RESUMO
Inherited disorders of platelet function are a heterogeneous group. For optimal prevention and management of bleeding, classification and diagnosis of the underlying defect are highly recommended. An interdisciplinary guideline for a diagnostic approach has been published (AWMF # 086-003 S2K; Hämostaseologie 2014; 34: 201-212). Underlying platelet disorder, platelet count, age and clinical situation modify treatment. Exclusive transfusion of platelet concentrates may be inappropriate as potentially adverse effects can outweigh its benefit. A stepwise and individually adjusted approach for restitution and maintenance of haemostasis is recommended. Administration of antifibrinolytics is generally endorsed, but is of particular use in Quebec disease. Restricted to older children, desmopressin is favourable in storage pool disease and unclassified platelet disorders. Although licensed only for patients with Glanzmann thrombasthenia and alloantibodies, in clinical practice rFVIIa is widely used in inherited platelet disorders with severe bleeding tendency. This guideline aims at presenting the best available advice for the management of patients with inherited platelet function disorders.
Assuntos
Antiarrítmicos/uso terapêutico , Transtornos Plaquetários/congênito , Transtornos Plaquetários/terapia , Desamino Arginina Vasopressina/uso terapêutico , Fator VIIa/uso terapêutico , Hemorragia/terapia , Transfusão de Plaquetas/normas , Antiarrítmicos/normas , Transtornos Plaquetários/diagnóstico , Criança , Pré-Escolar , Feminino , Alemanha , Hematologia/normas , Hemorragia/congênito , Hemorragia/diagnóstico , Hemostáticos/uso terapêutico , Humanos , Lactente , Recém-Nascido , Masculino , Pediatria/normas , Guias de Prática Clínica como AssuntoRESUMO
Congenital disorders of platelet function are a heterogeneous group of disorders that are often not detected until bleeding occurs. In clinical settings only a few methods have proven to be useful for identification and classification of inherited platelet disorders. For a rational diagnostic approach, a stepwise algorithm is recommended. Patient history and clinical investigation are mandatory. Von Willebrand disease and other coagulation disorders should always be ruled out prior to specific platelet testing. Platelet count, size, volume (MPV) and morphology may guide further investigations. The PFA-100® CT is suited for screening for severe platelet defects. Platelet aggregometry allows assessment of multiple aspects of platelet function. Flow cytometry enables diagnosis of thrombasthenia Glanzmann, Bernard-Soulier syndrome and storage pool defects. Molecular genetics may confirm a putative diagnosis or pave the way for identifying new defects. We present an unabridged version of the interdisciplinary guideline.
Assuntos
Transtornos Plaquetários/diagnóstico , Transtornos Plaquetários/genética , Testes Genéticos/normas , Hematologia/normas , Técnicas de Diagnóstico Molecular/normas , Testes de Função Plaquetária/normas , Guias de Prática Clínica como Assunto , Transtornos Plaquetários/sangue , Alemanha , Humanos , Pediatria/normasRESUMO
Recombinant activated factor VII (rFVIIa) has been available for the treatment of acute bleeding and for prevention of bleeding during surgery and invasive procedures in patients with congenital haemophilia with inhibitors (CHwI) and acquired haemophilia since 1996. The study objective was to assess the efficacy and safety of rFVIIa in patients with CHwI, acquired haemophilia, congenital FVII deficiency and Glanzmann's thrombasthenia, in a real-life clinical setting. There were no specific inclusion or exclusion criteria; participation was offered to all German haemophilia centres known to use rFVIIa to treat patients with the above indications. Data on rFVIIa use and efficacy for the treatment of acute bleeding episodes and invasive procedures were recorded. Adverse drug reactions and recurrent bleeding episodes were also monitored. In total, 64 patients (50.0% women) received rFVIIa treatment. Patients experienced 281 evaluable bleeding episodes and underwent 44 invasive procedures. In 252 of 281 (89.7%) bleeding episodes, a stop (66.5%) or a significant reduction (23.1%) in bleeding was observed. No bleeding complications were reported for 42 of 44 (95.5%) invasive procedures covered with rFVIIa. A clear positive association was observed between early initiation of rFVIIa treatment for acute bleeding and efficacy. The total cumulative dose and number of injections were 468.3 ± 545.8 µg kg(-1) and 3.6 ± 4.6 respectively. No drug-related adverse events were reported. rFVIIa use in Germany provided effective haemostatic cover without associated adverse events in the management of acute bleeds and invasive procedures across a range of bleeding disorders.
