RESUMEN
BACKGROUND AND OBJECTIVES: A postauthorization safety study was performed between 2009 and 2012 to describe the use of Clottafact® in acquired fibrinogen deficiency in real-life medical practice in France. MATERIALS AND METHODS: One hundred and fifty patients were planned for 28 days of prospective follow-up after infusion. The analysis of this observational study was descriptive and performed according to the type of treatment (curative or preventive) and the origin of the bleed. RESULTS: One hundred and fifty-six patients (16-87 years) were included in 13 centres and treated in five different medical bleeding situations: postpartum (59), other gynaecological/obstetrical (6), trauma (34), liver (13), cardiovascular (23) and other various bleeding situations (21). The mean follow-up time was 18·9 ± 12·3 days. Two patients presented adverse drug reactions: one a pulmonary embolism and the other a four-site venous thromboembolic episode. All were serious with a dubious causal relationship with the study treatment. Efficacy data were collected as a secondary objective. In 150 patients receiving curative treatment, 117 of 159 infusions (73·6%) were considered as successful by the investigators, 35 as moderate (22%) and seven as no response (4·4%). CONCLUSION: The Clottafact® safety profile observed during the study matched the known profile of fibrinogen during use.
Asunto(s)
Afibrinogenemia/tratamiento farmacológico , Coagulantes/efectos adversos , Fibrinógeno/efectos adversos , Hemostáticos/efectos adversos , Adulto , Anciano , Coagulantes/administración & dosificación , Coagulantes/uso terapéutico , Femenino , Fibrinógeno/administración & dosificación , Fibrinógeno/uso terapéutico , Hemostáticos/administración & dosificación , Hemostáticos/uso terapéutico , Humanos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
BACKGROUND AND PURPOSE: Whereas intravenous thrombolysis (IVT) is allowed for acute ischaemic stroke in patients on vitamin K antagonists with international normalized ratio ≤1.7, there are no similar recommendations for patients on direct oral anticoagulants (DOACs), notably due to the lack of coagulation tests to assess the therapeutic effects. Although the literature is scarce, consisting of small case series and retrospective studies, considering the frequency of this situation the French Vascular Neurology Society and the French Study Group on Haemostasis and Thrombosis have worked on a joint position paper to provide a practical position regarding the emergency management of ischaemic stroke in patients on DOACs. METHOD: Based on a review of the literature, the authors wrote a first text that was submitted to a broad panel of members from the two societies. The text was then amended by the authors to address experts' comments and to reach a consensus. RESULTS: In patients with normal renal function and who stopped the DOAC for at least 48 h, the management should not differ from that in patients without oral anticoagulant. In patients who are still on DOACs, mechanical thrombectomy is encouraged preferentially when applicable in first line. Otherwise, when specific tests are available, values <50 ng/ml indicate that IVT is allowed. In the absence of specific tests, standard tests (thrombin time, prothrombin time and activated partial thromboplastin time) can be used for dabigatran and rivaroxaban, although interpretation of these tests may be less reliable. In some patients on dabigatran, idarucizumab may be used before IVT. CONCLUSIONS: In this expert opinion paper, it is suggested that IVT can be performed in patients selected according to the time elapsed since the drug was last taken, renal function, type of hospital where the patient is admitted and plasma concentration of DOAC.
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Anticoagulantes/uso terapéutico , Isquemia Encefálica/tratamiento farmacológico , Fibrinolíticos/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Administración Intravenosa , Administración Oral , Antitrombinas/uso terapéutico , Fibrilación Atrial/tratamiento farmacológico , Pruebas de Coagulación Sanguínea , Dabigatrán/uso terapéutico , Inhibidores del Factor Xa/uso terapéutico , Humanos , Estudios Retrospectivos , Rivaroxabán , Terapia TrombolíticaRESUMEN
BACKGROUND AND OBJECTIVES: A new fibrinogen concentrate Clottafact® was developed according to European guidelines on plasma-derived products. A post-authorization safety study was set up in 2009 as part of the risk management plan. This was a non-interventional, prospective, non-comparative, multicenter study of the use of fibrinogen concentrate for congenital afibrinogenemia in real-life medical practice in France. MATERIALS AND METHODS: The analysis was descriptive and performed on 3 subgroups: prophylaxis vs. on-demand treatment, age (<6, <12 and ≥12) and severity of the deficiency. RESULTS: Fourteen patients [1-78 years] were included in 7 centres and followed for 1 year. Twenty-one adverse drug reactions (ADRs) classically reported with fibrinogen (pallor, chills, cough, vomiting, headache, urticaria and erythematous rash) were reported in 5 of 14 patients. Two ADRs were serious: an anaphylactic shock and a subclavian venous thrombosis with a favourable outcome without sequelae. In the nine patients under prophylaxis, 365 of 367 infusions were considered as successful (99·5%) and 2 as failures. For the five patients treated on-demand, the efficacy was rated as excellent for 27 of 48 infusions and good for the 21 others. CONCLUSION: This study confirms that the benefit/risk balance for this fibrinogen concentrate is favourable.
