RESUMO
INTRODUCTION: Monitoring replacement therapy with standard and extended half-life (EHL) products is challenging, since one-stage assay (OSA) and chromogenic substrate assay (CSA) results may differ significantly. Recent recommendations include local validation of each new product with recovery within 20-30%, depending on activity level. AIM: To validate factor VIII (FVIII) activity for monitoring products in clinical use on Atellica Coag and to correlate it with thrombin generation. METHODS: Plasma samples spiked with Advate® , Elocta® , Adynovi® , Nuwiq® , NovoEight® and Afstyla® (0.05, 0.20, 0.50 and 0.80 IU/ml) were analysed using Atellica Coag 360 with CSA-1 (Coatest SP) and CSA-2 (FVIII chromogenic), and OSA (Actin FS). Thrombin generation was performed using two thrombin generation assays (TGA-1 (Thrombinoscope) and TGA-2 (Technothrombin). RESULTS: All products at levels above 0.05 IU/ml, except Adynovi, showed acceptable recovery using CSA-1, whereas measurements using CSA-2 gave more results outside the target level. All products, except Afstyla, showed acceptable recovery using OSA. Correlation between CSA-1 and OSA was excellent (r2 =1.0) with biases of 6-3â2%, depending on FVIII product. A clear dose-response was seen for all thrombin generation parameters and products using both methods, except at low levels for lag time using TGA-1. With CSA-1 as an independent variable, the correlations to thrombin peak (measured with TGA-2) were good (r2 = .8-.9). CONCLUSION: Our data revealed good correlation and acceptable bias between CSA and OSA using our sets of reagents, methods and analyser in spiked samples. Thrombin generation gave good correlation to CSA-1 factor activity and is a possible complement to factor activity assays.
Assuntos
Fator VIII , Hemofilia A , Testes de Coagulação Sanguínea , Fator VIII/farmacocinética , Meia-Vida , Hemofilia A/tratamento farmacológico , Humanos , TrombinaRESUMO
INTRODUCTION: Genetic screening using high-throughput DNA sequencing has become a tool in diagnosing patients with suspected inherited bleeding disorders (IBD). However, its usefulness and diagnostic efficacy in children is unclear. AIM: To evaluate the diagnostic efficacy of genetic screening for IBD in children and downstream further testing. METHODS: After informed consent, children (<18 years) with suspected IBD underwent genetic screening with 94 selected genes. RESULTS: A total of 68 heterozygous class 3-5 variants were detected in 30 children, 2.3 variants per patient. Directed specific functional testing was performed after genetic screening in a subset of patients. Adhering to the ACMG guidelines, the results of functional testing together with family history and previous publications classified three variants as likely disease causing (class 4) and two variants as disease causing (class 5), all in children with thrombocytopenia. The overall diagnostic rate was 16.7% (5/30). Children with thrombocytopenia had a significantly higher rate of significant genetic findings, 5/9 (55.6%) vs. 0/21 (0%; P = .0009). CONCLUSION: We conclude that performing genetic screening in children is an effective tool especially for children with inherited thrombocytopenia and has the possibility to diagnose platelet disorders adequately early in life. Children with bleeding diathesis, normal coagulation work-up and without thrombocytopenia are unlikely to be diagnosed by genetic screening. Ethical issues such as incidental findings, variants associated with cancer and the interpretation of the genetic results into clinical practice remain problematic.
Assuntos
Transtornos Herdados da Coagulação Sanguínea/diagnóstico , Transtornos Herdados da Coagulação Sanguínea/genética , Predisposição Genética para Doença/genética , Testes Genéticos/métodos , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , MasculinoRESUMO
Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis.
