RESUMO
Emicizumab, a bispecific humanised monoclonal antibody restoring to some extent the function of activated FVIII deficient in haemophilia A, represents a major therapeutic advance in the management of haemophilia A patients. No dosage adjustment is required, which leads to a major change for patients used to regular biological monitoring which is particularly burdensome in the case of substitution therapy. In some circumstances, such as before an invasive procedure or in case of bleeding, biological monitoring will be necessary and emicizumab's interference with haemostasis tests, particularly those based on an activated partial thromboplastin times (aPTT), must be known to best interpret the tests and to select the most appropriate methods to guide therapy. The normalisation of aPTT in patients treated with emicizumab is not sufficient to consider haemostasis as normalised. In the event of administration of FVIII to a patient receiving emicizumab, the determination of FVIII should use a chromogenic method using non-human reagents. Coagulation global tests have been proposed to evaluate the biological response when using bypassing agents in patients treated with emicizumab, but the usefulness must be confirmed. The French group BIMHO presents proposals for biological monitoring of a patient treated with emicizumab according to clinical situations.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Hemofilia A/sangue , Hemofilia A/tratamento farmacológico , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/efeitos adversos , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Testes de Coagulação Sanguínea/métodos , Hemostasia/efeitos dos fármacos , Humanos , Resultado do TratamentoAssuntos
Anticorpos Biespecíficos , Hemofilia A , Humanos , Criança , Adolescente , Hemofilia A/complicações , Hemofilia A/tratamento farmacológico , Qualidade de Vida , Hemorragia/prevenção & controle , Hemorragia/tratamento farmacológico , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Biespecíficos/farmacologia , Percepção , Fator VIII/uso terapêuticoRESUMO
OBJECTIVE: Elastin is the major structural extracellular matrix component of the arterial wall that provides the elastic recoil properties and resilience essential for proper vascular function. Elastin-derived peptides (EDP) originating from elastin fragmentation during vascular remodeling have been shown to play an important role in cell physiology and development of cardiovascular diseases. However, their involvement in thrombosis has been unexplored to date. In this study, we investigated the effects of EDP on (1) platelet aggregation and related signaling and (2) thrombus formation. We also characterized the mechanism by which EDP regulate thrombosis. APPROACH AND RESULTS: We show that EDP, derived from organo-alkaline hydrolysate of bovine insoluble elastin (kappa-elastin), decrease human platelet aggregation in whole blood induced by weak and strong agonists, such as ADP, epinephrine, arachidonic acid, collagen, TRAP, and U46619. In a mouse whole blood perfusion assay over a collagen matrix, kappa-elastin and VGVAPG, the canonical peptide recognizing the elastin receptor complex, significantly decrease thrombus formation under arterial shear conditions. We confirmed these results in vivo by demonstrating that both kappa-elastin and VGVAPG significantly prolonged the time for complete arteriole occlusion in a mouse model of thrombosis and increased tail bleeding times. Finally, we demonstrate that the regulatory role of EDP on thrombosis relies on platelets that express a functional elastin receptor complex and on the ability of EDP to disrupt plasma von Willebrand factor interaction with collagen. CONCLUSIONS: These results highlight the complex nature of the mechanisms governing thrombus formation and reveal an unsuspected regulatory role for circulating EDP in thrombosis.