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Afibrinogenemia/tratamiento farmacológico , Coagulantes/uso terapéutico , Fibrinógeno/uso terapéutico , Adolescente , Adulto , Anciano , Niño , Preescolar , Coagulantes/efectos adversos , Femenino , Fibrinógeno/efectos adversos , Humanos , Lactante , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Medición de Riesgo , Resultado del Tratamiento , Adulto JovenRESUMEN
Direct oral anticoagulants (DOAC)s are often preferred to other anticoagulants as they are more practical and do not require routine laboratory monitoring. Less is known about their use in congenital thrombophilia. Efficacy of DOACs in congenital thrombophilia, effect of DOACs and other anticoagulants on diagnostic tests as well as efficacy and safety of anticoagulant use in this population is still a matter of debate. In this review we intended to analyze the potential pitfalls of testing for thrombophilia in patients using DOACs and vitamin K antagonists (VKA)s as well as to suggest strategies to improve diagnostic accuracy in this setting. We also reviewed the literature for evidence regarding the safety and efficacy of DOACs in patients with congenital thrombophilia. Some evidence was found supporting the use of DOACs in low risk thrombophilia, although evidence for their use in high risk thrombophilia is limited to small series and case reports. Our findings support the generation of better evidence to support DOAC use for congenital thrombophilia, especially in the high risk subgroups.
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Anticoagulantes , Trombofilia , Administración Oral , Anticoagulantes/efectos adversos , Humanos , Trombofilia/diagnóstico , Trombofilia/tratamiento farmacológicoRESUMEN
Antibody inhibitors against human thrombin are rare and have remained poorly characterized. We report the case of a 40-yr-old patient who developed a potent thrombin inhibitor revealed by mild bleeding symptoms and marked prolongation of most laboratory clotting times. After two years of evolution, he died from cerebral hemorrhage. The inhibitor, a polyclonal IgG, was associated with hematological and immunological criteria of autoimmune disorder. Antithrombin IgG was isolated from the patient's plasma by protein A- and thrombin-affinity chromatography. Fab fragments inhibited amidolytic activity of alpha thrombin, and thrombin-thrombomodulin catalyzed protein C activation with a Ki of approximately 10(-8) M in a noncompetitive manner. Alpha to gamma conversion of thrombin resulted in a moderate loss of affinity for the inhibitor. Upon complex formation of thrombin with staphylocoagulase or alpha 2-macroglobulin (alpha 2M), inhibition was decreased by two orders of magnitude and acquired an apparent competitive character. In Western blot experiments, the antibody reacted with active alpha-thrombin, did not react with chloromethylketone-inhibited thrombin and reacted with a lower affinity with iPr2P-thrombin. The inhibitor did not block thrombin binding to benzamidine-, heparin-, or fibrin-Sepharose, but displaced proflavin from its complex with thrombin. Taken together, these results indicate that the patient's autoantibody recognized a conformational structure which includes, at least in part, the apolar binding site adjacent to the catalytic site of thrombin.