Assuntos
Coagulação Sanguínea , Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Fosfatidilserinas/sangue , Sepse/sangue , Trombina/metabolismo , Animais , Anexina A5/sangue , Antitrombina III , Plaquetas/imunologia , Plaquetas/microbiologia , Plaquetas/ultraestrutura , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/microbiologia , Micropartículas Derivadas de Células/ultraestrutura , Quimiocinas CXC/metabolismo , Modelos Animais de Doenças , Inflamação/sangue , Inflamação/imunologia , Inflamação/microbiologia , Interleucina-6/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Masculino , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Peptídeo Hidrolases/sangue , Sepse/imunologia , Sepse/microbiologia , Sepse/patologia , Transdução de Sinais , Fatores de TempoRESUMO
Familial hemophagocytic lymphohistiocytosis (FHL) is caused by biallelic variants in genes regulating granule secretion in cytotoxic lymphocytes. In FHL3-5, the affected genes UNC13D, STX11 and STXBP2 have further been shown to regulate the secretion of platelet granules, giving rise to compromised platelet function. Therefore, we aimed to investigate platelet degranulation in patients heterozygous for variants in UNC13D, STX11 and STXBP2. During the work-up of patients referred to the Coagulation Unit, Skåne University Hospital, Malmö, Sweden and the Department of Hematology, Rigshospitalet, Copenhagen, Denmark due to bleeding tendencies, 12 patients harboring heterozygous variants in UNC13D, STX11 or STXBP2 were identified using targeted whole exome sequencing. Transmission electron microscopy (TEM) was used to assess the secretion of platelet dense granules following thrombin stimulation. Platelet degranulation, activation and aggregation were further assessed by flow cytometry (FC) and light transmission aggregometry (LTA) with lumi-aggregometry. In total, eight out of twelve (67%) patients showed impaired degranulation by at least one of the assays (TEM, FC and LTA). In the 12 patients, eight different heterozygous variants were identified. One variant was strongly associated with impaired degranulation, while four of the variants were associated with impaired granule secretion to a slightly lesser extent. One additional variant was found in six out of the twelve patients, and was associated with varying degrees of degranulation impairment. Accordingly, six out of the eight (75%) identified variants were associated with impaired platelet degranulation. Our results suggest that heterozygous variants in UNC13D, STX11 and STXBP2 are sufficient to cause platelet secretion defects resulting in increased bleeding.
Assuntos
Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Hemorragia/etiologia , Heterozigoto , Linfo-Histiocitose Hemofagocítica/complicações , Linfo-Histiocitose Hemofagocítica/genética , Mutação , Adolescente , Adulto , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Criança , Pré-Escolar , Comorbidade , Feminino , Citometria de Fluxo , Humanos , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfo-Histiocitose Hemofagocítica/metabolismo , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Modelos Biológicos , Proteínas Munc18/genética , Contagem de Plaquetas , Proteínas Qa-SNARE/genética , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
Rare inherited bleeding disorders (IBD) are a common cause of bleeding tendency. To ensure a correct diagnosis, specialized laboratory analyses are necessary. This study reports the results of an upfront diagnostic strategy using targeted whole exome sequencing. In total, 156 patients with a significant bleeding assessment tool score participated in the study, of which a third had thrombocytopenia. Eighty-seven genes specifically associated with genetic predisposition to bleeding were analysed by whole exome sequencing. Variants were classified according to the five-tier scheme. We identified 353 germline variants. Eight patients (5%) harboured a known pathogenic variant. Of the 345 previously unknown variants, computational analyses predicted 99 to be significant. Further filtration according to the Mendelian inheritance pattern, resulted in 59 variants being predicted to be clinically significant. Moreover, 34% (20/59) were assigned as novel class 4 or 5 variants upon targeted functional testing. A class 4 or 5 variant was identified in 30% of patients with thrombocytopenia (14/47) versus 11% of patients with a normal platelet count (12/109) (P < 0·01). An IBD diagnosis has a major clinical impact. The genetic investigations detailed here extricated our patients from a diagnostic conundrum, thus demonstrating that continuous optimization of the diagnostic work-up of IBD is of great benefit.
Assuntos
Transtornos Herdados da Coagulação Sanguínea/diagnóstico , Transtornos Herdados da Coagulação Sanguínea/genética , Exoma , Mutação em Linhagem Germinativa , Transtornos Herdados da Coagulação Sanguínea/epidemiologia , Dinamarca/epidemiologia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Suécia/epidemiologiaRESUMO
OBJECTIVE: Systemic inflammatory response syndrome is associated with severe coagulopathy. The purpose of this study was to examine thrombin generation in systemic inflammation triggered by the endotoxin lipopolysaccharide (LPS) and the exotoxin streptococcal M1 protein. METHODS: Thrombin generation, lung histology and myeloperoxidase (MPO) activity were determined 6 and 24 h after induction of systemic inflammation. Male C57BL/6 mice received the Rac1 inhibitor NSC23766 prior to challenge with bacterial toxins. RESULTS: LPS and M1 protein challenge increased neutrophil infiltration and caused damage in the lung. Time to peak thrombin formation was increased and peak and total generation of thrombin were decreased in plasma from LPS- and M1 protein-treated mice. Coincubation of samples from mice exposed to bacterial toxins with platelet poor plasma from healthy mice completely reversed the inhibitory effect of LPS and M1 protein on thrombin generation, suggesting that bacterial toxins decreased levels of plasma factors explaining the reduction of thrombin generating capacity of plasma from septic animals. NSC23766 treatment not only decreased LPS- and M1 protein-induced neutrophil accumulation as well as levels of interleukin-6 and CXCL2 in the lung, but also abolished bacterial toxin-induced changes in thrombin generation. For example, NSC23766 increased peak formation by 57% and total thrombin generation by 48% in LPS-treated animals at 6 h. CONCLUSIONS: Taken together, our novel findings show that bacterial toxins increase thrombin generation via consumption of plasma factors and that Rac1 signaling plays an important role in thrombin generation in response to bacterial toxins. Thus, targeting Rac1 activity might be a useful way not only to ameliorate pulmonary inflammation, but also inhibit pathological changes in coagulation in bacterial infections.