Assuntos
Elastina/fisiologia , Trombose/etiologia , Animais , Plaquetas/fisiologia , Catepsina A/sangue , Bovinos , Colágeno/sangue , Elastina/sangue , Elastina/química , Humanos , Camundongos , Neuraminidase/sangue , Oligopeptídeos/sangue , Oligopeptídeos/química , Oligopeptídeos/fisiologia , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Agregação Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteólise , Receptores de Superfície Celular/sangue , Transdução de Sinais , Trombose/sangue , Remodelação Vascular/fisiologia , Fator de von Willebrand/metabolismoRESUMO
Bernard-Soulier syndrome (BSS) is a rare autosomal recessive bleeding disorder characterized by defects of the GPIb-IX-V complex, a platelet receptor for von Willebrand factor (VWF). Most of the mutations identified in the genes encoding for the GP1BA (GPIbα), GP1BB (GPIbß), and GP9 (GPIX) subunits prevent expression of the complex at the platelet membrane or more rarely its interaction with VWF. As a consequence, platelets are unable to adhere to the vascular subendothelium and agglutinate in response to ristocetin. In order to collect information on BSS patients, we established an International Consortium for the study of BSS, allowing us to enrol and genotype 132 families (56 previously unreported). With 79 additional families for which molecular data were gleaned from the literature, the 211 families characterized so far have mutations in the GP1BA (28%), GP1BB (28%), or GP9 (44%) genes. There is a wide spectrum of mutations with 112 different variants, including 22 novel alterations. Consistent with the rarity of the disease, 85% of the probands carry homozygous mutations with evidence of founder effects in some geographical areas. This overview provides the first global picture of the molecular basis of BSS and will lead to improve patient diagnosis and management.
Assuntos
Síndrome de Bernard-Soulier/genética , Variação Genética , Mutação , Alelos , Síndrome de Bernard-Soulier/diagnóstico , Bases de Dados de Ácidos Nucleicos , Efeito Fundador , Humanos , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Polimorfismo de Nucleotídeo Único , Navegador , Doenças de von Willebrand/genéticaRESUMO
A rapid lateral flow immunoassay (LFIA) (STic Expert(®) HIT), recently developed for the diagnosis of heparin-induced thrombocytopenia (HIT), was evaluated in a prospective multicentre cohort of 334 consecutive patients. The risk of HIT was estimated by the 4Ts score as low, intermediate and high in 28·7%, 61·7% and 9·6% of patients, respectively. Definite HIT was diagnosed in 40 patients (12·0%) with positive results on both enzyme-linked immunosorbent assay (Asserachrom(®) HPIA IgG) and serotonin release assay. The inter-reader reproducibility of results obtained was excellent (kappa ratio > 0·9). The negative predictive value of LFIA with plasma samples was 99·6% with a negative likelihood ratio (LR) of 0·03, and was comparable to those of the particle gel immunoassay (H/PF4-PaGIA(®) ) performed in 124 cases. Positive predictive value and positive LR were 44·4% and 5·87, respectively, and the results were similar for serum samples. The probability of HIT in intermediate risk patients decreased from 11·2% to 0·4% when the LFIA result was negative and increased to 42·5% when it was positive. In conclusion, the STic Expert(®) HIT combined with the 4Ts score is a reliable tool to rule out the diagnosis of HIT.
Assuntos
Heparina/efeitos adversos , Imunoensaio/métodos , Nanopartículas/química , Trombocitopenia/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Trombocitopenia/induzido quimicamenteRESUMO
Background: Despite the wide use of bleeding scores and the reliability of clotting factor level measurement, bleeding risk stratification before surgery remains challenging in patients with rare inherited bleeding disorders. Objectives: This multicenter observational prospective study assessed in patients with rare coagulation factor deficiency, the perioperative hemostatic management choices by hemostasis experts and the bleeding outcomes after surgery. Methods: One hundred seventy-eight patients with low coagulation activity level (factor [F] II, FV, combined FV-FVIII, FVII, FX, or FXI <50%) underwent 207 surgical procedures. The bleeding outcome, Tosetto's bleeding score, and perioperative hemostatic protocols were collected. Results: Among the 81 procedures performed in patients with severe factor deficiency (level ≤10%), 27 were done without factor replacement (including 6 in patients at high bleeding risk), without any bleeding event. Factor replacement therapy was used mainly for orthopedic procedures. In patients with mild deficiency, 100/126 surgical procedures were carried out without perioperative hemostatic treatment. In patients with FVII or FXI deficiency, factor replacement therapy was in function of the procedure, bleeding risk, and to a lesser extent previous bleeding history. Tranexamic acid was used in almost half of the procedures, particularly in case of surgery in tissues with high fibrinolytic activity (76.8%). Conclusions: The current perioperative hemostatic management of patients with rare bleeding disorders appears to be adapted. Among the 207 procedures, only 6 were associated with excessive bleeding. Our findings suggest that rather than the bleeding score, factor level and surgery type are the most relevant criteria for perioperative factor replacement therapy.