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Autoanticuerpos/inmunología , Serina Endopeptidasas/inmunología , Trombina/inmunología , Adulto , Sitios de Unión , Trastornos de la Coagulación Sanguínea/etiología , Epítopos/análisis , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/clasificación , Inmunoglobulina G/aislamiento & purificación , Masculino , Conformación Proteica , Trombina/antagonistas & inhibidores , Trombina/metabolismoAsunto(s)
Neuropatía Femoral/complicaciones , Hematoma/complicaciones , Enfermedad de von Willebrand Tipo 2/complicaciones , Hematoma/cirugía , Humanos , Masculino , Modalidades de Fisioterapia , Músculos Psoas/cirugía , Tomografía Computarizada por Rayos X , Adulto Joven , Enfermedad de von Willebrand Tipo 2/tratamiento farmacológico , Factor de von Willebrand/uso terapéuticoRESUMEN
Whereas hormonal replacement/menopause therapy (HRT) in post-menopausal women increases the coronary artery risk, epidemiological studies (protection in pre-menopaused women) suggest and experimental studies (prevention of the development of fatty streaks in animals) demonstrate a major atheroprotective action of estradiol (E2). The understanding of the deleterious and beneficial effects of estrogens is thus required. The immuno-inflammatory system plays a key role in the development of fatty streak deposit as well as in the atherosclerotic plaque rupture. Whereas E2 favors an anti-inflammatory effect in vitro (cultured cells), it rather elicits pro-inflammation in vivo, at the level of several subpopulations of the immuno-inflammatory system, which could contribute to plaque destabilization. Endothelium is another important target for E2, since it stimulates endothelial NO and prostacyclin production, thus promoting beneficial effects of vasorelaxation and platelet aggregation inhibition. Prostacyclin, but not NO, appears to be involved in the atheroprotective effect of E2. Estradiol accelerates also endothelial regrowth, thus favoring vascular healing. Finally, most of these effects of E2 are mediated by estrogen receptor alpha, and are independent of estrogen receptor beta. In summary, a better understanding of the mechanisms of estrogen action is required not only on the normal and atheromatous arteries, but also on innate and adaptive immune responses. This should help cardiovascular disease prevention optimization after menopause. These mouse models should help to screen existing and future Selective Estrogen Receptor Modulators (SERMs).
Asunto(s)
Estradiol/farmacología , Terapia de Reemplazo de Estrógeno , Animales , Antiinflamatorios/farmacología , Enfermedad de la Arteria Coronaria/inducido químicamente , Enfermedad de la Arteria Coronaria/prevención & control , Modelos Animales de Enfermedad , Endotelio Vascular/efectos de los fármacos , Estradiol/efectos adversos , Femenino , Humanos , Mediadores de Inflamación/farmacología , Ratones , Posmenopausia/efectos de los fármacos , Premenopausia/efectos de los fármacos , Factores de Riesgo , Moduladores Selectivos de los Receptores de Estrógeno/farmacologíaRESUMEN
UNLABELLED: ESSENTIALS: The role of ATP-binding cassette transporter 1 (ABCA1) in platelet functions is poorly characterized. We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and two Tangier patients. ABCA1-deficient platelets exhibit reduced positive feedback loop mechanisms. This reduced reactivity is dependent on external environment and independent of hematopoietic ABCA1. BACKGROUND: The ATP-binding cassette transporter ABCA1 is required for the conversion of apolipoprotein A-1 to high-density lipoprotein (HDL), and its defect causes Tangier disease, a rare disorder characterized by an absence of HDL and accumulation of cholesterol in peripheral tissues. The role of ABCA1 in platelet functions remains poorly characterized. OBJECTIVE: To determine the role of ABCA1 in platelet functions and to clarify controversies concerning its implication in processes as fundamental as platelet phosphatidylserine exposure and control of platelet membrane lipid composition. METHODS AND RESULTS: We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and in two Tangier patients. We show that platelets in ABCA1-deficient mice are slightly larger in size and exhibit aggregation and secretion defects in response to low concentrations of thrombin and collagen. These platelets have normal cholesterol and major phospholipid composition, granule morphology, or calcium-induced phosphatidylserine exposure. Interestingly, ABCA1-deficient platelets display a reduction in positive feedback loop mechanisms, particularly in thromboxane A2 (TXA2) production. Hematopoietic chimera mice demonstrated that defective eicosanoids production, particularly TXA2, was primarily dependent on external environment and not on the hematopoietic ABCA1. Decreased aggregation and production of TXA2 and eicosanoids were also observed in platelets from Tangier patients. CONCLUSIONS: Absence of ABCA1 and low HDL level induce reduction of platelet reactivity by decreasing positive feedback loops, particularly TXA2 production through a hematopoietic ABCA1-independent mechanism.