Assuntos
Antígenos de Bactérias/farmacologia , Proteínas da Membrana Bacteriana Externa/farmacologia , Proteínas de Transporte/farmacologia , Lipopolissacarídeos/farmacologia , Neuropeptídeos/metabolismo , Trombina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Aminoquinolinas/farmacologia , Animais , Quimiocina CXCL2/metabolismo , Interleucina-6/metabolismo , Contagem de Leucócitos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Neuropeptídeos/antagonistas & inibidores , Infiltração de Neutrófilos/efeitos dos fármacos , Peroxidase/metabolismo , Contagem de Plaquetas , Protrombina/análise , Pirimidinas/farmacologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
Abdominal sepsis is associated with significant changes in systemic inflammation and coagulation. The purpose of the present study was to examine the role of peripheral blood monocytes for systemic coagulation, including thrombin generation and consumption of coagulation factors. Abdominal sepsis was induced by cecal ligation and puncture (CLP) in C57BL/6 mice. Plasma and lung levels of IL-6 and C-X-C motif (CXC) chemokines [chemokine CXC ligand (CXCL)1, CXCL2, and CXCL5], pulmonary activity of myeloperoxidase, thrombin generation, and coagulation factors were determined 6 h after CLP induction. Administration of clodronate liposomes decreased circulating levels of monocytes by 96%. Time to peak thrombin formation was increased and peak and total thrombin generation was decreased in plasma from CLP animals. Monocyte depletion decreased time to peak formation of thrombin and increased peak and total generation of thrombin in septic animals. In addition, monocyte depletion decreased the CLP-induced increase in the levels of thrombin-antithrombin complexes in plasma. Depletion of monocytes increased plasma levels of prothrombin, factor V, factor X, and protein C in septic mice. Moreover, depletion of monocytes decreased CLP-induced levels of IL-6 and CXC chemokines in the plasma and lung by >59% and 20%, respectively. CLP-induced myeloperoxidase activity in the lung was attenuated by 44% in animals depleted of monocytes. Taken together, our findings show, for the first time, that peripheral blood monocytes regulate systemic coagulation. The results of our study improve our understanding of the pathophysiology of sepsis and encourage further attempts to target innate immune cell functions in abdominal sepsis.
Assuntos
Coagulação Sanguínea , Monócitos/metabolismo , Sepse/sangue , Animais , Fatores de Coagulação Sanguínea/metabolismo , Quimiocinas CXC/sangue , Quimiocinas CXC/metabolismo , Inflamação/metabolismo , Interleucina-6/sangue , Interleucina-6/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peroxidase/sangue , Peroxidase/metabolismo , Trombina/metabolismoRESUMO
Sepsis causes severe derangements of the coagulation system. However, the signaling mechanisms regulating sepsis-induced thrombin generation remain elusive. Herein, we hypothesized that Rho-kinase might be an important regulator of thrombin generation in abdominal sepsis. Abdominal sepsis was induced by cecal ligation and puncture (CLP) in C57Bl/6 mice. Thrombin generation, coagulation factors, lung histology and myeloperoxidase (MPO) activity were determined 6 h and 24 h after induction of CLP. Induction of CLP triggered a systemic inflammatory response characterized by neutrophil accumulation and tissue injury in the lung as well as thrombocytopenia and leukocytopenia. Administration of Y-27632, a Rho-kinase inhibitor, attenuated these markers of systemic inflammation in CLP animals. Moreover, peak thrombin formation was decreased by 77% and 81% in plasma from mice 6 h and 24 h after induction of CLP. Total thrombin generation was reduced by 64% and 67% 6 h and 24 h after CLP induction, respectively. Notably, administration of Y-27632 increased peak formation by 99% and total thrombin generation by 66% in plasma from septic animals. In addition, CLP markedly decreased plasma levels of prothrombin, factor V and factor X at 6 h and 24 h. Interestingly, Rho-kinase inhibition significantly enhanced levels of prothrombin, factor V and factor X in plasma from septic mice. In addition, inhibition of Rho-kinase decreased CLP-induced elevations of CXCL2 by 36% and interleukin-6 by 38%. These novel findings suggest that sepsis-induced thrombin generation is regulated by Rho-kinase. Moreover, inhibition of Rho-kinase reverses sepsis-evoked consumption of coagulation factors. Thus, our results show that targeting Rho-kinase signaling might protect against coagulation dysfunction in abdominal sepsis.