RESUMO
In addition to its established immuno-regulating capacity, the anti-inflammatory cytokine interleukin (IL)-10 exerts direct effects on coagulation. IL-10 down regulates the expression of tissue factor (TF) and thrombin generation (TG). Thus, we hypothesised that IL-10 could enhance the effect of anticoagulants. To evaluate in vitro the potential additive effect of IL-10 on fondaparinux-induced anticoagulation. Human monocytes were purified by elutriation, and were activated by factor Xa (FXa). Real-time RT-PCR and Western blotting were used to evaluate FXa-induced TF synthesis. TG test was used as a functional test to assess TF-dependent monocyte procoagulation, and to evaluate the effects of IL-10 (200 and 500 pg/ml) and fondaparinux (0.0, 0.1, 0.4, 0.7 and 1.2 µg/ml), separately and in combination. We confirmed that FXa induced TF mRNA and protein synthesis by monocyte in a concentration dependent manner. We showed that FXa-activated monocytes triggered TG via TF expression. We reported that IL-10 inhibited TG with a marginal effect seen at 200 pg/ml. Results with fondaparinux showed a concentration-dependent TG inhibition. The combination of IL-10 and fondaparinux effects demonstrated that IL-10: (i) potentiates the inhibitory effect of fondaparinux on TG by 10-30%, and (ii) dramatically modifies fondaparinux IC50 for each TG parameter. IL-10 enhances in vitro the extent of anticoagulation induced by fondaparinux.
Assuntos
Anticoagulantes/farmacologia , Interleucina-10/farmacologia , Monócitos/metabolismo , Polissacarídeos/farmacologia , Trombina/biossíntese , Adulto , Relação Dose-Resposta a Droga , Fator Xa/farmacologia , Feminino , Fondaparinux , Humanos , Masculino , Monócitos/citologia , RNA Mensageiro/biossínteseRESUMO
BACKGROUND: Coagulation factor V (FV), present in plasma and platelets, has both pro- and anticoagulant functions. OBJECTIVE: We investigated an FV-deficient patient (FV:C 3%, FV:Ag 4%) paradoxically presenting with recurrent venous thrombosis (11 events) instead of bleeding. METHODS/RESULTS: Thrombophilia screening revealed only heterozygosity for the F2 20210G>A mutation. Although thrombin generation in the patient's platelet-poor plasma was suggestive of a hypocoagulable state, thrombin generation in the patient's platelet-rich plasma (PRP) was higher than in control PRP and extremely resistant to activated protein C (APC). This was partially attributable to the complete abolition of the APC-cofactor activity of FV and a marked reduction of plasma tissue factor pathway inhibitor antigen and activity. The patient was homozygous for a novel missense mutation (Ala2086Asp, FVBesançon ) that favors a "closed conformation" of the C2 domain, predicting impaired binding of FV(a) to phospholipids. Recombinant FVBesançon was hardly secreted, indicating that this mutation is responsible for the patient's FV deficiency. Model system experiments performed using highly diluted plasma as a source of FV showed that, compared with normal FVa, FVaBesançon has slightly (≤1.5-fold) unfavorable kinetic parameters (Km , Vmax ) of prothrombin activation, but also a lower rate of APC-catalyzed inactivation in the presence of protein S. CONCLUSIONS: FVBesançon induces a hypercoagulable state via quantitative (markedly decreased FV level) and qualitative (phospholipid-binding defect) effects that affect anticoagulant pathways (anticoagulant activities of FV, FVa inactivation, tissue factor pathway inhibitor α level) more strongly than the prothrombinase activity of FVa. A possible specific role of platelet FV cannot be excluded.