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/deficiencia , Plaquetas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Enfermedad de Tangier/sangre , Transportador 1 de Casete de Unión a ATP/sangre , Transportador 1 de Casete de Unión a ATP/genética , Animales , Plaquetas/patología , Tamaño de la Célula , Modelos Animales de Enfermedad , Retroalimentación Fisiológica , Femenino , Predisposición Genética a la Enfermedad , Trasplante de Células Madre Hematopoyéticas , Hemostasis , Humanos , Lipoproteínas HDL/sangre , Masculino , Ratones Endogámicos DBA , Ratones Noqueados , Persona de Mediana Edad , Fenotipo , Agregación Plaquetaria , Enfermedad de Tangier/genética , Enfermedad de Tangier/patología , Trombosis/sangre , Trombosis/genética , Tromboxano A2/metabolismo , Factores de TiempoRESUMEN
The structural requirements of heparin for the catalysis of thrombin inhibition by heparin cofactor II (HC II) were investigated. A series of well characterized heparin derivatives were prepared and their activities were measured using human thrombin in the presence of an excess of purified human HC II and, for comparison, antithrombin III (AT III). The 50% inhibitory concentrations of each derivative were calculated and compared with those of unmodified heparin. Heparin activity was strongly dependent on molecular weight (Mr) in a manner grossly comparable for the two inhibitors. High-Mr fractions were the most active. Below 10 kDa, the activity dropped rapidly. A minimum size of 26 residues appeared to be required for HC II activation (against 16-18 for AT III). Below 5 kDa, a residual activity two orders of magnitude lower than that of high-Mr species remained with HC II (but not with AT III). Heparin was selectively desulfated or oversulfated in the O- and/or N-position. When an N-acetyl group was substituted for the original N-sulfate in the glucosamine and the derivative was oversulfated in the O-position, a strong activity with HC activities with both inhibitors decreased when the overall sulfate content (i.e., the charge density) was reduced, and vice-versa. Carboxyl-reduced heparin was also inactive but activity could be restored after O-sulfation. Our results thus suggest that, unlike the case of AT III, no functional group in heparin is critical for optimal thrombin inhibition by HC II. Sulfate and carboxylate are important in as much as they contribute to the global charge of the molecule.
Asunto(s)
Glicoproteínas/fisiología , Heparina/fisiología , Trombina/antagonistas & inhibidores , Antitrombina III/fisiología , Catálisis , Heparina/análogos & derivados , Heparina/metabolismo , Cofactor II de Heparina , Humanos , Peso Molecular , Relación Estructura-ActividadRESUMEN
Recent reports have suggested that basic fibroblast growth factor (bFGF) could play a permissive role in hematopoiesis, in combination with specific colony-stimulating factors. We investigated the expression of bFGF and FGF-receptors (FGF-Rs) in leukemic cell lines of various hematopoietic lineages. Three protein isoforms of bFGF of approximately 18, 22 and 24 kDa were detected in the myeloid cell line K562, but not in myelomonocytic or lymphoid (T or B) cell lines. In vitro-induced differentiation of K562 cells did not change the pattern of expression of the different bFGF isoforms. Accordingly, the mRNA of bFGF was found expressed in K562, but not in other leukemic lines tested, as assayed by reverse transcript amplification (RT-PCR). Using the same technique, we searched for the presence of high affinity FGF-Rs on these cells: in eight out of ten cell lines tested, mRNA for at least one FGF-R among FGF-R1, FGF-R3 or FGF-R4 was expressed, whereas FGF-R2 was never detected. We found that two cell lines were responsive to bFGF in different biological assays: (i) in K562 myeloid cells induced to differentiate by hemin, preincubation with bFGF and heparin increased cell viability and decreased hemin-induced DNA fragmentation, without affecting erythroid differentiation; and (ii) in U937 monocytic cells, the production of plasminogen activator was increased by bFGF or aFGF in combination with heparin. Binding experiments with 125I-bFGF (up to 200 pM) in the presence of heparin revealed high affinity receptors on the K562 and U937 cell lines (1177 +/- 440 and 392 +/- 184 sites/cell, Kd = 61.7 +/- 8.6 and 43.1 +/- 13.5 pM, respectively). Thus our results strongly suggest that cells of hematopoietic origin could express functional FGF-receptors.