Assuntos
Abdome/patologia , Sepse/metabolismo , Trombina/biossíntese , Quinases Associadas a rho/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Peroxidase/metabolismo , Sepse/enzimologia , Sepse/patologiaRESUMO
Sphingosine 1-phosphate (S1P) is a signalling sphingolipid affecting multiple cellular functions of vascular and immune systems. It circulates at submicromolar levels bound to HDL-associated apolipoprotein M (apoM) or to albumin. S1P in blood is mainly produced by platelets and erythrocytes, making blood sampling for S1P quantification delicate. Standardisation of sampling is thereby of great importance to obtain robust data. By optimising and characterising the extraction procedure and the LC-MS/MS analysis, we have developed and validated a highly specific and sensitive method for S1P quantification. Blood was collected from healthy individuals (n = 15) to evaluate the effects of differential blood sampling on S1P levels. To evaluate correlation between S1P and apoM in different types of plasma and serum, apoM was measured by ELISA. The method showed good accuracy and precision in the range of 0.011 to 0.9 µM with less than 0.07 % carryover. We found that the methanol precipitation used to extract S1P co-extracted apoM and several other HDL-proteins from plasma. The platelet-associated S1P was released during coagulation, thus increasing the S1P concentration to double in serum as compared to that in plasma. Gel filtration chromatography revealed that the platelet-released S1P was mainly bound to albumin. This explains why the strong correlation between S1P and apoM levels in plasma is lost upon the clotting process and hence not observed in serum. We have developed, characterised and validated an efficient, highly sensitive and specific method for the quantification of S1P in biological material.
Assuntos
Cromatografia Líquida/normas , Lisofosfolipídeos/sangue , Plasma/química , Soro/química , Esfingosina/análogos & derivados , Espectrometria de Massas em Tandem/normas , Apolipoproteínas/química , Apolipoproteínas M , Coagulação Sanguínea/fisiologia , Plaquetas/química , Plaquetas/metabolismo , Cromatografia Líquida/métodos , Humanos , Lipocalinas/química , Lipoproteínas HDL/química , Extração Líquido-Líquido/métodos , Lisofosfolipídeos/biossíntese , Ativação Plaquetária/fisiologia , Ligação Proteica , Albumina Sérica/química , Esfingosina/biossíntese , Esfingosina/sangue , Espectrometria de Massas em Tandem/métodosRESUMO
BACKGROUND: Despite advances in anti-platelet treatments, there still exists an early increase in both ischemic as well as bleeding events following primary PCI in patients with ST-elevation myocardial infarction (STEMI). Platelet inhibition data of different anti-platelet treatments in the acute phase of a myocardial infarction might offer some insight into these problems. The aim of this study was to evaluate the pharmacodynamic profile of 5 different anti-platelet treatments in the acute phase of STEMI in patients undergoing primary PCI. METHODS: A total of 223 STEMI patients undergoing primary PCI were prospectively included. Patients received either pre-hospital clopidogrel only, pre-hospital clopidogrel followed by prasugrel switch in the cath lab, prasugrel treatment only, pre-hospital clopidogrel followed by ticagrelor switch in the cath lab or pre-hospital ticagrelor only. Platelet reactivity was measured serially using vasodilator-stimulated phosphoprotein (VASP). RESULTS: Patients receiving pre-hospital clopidogrel followed by prasugrel switch showed similar platelet inhibition data as patients receiving prasugrel only, with more than 90% being good responders the day after PCI. Average time from prasugrel administration to a VASP value of <50% was 1.5 hours. In patients receiving pre-hospital ticagrelor, 50% were good responders at completion of PCI and average time to a VASP-value of <50% was 2.3 hours. Only 32% of patients receiving clopidogrel only were responders the day after PCI. CONCLUSIONS: Switching from an upstream bolus dose of clopidogrel to prasugrel at the time of PCI, appeared as a safe and feasible option with no tendency for overshoot or attenuation of platelet inhibition. Pre-hospital administration of ticagrelor was associated with a 50% good responder rate at completion of PCI.