Assuntos
Fator V , Trombofilia , Testes de Coagulação Sanguínea , Fator V/genética , Homozigoto , Humanos , Mutação , Trombofilia/genéticaRESUMO
Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation due to defects in integrin alphaIIbbeta3 (ITGA2B, ITGB3), a fibrinogen receptor. Mutations from 24 GT patients and two carriers of various origins, Caucasian, North-African and Asian were characterized. Promoter and exon sequences of alphaIIb and beta3 genes were amplified and directly sequenced. Among 29 identified mutations, 17 new allelic variants resulting from nonsense, missense and deletion/insertion mutations were described. RNA alterations were evaluated by using Web servers. The alphaIIb p.S926L, p.V903F, and beta3 p.C38Y, p.M118R, p.G221D substitutions prevented complex expression at the surface of COS-7 cells by altering the alphaIIb or the beta3 subunit structure. As shown by free energy analyses applied on the resolved structure of alphaIIbbeta3 and structural modeling of the mutant, the p.K253M substitution of beta3 helped to define a key role of the K253 in the interaction of the alphaIIb beta-propeller and the beta3 beta-I domains. finally, the alphaIIb p.Q595H substitution allowed cell surface expression of the complex but its corresponding c.2800G>T mutation is predicted to alter normal RNA splicing. In conclusion, our study yielded the discovery of 17 new GT allelic variants, revealed the key role of K253 of alphaIIb for the alphaIIbbeta3 complex formation and provides an additional example of an apparently missense mutation causing a splicing defect.
Assuntos
Alelos , Processamento Alternativo , Integrina alfa2/genética , Integrina beta3/genética , Trombastenia/genética , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Análise Mutacional de DNA , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Receptores de Fibrinogênio/genética , Relação Estrutura-AtividadeRESUMO
Monocytes and monocyte-derived microparticles (MMPs) play a major role in acute coronary syndrome (ASC). Activated monocytes (ac-M) and MMPs support thrombin generation via tissue factor (TF). The aim of this study was to evaluate the inhibitory effect of fondaparinux, a selective Xa inhibitor, on thrombin generation supported by activated monocytes and MMPs. Monocytes were purified by elutriation. They were activated by LPS, allowing to obtain both ac-M and MMPs. Thrombin generation was performed using Fluoroscan(®) in these two cell models, in comparison with a cell-free model (TF 5 pM final). Two concentrations of ac-M (0.2 × 106 and 1 × 106/well) and four concentrations of MMPs (40,000; 80,000; 120,000 and 160,000/well) were tested. TGT was evaluated for increasing fondaparinux concentrations (0, 0.1, 0.4, 0.7 and 1.2 µg/ml). Without fondaparinux, 0.2 × 106 ac-M and 160,000 MMPs induced comparable results. Fondaparinux inhibited thrombin generation in the three models. Inhibition was fondaparinux concentration dependent. Rate index was the most sensitive parameter, compared to lag-time, peak and endogenous thrombin potential. The rate index IC(50) were 0.69 ± 0.03 µg/ml for ac-M, 0.20 ± 0.03 µg/ml for MMPs, and 0.22 ± 0.02 µg/ml for cell-free model. Fondaparinux exerted an inhibitory effect at all concentrations, including the lowest (0.1 µg/ml). The extend of inhibition was similar between MMPs and cell-free models, and stronger than ac-M model. We assume that the efficacy of fondaparinux 2.5 mg once daily in ACS patients may be in part attributed to its inhibitory effect on MMPs.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Micropartículas Derivadas de Células/efeitos dos fármacos , Polissacarídeos/farmacologia , Adulto , Coagulação Sanguínea/fisiologia , Micropartículas Derivadas de Células/fisiologia , Relação Dose-Resposta a Droga , Feminino , Fondaparinux , Humanos , Masculino , Monócitos , Polissacarídeos/sangue , Trombina/antagonistas & inibidores , Trombina/fisiologia , Adulto JovemRESUMO