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Factor 2 de Crecimiento de Fibroblastos/análisis , Leucemia/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/análisis , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , ADN/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Heparina/farmacología , Humanos , Leucemia/patología , Datos de Secuencia Molecular , ARN Mensajero/análisis , Receptores de Factores de Crecimiento de Fibroblastos/genética , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesisRESUMEN
BACKGROUND: A low-molecular-weight heparin, enoxaparin sodium, has been shown to be effective and safe in preventing deep vein thrombosis both in general surgery and in high-risk orthopedic surgery. We conducted a controlled, randomized trial with enoxaparin in the treatment of established deep vein thrombosis. METHODS: In a multicenter trial, we compared fixed-dose subcutaneous enoxaparin, given twice daily, with adjusted-dose intravenous unfractionated heparin (UFH) given by continuous intravenous infusion for the initial 10 days of treatment of patients with proximal vein thrombosis. The primary efficacy outcome was the change of the size of the thrombus assessed by repeated venograms between day 0 and day 10. The primary analysis of safety was based on the incidence of major bleeding during 10 days of treatment. RESULTS: There were 67 patients in each group. Venographic assessment of clot size evolution between day 0 and day 10 showed a statistically significant superiority (P < .002) of enoxaparin over the reference treatment with UFH. Moreover, the incidence of overall recurrent thromboembolic events during 10 days of treatment was significantly higher (P < .002) in the UFH group (seven of 67) than in the enoxaparin group (one of 67). There were no serious bleeding complications in either group. CONCLUSIONS: Enoxaparin is at least as effective and safe as UFH under the conditions of this study. Moreover, it is more comfortable for patients and less time-consuming for nurses and laboratories. Thus, our study confirmed, with the use of enoxaparin, other observations that low-molecular-weight heparin provides a real therapeutic advance in the treatment of deep vein thrombosis.
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Heparina de Bajo-Peso-Molecular/uso terapéutico , Heparina/uso terapéutico , Tromboflebitis/tratamiento farmacológico , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Heparina/administración & dosificación , Heparina de Bajo-Peso-Molecular/administración & dosificación , Humanos , Infusiones Intravenosas , Inyecciones Subcutáneas , Masculino , Persona de Mediana Edad , Recurrencia , Resultado del TratamientoRESUMEN
The relationships between plasminogen activator inhibitor (PAi) activity and lipoprotein(a) [Lp(a)] and insulin, testosterone, 17 beta-estradiol, and testosterone binding globulin (TEBG) were assessed in 42 myocardial infarction male patients and 74 healthy controls. Patients had higher levels of insulin than did controls (87 +/- 30 vs. 75 +/- 28 pmol/L, respectively; P < 0.04), and no differences were found in levels of PAi activity, testosterone, 17 beta-estradiol, and TEBG. Lp(a) levels greater than 0.3 g/L were more frequent in patients than in controls (P < 0.002). In all subjects, PAi activity levels were significantly and positively correlated with body mass index (r = 0.20, P < 0.05), triglycerides (r = 0.38, P < 0.0001), and insulin (r = 0.27, P < 0.005) and were negatively correlated with testosterone (r = -0.28, P < 0.005) and TEBG (r = -0.42, P < 0.001). Stepwise multiple regression analysis showed triglyceride, insulin, and TEBG levels to be significantly related to PAi activity. No significant correlations were found between Lp(a) levels and all hormonal variables studied and between Lp(a) and PAi activity (r = -0.06, P < 0.58). These results suggest that TEBG is significantly and independently related to PAi levels.