Assuntos
Plaquetas/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/cirurgia , Intervenção Coronária Percutânea , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Adenosina/efeitos adversos , Adenosina/análogos & derivados , Adenosina/farmacologia , Adenosina/uso terapêutico , Idoso , Protocolos Clínicos , Clopidogrel , Esquema de Medicação , Eletrocardiografia , Feminino , Hemorragia/induzido quimicamente , Humanos , Masculino , Pessoa de Meia-Idade , Piperazinas/efeitos adversos , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores da Agregação Plaquetária/efeitos adversos , Cloridrato de Prasugrel , Estudos Prospectivos , Tiofenos/efeitos adversos , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Ticagrelor , Ticlopidina/efeitos adversos , Ticlopidina/análogos & derivados , Ticlopidina/farmacologia , Ticlopidina/uso terapêuticoRESUMO
BACKGROUND: Patients with bone marrow failure and severe thrombocytopenia are frequently given prophylactic platelet transfusion before interventions. The clinical effects of such transfusions, however, are poorly defined. We performed a prospective observational study on patients with bone marrow failure scheduled for prophylactic platelet transfusion before the insertion of a central venous catheter. The objectives were to evaluate the effect and duration of prophylactic platelet transfusions on central venous catheter insertion in thrombocytopenic patients with bone marrow failure. METHODS: Thirty-nine adult patients with bone marrow failure and platelet counts below 50 × 10/L were consecutively enrolled before prophylactic platelet transfusion for subclavian central venous catheter insertion. Blood samples were drawn from the patients before platelet transfusion, 1 hour, and 4 hours after completion of the transfusion. The coagulation profile was assessed by conventional hematological tests, thromboelastometry (ROTEM) assays (EXTEM and FIBTEM), multiple electrode aggregometry (Multiplate) assays including adenosine diphosphate, collagen, and thrombin receptor agonist peptide, and by flow cytometry for the platelet expression of P-selectin (CD62P) and activated glycoprotein IIb-IIIa (PAC-1). Bleeding complications were classified with a 5-grade scale, according to the Common Terminology Criteria for Adverse Events. RESULTS: Seventeen women and 22 men were included in the study. Platelet count was increased from 24 × 10/L (18-32) before to 42 × 10/L (31-50) 1 hour after transfusion (P < 0.0001) and was not significantly different 4 hours after transfusion (40 × 10/L (29-50), P = 0.047). Maximal clot firmness EXTEM was increased from 38 mm (32-45) before to 46 mm (41-52) 1 hour after transfusion (P < 0.0001) and did not change 4 hours after transfusion. Clotting time EXTEM was decreased from 58.5 seconds (50-78) beforehand to 53 seconds (45-61) 1 hour after transfusion (P = 0.0006) and was not significantly different 4 hours after transfusion (57 seconds (52-70, P = 0.025). FIBTEM results were all unchanged after transfusion. All Multiplate analyses were significantly increased after 1 hour and were not diminished 4 hours after transfusion. Four grade 1 bleeding episodes occurred, but no grade 2 to 5 bleeding could be detected. Flow cytometry analyses showed mixed results with no overall trend. CONCLUSIONS: Prophylactic platelet transfusions in thrombocytopenic patients with bone marrow failure improve hemostatic parameters on ROTEM and Multiplate by increasing the number of platelets, and not through enhancement of platelet function. Improved clotting parameters on ROTEM and platelet aggregation on Multiplate appear to persist between 1 and 4 hours after transfusion.
Assuntos
Transplante de Medula Óssea/normas , Cateteres Venosos Centrais/normas , Citometria de Fluxo/normas , Transfusão de Plaquetas/normas , Sistemas Automatizados de Assistência Junto ao Leito/normas , Adulto , Idoso , Transplante de Medula Óssea/métodos , Feminino , Citometria de Fluxo/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas/métodos , Contagem de Plaquetas/normas , Transfusão de Plaquetas/métodos , Fatores de Tempo , Falha de Tratamento , Resultado do TratamentoRESUMO
Protein S has an important anticoagulant function by acting as a cofactor for activated protein C (APC). We recently reported that the EGF1 domain residue Asp95 is critical for APC cofactor function. In the present study, we examined whether additional interaction sites within the Gla domain of protein S might contribute to its APC cofactor function. We examined 4 residues, composing the previously reported "Face1" (N33S/P35T/E36A/Y39V) variant, as single point substitutions. Of these protein S variants, protein S E36A was found to be almost completely inactive using calibrated automated thrombography. In factor Va inactivation assays, protein S E36A had 89% reduced cofactor activity compared with wild-type protein S and was almost completely inactive in factor VIIIa inactivation; phospholipid binding was, however, normal. Glu36 lies outside the ω-loop that mediates Ca(2+)-dependent phospholipid binding. Using mass spectrometry, it was nevertheless confirmed that Glu36 is γ-carboxylated. Our finding that Gla36 is important for APC cofactor function, but not for phospholipid binding, defines a novel function (other than Ca(2+) coordination/phospholipid binding) for a Gla residue in vitamin K-dependent proteins. It also suggests that residues within the Gla and EGF1 domains of protein S act cooperatively for its APC cofactor function.
Assuntos
Ácido 1-Carboxiglutâmico/fisiologia , Proteína C/metabolismo , Proteína S/metabolismo , Proteína S/fisiologia , Ácido 1-Carboxiglutâmico/química , Sequência de Aminoácidos , Substituição de Aminoácidos/fisiologia , Sítios de Ligação/genética , Domínio Catalítico/genética , Células Cultivadas , Fator VIIIa/metabolismo , Fator Va/metabolismo , Humanos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , Proteína C/agonistas , Proteína C/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteína S/química , Proteína S/genética , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
This article analyzes the migration experiences of thirteen separated minors who arrived in Sweden between 1943 and 2008. Using the framework of "dislocation" and the "liberated self," this chapter shows that the experiences of separated minors are shaped in the intersection between contexts and conditions of transnational migration and the Swedish reception system. Their efforts to continue living based on the past and building a new life during a period of transition between different countries and between childhood and adulthood can be described as "a life on hold." The paradox that migration serves simultaneously to empower and render children powerless is discussed.