Platelets play a major role in primary hemostasis, where activated platelets form plugs to stop hemorrhaging in response to vessel injuries. Defects in any step of the platelet activation process can cause a variety of platelet dysfunction conditions associated with bleeding. To make an accurate diagnosis, constitutional platelet dysfunction (CPDF) should be considered once von Willebrand disease and drug intake are ruled out. CPDF may be associated with thrombocytopenia or a genetic syndrome. CPDF diagnosis is complex, as no single test enables the analysis of all aspects of platelet function. Furthermore, the available tests lack standardization, and repeat tests must be performed in specialized laboratories especially for mild and moderate forms of the disease. In this review, we provide an overview of the laboratory tests used to diagnose CPDF, with a focus on light transmission platelet aggregation (LTA), flow cytometry (FC), and granules assessment. Global tests, mainly represented by LTA, are often initially performed to investigate the consequences of platelet activation on platelet aggregation in a single step. Global test results should be confirmed by additional analytical tests. FC represents an accurate, simple, and reliable test to analyze abnormalities in platelet receptors, and granule content and release. This technique may also be used to investigate platelet function by comparing resting- and activated-state platelet populations. Assessment of granule content and release also requires additional specialized analytical tests. High-throughput sequencing has become increasingly useful to diagnose CPDF. Advanced tests or external research laboratory techniques may also be beneficial in some cases.
Assuntos
Transtornos Plaquetários/diagnóstico , Laboratórios/normas , Agregação Plaquetária/fisiologia , Testes de Função Plaquetária/métodos , Citometria de Fluxo , HumanosRESUMO
BACKGROUND: Chronic kidney disease (CKD) is associated with a high cardiovascular mortality due to increased rates of vascular lesions and thrombotic events, as well as serum accumulation of uremic toxins. A subgroup of these toxins (advanced glycation end products [AGEs] and S100 proteins) can interact with the receptor for AGEs (RAGE). In this study, we analyzed the impact of CKD on platelet function and arterial thrombosis, and the potential role of RAGE in this process. METHODS: Twelve weeks after induction of CKD in mice, platelet function and time to complete carotid artery occlusion were analyzed in four groups of animals (sham-operated, CKD, apolipoprotein E [Apoe]-/-, and Apoe-/-/Ager-/- mice). RESULTS: Analysis of platelet function from whole blood and platelet-rich plasma showed hyperactivation of platelets only in CKD Apoe-/- mice. There was no difference when experiments were done on washed platelets. However, preincubation of such platelets with AGEs or S100 proteins induced RAGE-mediated platelet hyperactivation. In vivo, CKD significantly reduced carotid occlusion times of Apoe-/- mice (9.2 ± 1.1 vs. 11.1 ± 0.6 minutes for sham, p < 0.01). In contrast, CKD had no effect on occlusion times in Apoe-/-/Ager-/- mice. Moreover, carotid occlusion in Apoe-/- CKD mice occurred significantly faster than in Apoe-/-/Ager-/- CKD mice (p < 0.0001). CONCLUSION: Our results show that CKD induces platelet hyperactivation, accelerates thrombus formation in a murine model of arterial thrombosis, and that RAGE deletion has a protective role. We propose that RAGE ligands binding to RAGE is involved in CKD-induced arterial thrombosis.