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Estradiol/sangre , Insulina/sangre , Lipoproteína(a)/sangre , Infarto del Miocardio/sangre , Inactivadores Plasminogénicos/sangre , Testosterona/sangre , Adulto , Anciano , Índice de Masa Corporal , Humanos , Lípidos/sangre , Masculino , Persona de Mediana Edad , Globulina de Unión a Hormona Sexual/metabolismo , Triglicéridos/sangreRESUMEN
The complement inhibitor S protein, which is identical to the adhesive protein vitronectin, functions as heparin-neutralizing factor by protecting thrombin as well as factor Xa against fast inactivation by antithrombin III. The interference of S protein with glycosaminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated in these studies. S protein significantly counteracted the anticoagulant activity of heparin and pentosan polysulfate but not of dermatan sulfate. In the presence of 0.3 micrograms/ml heparin, 0.5 micrograms/ml pentosan polysulfate, or 2 micrograms/ml dermatan sulfate, S protein induced a concentration-dependent reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted in a decrease of the apparent pseudo first-order rate constants by about 17-fold (heparin), or about 7-fold (pentosan polysulfate), whereas no neutralization of dermatan sulfate was demonstrable at a physiological ratio of S protein to heparin cofactor II. Exposure of the glycosaminoglycan-binding region of S protein by reduction and carboxymethylation of the protein increased the neutralizing activity of S protein towards heparin and pentosan polysulfate. The results of these functional experiments correlated well with the demonstration of direct binding of S protein to both polysaccharides but not to dermatan sulfate. While reduced/carboxymethylated S protein remained also ineffective in neutralizing other dermatan sulfate compounds with varying degree of sulfation, a synthetic highly basic tridecapeptide, representing a portion of the glycosaminoglycan-binding domain of S protein, counteracted their anticoagulant activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Antitrombinas/antagonistas & inhibidores , Glicoproteínas/antagonistas & inhibidores , Glicoproteínas/farmacología , Trombina/metabolismo , Antitrombinas/metabolismo , Unión Competitiva , Glicoproteínas/metabolismo , Glicosaminoglicanos/metabolismo , Cofactor II de Heparina , Humanos , Proteína S , Trombina/antagonistas & inhibidoresRESUMEN
Heparin cofactor II (HC II) is a heparin-dependent inhibitor of thrombin, distinct from antithrombin III (AT III). This study was designed to evaluate its metabolism in healthy subjects. Purified HC II was labelled with 125I by the lactoperoxidase-glucose oxidase technique. The biological activity of the HC II was unchanged after labelling as was its migratory pattern by crossed immunoelectrophoresis in the presence of heparin or dermatan sulfate. Three healthy volunteers were injected with 10 microCi and the plasma radioactivity was measured daily. The data were approximated by a sum of two exponential terms and the metabolism of HC II was described by a two compartment mamillary system. The mean values of fractional catabolic rate, intravascular fraction and half-life of the elimination phase were respectively: 0.44 d-1, 0.60 and 2.53 d. These parameters are of the same order of magnitude as those reported in the literature for AT III. The plasma HC II concentration in the 3 subjects ranged from 61 to 82 micrograms/ml as estimated using our purified preparation. Accordingly, the absolute catabolic rate ranged from 1.17 to 1.36 mg X kg-1 X d-1.
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Antitrombinas/metabolismo , Glicoproteínas/metabolismo , Adulto , Cofactor II de Heparina , Humanos , Masculino , Tasa de Depuración MetabólicaRESUMEN
A simple method for biological assay of dermatan sulfate (DS) in plasma is described. DS accelerates thrombin inhibition by heparin cofactor II (HC II). The principle of the assay is to measure the residual amidolytic thrombin activity after a short period of incubation with HC II in defibrinated plasma at low ionic strength. For this method we take advantage of two observations. Firstly, at fixed concentrations of DS and of HC II, the rate of thrombin inhibition increases when the ionic strength of the medium decreases. Secondly, defibrination by bentonite absorption also removes antithrombin III, HC II and for a large part alpha-2 macroglobulin from the plasma, so that no other thrombin inhibitor competes with HC II added as a reagent in a second step. In the conditions described, there is a linear relationship between DS concentrations in plasma from 0 to 2 micrograms/ml and the log of residual thrombin activity. The limit of sensitivity is 0.1 micrograms/ml. The assay displays an acceptable reproducibility in intra-assay, inter-assay and inter-individual experiments. It can be used to measure DS in human, rabbit and rat plasmas. The assay is also sensitive to other HC II activators such as heparin and pentosan polysulfate. DS is effective in experimental thrombosis without any detectable anticoagulant effect ex vivo. Pharmacological concentrations of DS in plasma fall into the range of sensitivity of this assay, which would be helpful in experimental or clinical studies of DS and related glycosaminoglycans.
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Condroitín/análogos & derivados , Dermatán Sulfato/sangre , Antitrombina III/análisis , Antitrombinas/análisis , Análisis Químico de la Sangre/métodos , Fibrina/metabolismo , Glicoproteínas/análisis , Cofactor II de Heparina , Humanos , Iones , Reproducibilidad de los ResultadosRESUMEN
An assay for the quantification of pentosan polysulphate (PPS) in plasma is described. As PPS has been shown to potentiate thrombin inhibition by the second heparin cofactor (HC II), the principle of this assay was to measure the formation of covalent complexes between HC II and the thrombin generated in plasma after contact activation and recalcification. The complexes were quantified by using purified 125I-HC II added to the plasma as a tracer and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The assay was sensitive to low PPS concentrations (limit of detection: 0.1 microgram/ml) and therefore suitable for the measurement of PPS in plasma after its administration to man. The clearance of PPS was studied in 3 subjects receiving respectively 10, 50 and 100 mg intravenously (IV) and in 3 subjects receiving 35 mg subcutaneously (SC). PPS was still detectable 8 h after 50 and 100 mg IV and 6 h after 35 mg SC. The activated partial thromboplastin time (APTT) was, in comparison, relatively insensitive but for concentrations above 1 microgram/ml the values derived from the APTT and from the SDS-PAGE method fitted. The results were also in general agreement with those reported by McGregor et al. (5) who used a sensitive competitive binding assay. This indicates that low concentrations of PPS previously measured chemically are also pharmacologically active in plasma.