Assuntos
Menores de Idade/psicologia , Relações Pais-Filho , Poder Psicológico , Refugiados/psicologia , Isolamento Social , Serviço Social/normas , Migrantes/psicologia , Adolescente , Relações Familiares , Feminino , Humanos , Controle Interno-Externo , Cooperação Internacional , Entrevistas como Assunto , Luxações Articulares , Masculino , Menores de Idade/legislação & jurisprudência , Refugiados/legislação & jurisprudência , Isolamento Social/psicologia , Apoio Social , Serviço Social/métodos , Fatores Socioeconômicos , Inquéritos e Questionários , Sobrevida/psicologia , Suécia , Migrantes/legislação & jurisprudência , Nações UnidasRESUMO
Apoptotic cells are opsonized by complement components such as C1q and C3b, which increases their susceptibility to phagocytosis. Soluble complement inhibitors such as factor H (fH) also recognize apoptotic cells to minimize the pro-inflammatory effects of downstream complement activation. We used four radiolabeled protein constructs that span different regions of the 20 complement control protein (CCP) modules that make up fH and found that fragments comprising CCPs 6-8, CCPs 8-15, and CCPs 19-20 but not CCPs 1-4, bound to apoptotic Jurkat T cells. There are four possible ligand types on apoptotic cells that could recruit fH: proteins, carbohydrates, lipids, and DNA. We found that CCPs 6-8 of fH bind to annexin-II, a trypsin-insensitive protein that becomes exposed on surfaces of apoptotic cells. The second ligand of fH, which interacts with CCPs 6-8 and 19-20, is DNA. Confocal microscopy showed co-localization of fH with antibodies specific for DNA. fH also binds to histones devoid of DNA, and CCPs 1-4, 6-8, and 8-15 mediate this interaction. Treatment of apoptotic cells with neuraminidase, chondroitinase, heparitinase, and heparinase did not change fH binding. Treatment of apoptotic cells with phospholipase A(2) dramatically increased both binding of fH and cell-surface DNA. We also excluded the possibility that fH interacts with lysophospholipids using surface plasmon resonance and flow cytometry with lipid-coated beads. Identification of annexin-II as one of the fH ligands on apoptotic cells together with the fact that autoantibodies against annexin-II are found in systemic lupus erythematosus provides further insight into understanding the pathogenesis of this disease.
Assuntos
Anexina A2/metabolismo , Apoptose , Membrana Celular/metabolismo , Fator H do Complemento/metabolismo , DNA de Neoplasias/metabolismo , Histonas/metabolismo , Sítios de Ligação , Proteína de Ligação ao Complemento C4b/metabolismo , Citometria de Fluxo , Glicosaminoglicanos/metabolismo , Humanos , Imunoprecipitação , Células Jurkat , Ligantes , Microscopia Confocal , Ácido N-Acetilneuramínico/metabolismo , Necrose , Fosfolipases A2/metabolismo , Fosfolipases A2/farmacologia , Ligação Proteica/efeitos dos fármacos , Ressonância de Plasmônio de SuperfícieRESUMO
BACKGROUND: Monitoring hemophilia treatment with extended half-life products is challenging for coagulation laboratories since factor assays may show substantial differences between results obtained with the one-stage assay (OSA) and the chromogenic substrate assay (CSA). OBJECTIVES: The aim of this study was to evaluate and compare different factor assays and global coagulation methods. METHODS: Factor VIII (FVIII) and IX (FIX) activities and global assay parameters were analyzed in pre- and postinfusion samples (5 patients 2 samples/product/method). RESULTS: Samples containing FVIII products (NovoEight, Elocta, and Nuwiq) gave higher levels when measured with CSA compared to OSA. The correlation was excellent (r 2 ≥ .97) while biases of 42%-72% of mean (CSA-OSA) were obtained. With FVIII (OSA) as independent variable, the correlations to kaolin clot time (CT) and thrombin generation assay (TGA) peak were modest (r2 = .71-.72 and .64-.65, respectively), except for Nuwiq for which there was a poor correlation to TGA peak (r 2 = .08). Samples containing Alprolix, a FIX product, gave a smaller difference between activity levels (CSA-OSA), and the correlation was excellent (r 2 = .96). With FIX (CSA) as independent variable for both Alprolix and Refixia, the correlations to Innovin CT and TGA peaks were weak (r 2 = .33-.45 and .44-.76, respectively). CONCLUSIONS: Our data show that factor activity assays differ between methods used and agents. These discrepancies indicate the value of having more than one type of assay available in the coagulation laboratory when monitoring hemophilia treatment with extended half-life products. Global assays gave complementary information indicated by the modest correlations to factor activities.