Assuntos
Plaquetas/patologia , Ativação Plaquetária , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Insuficiência Renal Crônica/complicações , Trombose/complicações , Animais , Plaquetas/metabolismo , Deleção de Genes , Camundongos Endogâmicos C57BL , Receptor para Produtos Finais de Glicação Avançada/genética , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Trombose/genética , Trombose/metabolismo , Trombose/patologiaAssuntos
Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Proteínas Motores Moleculares/genética , Cadeias Pesadas de Miosina/genética , Trombocitopenia/genética , Trombocitopenia/metabolismo , Adolescente , Adulto , Catarata/genética , Catarata/metabolismo , Criança , Feminino , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Nefrite/genética , Nefrite/metabolismo , Síndrome , Adulto JovemRESUMO
The human interleukin-10 promoter gene is highly polymorphic. IL-10 polymorphisms have been associated with various autoimmune and lymphoproliferative disorders. Although IL-10 has been shown to modulate thrombin generation in several experimental models, it is not known whether IL-10 polymorphisms could be a risk factor for venous thrombosis. We therefore conducted a case-control study comparing 74 consecutive patients who experienced at least one episode of documented venous thromboembolic event and 100 healthy controls. All subjects were Caucasians. Five polymorphisms of the IL-10 promoter gene were studied: two highly polymorphic dinucleotide repeats, IL-10 R and IL-10G, and three single nucleotide polymorphisms at position -1082, -819 and -592. Factor VG1691C Leiden mutation was systematically determined. Multivariate logistic regression analysis showed that IL-10 G13 and G10 alleles are independent risk factors for venous thrombosis (Odds ratio:OR = 3.33, p = 0.003 and OR = 2.83, p = 0.03, respectively). Furthermore, IL-10 G10 allele is more frequent in recurrent disease.
Assuntos
Interleucina-10/genética , Polimorfismo Genético , Regiões Promotoras Genéticas/genética , Trombose Venosa/genética , Adulto , Idoso , Estudos de Casos e Controles , Fator V , Feminino , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Recidiva , Análise de Regressão , Fatores de Risco , Tromboembolia , Trombose Venosa/etiologia , População BrancaRESUMO
Previous studies, using flow cytometry, have reported a lower platelet reactivity in neonates compared to adults. Only few studies were carried out in older children, and results were controversial in terms of age to reach adult platelet function. We studied a total of 125 healthy neonates, infants and older children, and 15 adults. alphaIIbbeta3 expression on resting and activated platelets was lower in all children, with an impaired capability of alphaIIbbeta3 activation (PAC1 and bound fibrinogen). This defect was observed until the age of fifteen with a gradual recovery with age. In neonates, we observed a defect of GPIalpha internalization, and demonstrated that this defect persisted in older children as well. In contrast with alphaIIbbeta3 integrin activation, we did not observe a gradual age-dependent recovery. These unexpected results point out the need for reference values in childhood.
Assuntos
Envelhecimento/sangue , Plaquetas/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Fibrinogênio/metabolismo , Citometria de Fluxo , Humanos , Lactente , Recém-Nascido , Masculino , Fragmentos de Peptídeos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária , Receptores de Trombina/fisiologia , Valores de ReferênciaRESUMO
INTRODUCTION: Platelet response to inhibitors varies widely, leading to a higher risk of abrupt closure events in insufficiently treated-coronary heart disease patients. The aim of this study was to compare, in patients under various antiplatelet regimens, three platelet function assays: aggregometry, PFA-100 and flow cytometry. These assays stand for available tests, as "ready-to-use" device (PFA-100) and sophisticated assay (cytometry). We chose the setting of percutaneous coronary intervention as a standardized procedure to determine which test was appropriate to detect the effect of (1) an aspirin bolus in patients under long-term aspirin treatment, and (2) ticlopidin in case of stent implantation. METHODS: Fifty patients under oral aspirin treatment were randomized to receive a bolus of 500 mg aspirin before angioplasty (n=25). Ticlopidin was given at a 500 mg loading dose in the case of stent implantation (n=38). Platelet function was assessed before, at 2 and 24 h after angioplasty. RESULTS: Considering aspirin antiplatelet effect, the following was observed: (1) a lack of further inhibition after the bolus whatever assay was used and (2) a disagreement between aggregometry and PFA-100 to classify patients as being poor or good aspirin responders (kappa were 0.11 and 0.28 between ADP 4 or 6 microM aggregation, respectively, and PFA-100). Another finding was the good performance of flow cytometry, which evaluated GPIIbIIIa activation, and aggregometry, to detect ticlopidin the day after the loading dose. In contrast, PFA-100 was insensitive to ticlopidin. CONCLUSION: Current assays are not interchangeable to monitor antiplatelet treatment in daily practice.
