPIb). Apoptosis was assessed in platelets from two patients with a severe VWD-2B mutation VWF/p.V1316M and from mice transiently expressing VWF/p.V1316M. We now report that the VWD-2B mutation VWF/p.V1316M which binds spontaneously to its receptor GPIbα does not induce apoptosis. In 2 unrelated patients (P1 and P2) exhibiting different VWF plasma levels (70% and 36%, respectively, compared with normal pooled human plasma given as 100%), inner transmembrane depolarization of mitochondria, characteristic of apoptotic events was undetectable in platelets, whether washed or in whole blood. No or a moderate phosphatidyl serine (PS) exposure as measured by annexin-V staining was observed for P1 and P2, respectively. Expression of pro-apoptotic proteins Bak and Bax, and caspase-3 activity were similar to control platelets. In the VWD-2B mouse model expressing high levels of mVWF/p.V1316M (423%), similar to what is found in inflammatory pathologies, no significant difference was observed between mice expressing mVWF/WT and mVWF/p.V1316M. These results strongly argue against apoptosis as a mechanism for the thrombocytopenia of severe VWD-2B exhibiting the VWF/p.V1316M mutation.