RESUMO
: Fibrinogen is essential for normal hemostasis. Congenital fibrinogen disorders (afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia and hypodysfibrinogenemia), caused by pathogenic variants in the genes FGA, FGB and FGG, have the potential of causing bleeding diathesis and/or thrombotic events of variable severity. We describe a case of familial hypofibrinogenemia in a Swedish family. The proband is a 27-year-old woman, with a history of significant bleeding diathesis. She was diagnosed with moderate hypofibrinogenemia (0.8âg/l), and genetic screening identified a rare heterozygous missense variant in FGB (c.854G>A, p.Arg285His) (Fibrinogen Merivale) previously described in a New Zealand European family with symptomatic hypofibrinogenemia. The father, sister and brother of the proband also harbored the FGB variant, segregating with hypofibrinogenemia (0.9-1.2âg/l). The proband showed a more severe bleeding phenotype compared with her other hypofibrinogenemic family members; this was attributed to a concomitant platelet dysfunction, also present in her normofibrinogenemic mother.
Assuntos
Afibrinogenemia/genética , Fibrinogênio/genética , Adulto , Feminino , Humanos , SuéciaRESUMO
BACKGROUND: Flow cytometric platelet activation has emerged as an alternative diagnostic test for inherited platelet disorders. It is, however, labor intensive and few studies have directly compared the performance of flow cytometric platelet activation (PACT) to light transmission aggregometry (LTA). The aims of this study were 1/ to develop a simplified flow cytometric platelet activation assay using microtiter plates and 2/ to correlate the outcome to gold standard method LTA, and to clinical bleeding assessment tool scores (BAT score). METHODS: The PACT method was developed in microtiter plates using adenosine diphosphate (ADP), collagen-derived peptide (CRP-XL) and thrombin receptor activator for peptide 6 (TRAP-6) as agonists. Antibodies against GPIIb-IIIa activation epitope (PAC1), P-selectin (CD62P) and lysosome-associated membrane glycoprotein 3 (LAMP3; CD63) were used as platelet activation markers. Sixty-six patients referred to the coagulation unit for bleeding symptoms were included in this single-center observational study. Platelet activation was determined by PACT and LTA. The results of both methods were correlated to BAT score. RESULTS: A two-by-two analysis using Cohen's kappa analysis gave moderate agreement between LTA and PACT (82%, kappa = 0.57), when PACT analysis with ADP and CRP-XL was compared to LTA. Using LTA as reference method, positive predictive value was 70% and negative predictive value was 87%. A substantial number of patients had high BAT score and normal LTA and PACT results. Patients with abnormal LTA or PACT results had higher BAT score than patients with normal results, but the difference was not significant. CONCLUSIONS: The performance in microtiter plates simplified the PACT method and enabled analysis of more patients at the same time. Our results indicate that with modification of the current PACT assay, a higher negative predictive value can be obtained. Furthermore, with comparable result to LTA the PACT could be used as a screening assay for inherited platelet disorders.
Assuntos
Transtornos Plaquetários , Plaquetas/metabolismo , Citometria de Fluxo/métodos , Doenças Genéticas Inatas , Ativação Plaquetária , Transtornos Plaquetários/sangue , Transtornos Plaquetários/diagnóstico , Feminino , Doenças Genéticas Inatas/sangue , Doenças Genéticas Inatas/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Função Plaquetária/métodosRESUMO
INTRODUCTION: Platelets are the main source of microparticles in plasma and the concentration of microparticles is increased in many diseases. As microparticles expose negatively charged phospholipids, they can bind and assemble the procoagulant enzyme-cofactor complexes. Our aim was to elucidate possible regulation of these complexes on microparticles by the anticoagulant protein C system. MATERIALS AND METHODS: Platelets were activated with thrombin ± collagen or the calcium ionophore A23187 ± thrombin to generate microparticles. The microparticles were analyzed using flow cytometry and functional coagulation assays to characterize parameters with importance for the activated protein C system. RESULTS: Activation with A23187+thrombin was most efficient, fully converting the platelets to microparticle-like vesicles, characterized by high lactadherin and protein S binding capacity. Suppression of thrombin generation by activated protein C in plasma spiked with these microparticles was dependent on the presence of plasma protein S. Experiments with purified components showed that activated protein C inhibited both factor Va and factor VIIIa on the microparticle surface. Inhibition of factor Va was stimulated by, but not fully dependent on, the presence of protein S. In the factor VIIIa-degradation, activated protein C was dependent on the addition of protein S, and exogenous factor V further increased the efficiency. CONCLUSIONS: Protein S is crucial for activated protein C-mediated inhibition of thrombin generation on platelet-derived microparticles in plasma. Moreover, protein S and factor V are synergistic cofactors in the inhibition of factor VIIIa. The results demonstrate that the activated protein C system has the capacity to counterbalance the procoagulant ability of microparticles.