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Pruebas de Coagulación Sanguínea , Glicoproteínas/metabolismo , Tiempo de Tromboplastina Parcial , Poliéster Pentosan Sulfúrico/farmacología , Polisacáridos/farmacología , Trombina/metabolismo , Adulto , Unión Competitiva , Femenino , Cofactor II de Heparina , Humanos , Inyecciones Intravenosas , Inyecciones Subcutáneas , Masculino , Tasa de Depuración Metabólica , Métodos , Persona de Mediana Edad , Poliéster Pentosan Sulfúrico/administración & dosificación , Poliéster Pentosan Sulfúrico/sangreRESUMEN
Recombiplastin, a recombinant a human tissue factor, elaborated by Ortho Diagnostic Systems, produced by Baculovirus and relipidated with highly purified phospholipids, was tested as a new reagent for determining prothrombin time (PT) in a French multicentric study. Its intralaboratory performances, including sensitivity, repeatability, reproducibility and stability, were explored to establish whether its use would reduce the interlaboratory dispersion of PT values, and therefore improve the standardization of oral anticoagulant treatment. The 9 university hospital hematology laboratories involved in this study used the same type of instrument (KC 10). For 10 consecutive days, they determined PTS on a normal plasma pool, plasma dilutions of 1/2, 1/3 and 1/8, 3 identical lyophilized calibrated plasmas, as well as plasmas from 20 normal subjects, 50 patients on oral anticoagulant therapy with Recombiplastin which has an International Sensitivity Index (ISI) of 1, and 2 commercial thromboplastin extracts (ISI #1 or 2). In the patients on anticoagulants, factors VII, X and V were measured when results were conflicting. The intra and interlaboratory reproducibilities of Recombiplastin, calculated on the basis of either PTS expressed in seconds, or of the International Normalized Ratio (INR), were good, with coefficients of variation (CV) similar to those observed with the 5 other reagents used by the different laboratories (2% < CV < 8%). The stability of Recombiplastin was excellent, with no variation in PT after 72 h of incubation at 37 degrees C. A normal PT of 12 s was obtained with Recombiplastin, similar to the values found for the reagents with ISI #2.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Tromboplastina , Administración Oral , Anticoagulantes/uso terapéutico , Estabilidad de Medicamentos , Estudios de Evaluación como Asunto , Francia , Liofilización , Humanos , Modelos Lineales , Tiempo de Protrombina , Proteínas Recombinantes , Valores de Referencia , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Heparin and pentosan polysulfate (PPS) interact in plasma with antithrombin III (AT III) and Heparin cofactor II (HC II) respectively. To assess the influence of heparin or PPS treatment on the metabolism of their respective cofactors, we performed a double tracer study in baboons receiving heparin or PPS. Purified AT III and HC II from human plasma were labelled with 131I and 125I respectively by the lactoperoxidase-glucose oxidase technique. The tracers had unchanged biological activities, were homogeneous in SDS-PAGE, migrated as native proteins by crossed immunoelectrophoresis in the presence of heparin or PPS and virtually coeluted with endogenous baboon proteins from heparin-agarose. Nine animals were randomly allocated to receive, during the metabolic study, heparin (500 IU/kg/d, n = 3), PPS (5 mg/kg,d, n = 3) or a placebo (n = 3) given in 2 daily subcutaneous injections. Heparin levels and anticoagulant effects were similar in extent and duration to those usually achieved in man. The plasma concentrations of AT III and HC II did not vary under treatment. The half-life of the elimination phase in the placebo group ranged from 1.95 to 2.33 d for AT III and from 1.96 to 2.21 d for HC II. There was no significant difference in the half-lives of the 2 inhibitors between the placebo group and the animals receiving heparin or PPS. This suggest that clinical conditions associated with heparin treatment may be important for the effect of heparin on AT III metabolism previously reported in patients.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Antitrombina III/metabolismo , Glicoproteínas/metabolismo , Heparina/farmacología , Poliéster Pentosan Sulfúrico/farmacología , Polisacáridos/farmacología , Animales , Antitrombina III/administración & dosificación , Carga Corporal (Radioterapia) , Cromatografía en Gel , Glicoproteínas/administración & dosificación , Heparina/administración & dosificación , Cofactor II de Heparina , Humanos , Inmunoelectroforesis Bidimensional , Inyecciones Intravenosas , Radioisótopos de Yodo , Papio , Poliéster Pentosan Sulfúrico/administración & dosificaciónRESUMEN