Assuntos
Angioplastia Coronária com Balão , Inibidores da Agregação Plaquetária/uso terapêutico , Testes de Função Plaquetária/métodos , Aspirina/administração & dosagem , Aspirina/uso terapêutico , Plaquetas/efeitos dos fármacos , Doença das Coronárias/sangue , Doença das Coronárias/tratamento farmacológico , Doença das Coronárias/terapia , Feminino , Citometria de Fluxo/métodos , Humanos , Masculino , Agregação Plaquetária , Inibidores da Agregação Plaquetária/administração & dosagem , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Ticlopidina/administração & dosagem , Ticlopidina/uso terapêuticoRESUMO
The ristocetin cofactor activity assay (VWF:RCo) is the reference method for assessing von Willebrand factor (VWF) activity but remains difficult to perform, and the coefficient of variation of the method is high (about 20-30%). This study evaluated and compared the performance for measuring the VWF activity of two newly commercialised assays [VWF:Ac Innovance (VWF:Ac) and VWF:RCo Acustar (VWF:RCo Acu)] with the reference VWF:RCo aggregation in 123 pathological plasma samples. The correlation and concordance between both new tests (VWF:RCo-Acu and VWF:Ac) and the reference VWF:RCo were good. The results of the VWF activity to VWF antigen ratio were also comparable whatever the method for the classification of VWF deficiency in all patients. Our results showed that both new tests could replace the "gold standard" VWF:RCo in aggregometry with several benefits: they are fully automated, easier and faster to perform, better adapted to emergency situations if necessary.
Assuntos
Testes de Coagulação Sanguínea/métodos , Doenças de von Willebrand/sangue , Fator de von Willebrand/análise , Fator de von Willebrand/imunologia , Automação , Coagulação Sanguínea , Calibragem , Estudos de Casos e Controles , Colágeno/química , Ensaio de Imunoadsorção Enzimática , Humanos , Agregação Plaquetária , Estudos Prospectivos , Valores de Referência , Reprodutibilidade dos Testes , Ristocetina/sangue , Sensibilidade e Especificidade , Doenças de von Willebrand/diagnóstico , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Monocytes and factor Xa (FXa) are procoagulant agents implicated in the physiopathological processes of atherosclerosis and thrombosis. OBJECTIVE: we evaluated the anticoagulant effect of the anti-inflammatory cytokine IL-10 on an FXa-activated human monocyte (Hu-monocyte) procoagulant phenotype. METHODS: Hu-monocytes were purified by elutriation and activated by FXa. The effect of IL-10 was assessed by means of a 2 h pre-incubation step with recombined human IL-10 (0.5 and 1 ng/mL). Real-time RT-PCR and Western blotting were used to evaluate the effect of IL-10 on tissue factor (TF) mRNA and protein synthesis. A thrombin generation (TG) assay was used as a functional test to assess the effect of IL-10 on TF-dependent TG. RESULTS: we showed that IL-10 inhibited both TFmRNA and TF protein expression in a dose-dependant manner.We showed, as a functional consequence, that IL-10 inhibited Hu-monocyte-triggered TG and that this inhibition was concentration-dependant, and significant for all TG phases. The rate index of the propagation phase (rate index) was the most sensitive parameter while the endpoint of TG decay (S-tail) and the endogenous thrombin potential (ETP) were the least sensitive (inhibition of 80, 40 and 30% respectively). The IL-10 pattern of TG inhibition was similar to TF-Ab-induced inhibition: IC(50) were not reached by ETP and S-tail, and the lowest IC(50) values were reached by the rate index (0.61 ± 0.12 ng/mL and 1.87 ± 0.35 µg/mL respectively). CONCLUSION: the anticoagulant effect of the anti-inflammatory cytokine IL-10 in an FXa-activated Hu-monocyte model is an additional illustration of the cross-talk between inflammation and coagulation, opening new approaches in the field of arteriosclerosis and thrombosis.