Assuntos
Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Fator V/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Proteína C/metabolismo , Proteína S/metabolismo , Trombina/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Humanos , CinéticaRESUMO
Elevated levels of erythrocyte-derived microparticles are present in the circulation in medical conditions affecting the red blood cells. Erythrocyte-derived microparticles expose phosphatidylserine thus providing a suitable surface for procoagulant reactions leading to thrombin formation via the tenase and prothrombinase complexes. Patients with elevated levels of circulating erythrocyte-derived microparticles have increased thrombin generation in vivo. The aim of the present study was to investigate whether erythrocyte-derived microparticles are able to support the anticoagulant reactions of the protein C system. Erythrocyte-derived microparticles were isolated using ultracentrifugation after incubation of freshly prepared erythrocytes with the ionophore A23187 or from outdated erythrocyte concentrates, the different microparticles preparations yielding similar results. According to flow cytometry analysis, the microparticles exposed phoshatidylserine and bound lactadherin, annexin V, and protein S, which is a cofactor to activated protein C. The microparticles were able to assemble the tenase and prothrombinase complexes and to stimulate the formation of thrombin in plasma-based thrombin generation assay both in presence and absence of added tissue factor. The addition of activated protein C in the thrombin generation assay inhibited thrombin generation in a dose-dependent fashion. The anticoagulant effect of activated protein C in the thrombin generation assay was inhibited by a monoclonal antibody that prevents binding of protein S to microparticles and also attenuated by anti-TFPI antibodies. In the presence of erythrocyte-derived microparticles, activated protein C inhibited tenase and prothrombinase by degrading the cofactors FVIIIa and FVa, respectively. Protein S stimulated the Arg306-cleavage in FVa, whereas efficient inhibition of FVIIIa depended on the synergistic cofactor activity of protein S and FV. In summary, the erythrocyte-derived microparticle surface is suitable for the anticoagulant reactions of the protein C system, which may be important to balance the initiation and propagation of coagulation in vivo.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Micropartículas Derivadas de Células/efeitos dos fármacos , Proteína C/farmacologia , Trombina/biossíntese , Anexina A5/farmacologia , Anticorpos/farmacologia , Antígenos de Superfície/metabolismo , Testes de Coagulação Sanguínea , Calcimicina/farmacologia , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Eritrócitos/química , Fator V/metabolismo , Fator VIIIa/metabolismo , Fator VIIIa/farmacologia , Fator Va/metabolismo , Fator Va/farmacologia , Fator Xa/metabolismo , Humanos , Lipoproteínas/antagonistas & inibidores , Lipoproteínas/metabolismo , Proteínas do Leite/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Proteína C/metabolismo , Proteína S/metabolismo , Proteína S/farmacologia , Tromboplastina/metabolismo , Tromboplastina/farmacologia , UltracentrifugaçãoRESUMO
Systemic inflammatory response syndrome and severe infections are associated with major derangements in the coagulation system. The purpose of this study was to examine the dynamic alterations in thrombin generation in abdominal sepsis. Abdominal sepsis was induced by cecal ligation and puncture (CLP) in C57/Bl6 mice. Cecal ligation and puncture caused a systemic inflammatory response, with neutrophil recruitment and tissue damage in the lung as well as thrombocytopenia and leukocytopenia. Thrombin generation, coagulation factors, lung histology, and myeloperoxidase activity was determined 1, 3, 6, and 24 h after induction of CLP. It was found that thrombin generation was increased 1 h after CLP and that thrombin generation started to decrease at 3 h and was markedly reduced 6 and 24 h after CLP induction. Platelet-poor plasma from healthy mice could completely reverse the inhibitory effect of CLP on thrombin generation, suggesting that sepsis caused a decrease in the levels of plasma factors regulating thrombin generation in septic animals. Indeed, it was found that CLP markedly decreased plasma levels of prothrombin, factor V, and factor X at 6 and 24 h. Moreover, we observed that CLP increased plasma levels of activated protein C at 6 h, which returned to baseline levels 24 h after CLP induction. Finally, pretreatment with imipenem/cilastatin attenuated the CLP-evoked decrease in thrombin generation and consumption of prothrombin 24 h after CLP induction. Our novel findings suggest that thrombin generation is initially increased and later decreased in abdominal sepsis. Sepsis-induced reduction in thrombin generation is correlated to changes in the plasma levels of coagulation factors and activated protein C. These findings help explain the dynamic changes in global hemostasis in abdominal sepsis.