Tissue Factor (TF), the receptor for plasma VII/VIIa and the initiator of blood coagulation, is inducible in vascular smooth muscle cells (SMCs) by growth factors and bacterial lysopolysaccharides (LPS) and is expressed in vivo after vascular injury. As TF expression is a determinant of the thrombogenicity of vascular lesions, we investigated the signal pathways involved in this process. Human vascular SMCs were obtained from normal arteries and made quiescent by serum deprivation. Baseline TF antigen and activity were up-regulated by various agonists: fetal calf serum (FCS), LPS, and platelet derived growth factor (PDGF) being the most effective but with different kinetics. TF expression induced by LPS was transient with a maximum 6 h after stimulation and returned to baseline levels after 24 h whereas TF expression induced by serum or PDGF was sustained for at least 24 h. Rapid and transient activation of Extracellular signal-Regulated Kinase (ERK) was observed after stimulation by PDGF and FCS, but not by LPS. The role of ERK, Ras and protein kinase C activities were investigated using specific inhibitors, PD 98059, manumycin A and calphostin C respectively. For TF induction by LPS, PKC activity was required and the ERK/Ras pathway was not involved. In contrast, the effect of PDGF was strictly ERK and Ras dependent, but partially prevented by PKC inhibitors. TF induction by FCS was ERK dependent but partially Ras and PKC dependent. In conclusion, TF expression appears to be a non-specific response of SMCs to numerous stimuli through multiple signal pathways which differ according to the inducing agent.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Músculo Liso Vascular/efectos de los fármacos , Proteína Quinasa C/fisiología , Tromboplastina/biosíntesis , Adulto , Animales , Bovinos/sangre , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Medios de Cultivo/farmacología , Medio de Cultivo Libre de Suero , Activación Enzimática/efectos de los fármacos , Sangre Fetal/fisiología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Flavonoides/farmacología , Genes Inmediatos-Precoces , Humanos , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Arterias Mamarias/citología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Músculo Liso Vascular/metabolismo , Naftalenos/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Polienos/farmacología , Alcamidas Poliinsaturadas , Proteína Quinasa C/antagonistas & inhibidores , Trombina/farmacologíaRESUMEN
This study was performed to determine the accuracy of D-Dimer fibrin derivatives, thrombin-antithrombin III (TAT) complexes and prothrombin fragments 1 + 2 (F 1 + 2) determinations for the diagnosis of deep vein thrombosis (DVT). One hundred and sixteen consecutive patients referred to the angiology unit of our hospital for a clinically suspected DVT were investigated. They were submitted to mercury strain gauge plethysmography and to ultrasonic duplex scanning examination; in cases of inconclusive results or of proximal DVT (n = 35), an ascending phlebography was performed. After these investigations were completed, the diagnosis of DVT was confirmed in 34 and excluded in 82. One half of the patients were already under anticoagulant therapy at the time of investigation. The 3 biological markers were assayed using commercially available ELISA techniques and the D-Dimer was also assayed with a fast latex method. The normal distribution of these markers was established in 40 healthy blood donors. The most accurate assay for the diagnosis of DVT was the D-Dimer ELISA which had both a high sensitivity (94%) and a high negative predictive value (95%). The D-Dimer latex, TAT complexes and F 1 + 2 were far less sensitive and provided negative predictive values which ranged between 78 and 85%. In spite of positive and significant correlations between the levels of the 3 markers, their association did not improve their overall accuracy for detecting DVT. Therefore, with the exception of the D-Dimer ELISA, these markers were of little value for the diagnosis of DVT in this specific population.