Assuntos
Fator VIIa/uso terapêutico , Hemofilia A/tratamento farmacológico , Hemorragia/tratamento farmacológico , Adulto , Fator VIIa/efeitos adversos , Feminino , Hemofilia A/genética , Hemofilia A/imunologia , Hemorragia/prevenção & controle , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Observacionais como Assunto , Vigilância de Produtos Comercializados , Estudos Prospectivos , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Adulto JovemRESUMO
OBJECTIVES: Platelet releasate has been shown to promote osteogenetic cell proliferation and differentiation. Topography and chemistry of biomaterials have high impact on platelet activation. More specifically, the bioactive cell adhesive peptide sequence Arg-Gly-Asp (RGD) triggers platelet activation mediated by the α(IIb) ß(3) integrin receptor. Accordingly, topographical, chemical and biomimetical (immobilized RGD peptide) modifications of titanium (Ti) surfaces may enhance early platelet activation and bony healing of implants. Therefore, the aim of the study was to evaluate platelet activation with subsequent platelet-derived cytokine release by accordingly modified Ti surfaces. MATERIALS AND METHODS: Pre-treated (PT; mean roughness [R(a)]=0.04 µm, contact angle [CA]=91°), acid-etched (A, R(a) =0.83 µm, CA=106°), large grit-sandblasted, acid-etched (SLA, R(a) =3.2 µm, CA=109°) as well as hydrophilically modified acid-etched (modA, R(a) =0.83 µm, CA=0) and modified large grit-sandblasted, acid-etched (modSLA, R(a) =3.2 µm; CA=0°) titanium surfaces were investigated. Additionally, RGD peptides were chemically immobilized on PT, A and SLA surfaces (PT-RGD [CA=18°], A-RGD [CA=0°], SLA-RGD [CA=0°]). The different Ti surfaces were incubated with platelet concentrate of three healthy volunteers at room temperature for 15 min and for 30 min. High thrombogenous collagen served as the control group. Out of the supernatant, platelet consumption was assessed via platelet count (PC). Cytokine release was quantified via the level of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). RESULTS: After 15 min, especially the rough SLA surface showed a strong decrease in PC and a strong increase in VEGF and PDGF levels. After 30 min, high platelet consumption as well as high levels of VEGF and PDGF were measured for unspecifically modified (modA) and especially for biomimetic, specifically modified (PT-RGD, A-RGD) surfaces, indicating a delayed effect of the surface modifications on platelet activation. DISCUSSION: Modifications of surface roughness modifications appear to influence early platelet activation and cytokine release after 15 min whereas surface chemistry modifications with increased hydrophilic properties and surface modifications via RGD peptide on plainer surfaces lead to a further, more specific promotion of platelet activation and degranulation after 30 min. The observed effect could be valuable for critical clinical situations like compromised bone sites.
Assuntos
Implantes Dentários , Ativação Plaquetária , Fator de Crescimento Derivado de Plaquetas/metabolismo , Titânio/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologia , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Osteogênese , Propriedades de SuperfícieRESUMO
Recombinant factor VIIa is indicated for treatment of bleeding episodes in patients with haemophilia A or B with inhibitors; in FVII deficiency and in Glanzmann's thrombasthenia. The aim of the study reported here was to compare the pharmacokinetic profiles between two formulations of rFVIIa that are produced in two different cell lines and media: Chinese hamster ovary cells cultured in a serum-free medium (CHO-rFVIIa) and baby hamster kidney cells cultured in a non-human serum-based medium (BHK-rFVIIa). Two clinical trials were performed; one in healthy subjects and the other in patients with congenital haemophilia A or B, with or without inhibitors. Subjects were recruited into a two-way crossover trial and were randomized to receive a dose of CHO-rFVIIa and BHK-rFVIIa. Healthy subjects received one dose of 90 µg CHO-rFVIIa kg(-1) bodyweight (bw) in the newly developed room-temperature stable rFVIIa formulation and one dose of 90 µg BHK-rFVIIa kg(-1) bw, in the original rFVIIa formulation. Patients with haemophilia received one dose of 270 µg CHO-rFVIIa kg(-1) and one dose of 270 µg BHK-rFVIIa kg(-1), both in the room-temperature stable formulation. The trials showed higher FVII activity levels [higher area under the plasma concentration-time curve (AUC)] following administration of CHO-rFVIIa than after BHK-rFVIIa. Therefore, bioequivalence could not be established. The difference in FVII activity levels is believed to be a result of different glycosylation patterns between the two products. Neither the use of CHO-rFVIIa nor the use of one single dose of 270 µg kg(-1) of the newly developed room-temperature stable rFVIIa raised any safety concerns.
Assuntos
Coagulantes/farmacocinética , Fator VIIa/farmacocinética , Hemofilia A/metabolismo , Hemofilia B/metabolismo , Adulto , Área Sob a Curva , Técnicas de Cultura de Células , Células Cultivadas , Estudos Cross-Over , Hemofilia A/tratamento farmacológico , Hemofilia B/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/farmacocinética , Equivalência Terapêutica , Adulto JovemRESUMO
Inherited thrombocytopathies are much less frequent in comparison to acquired platelet function disorders. However, congenital disorders can lead to severe bleeding tendency and are often not diagnosed. They are induced by different platelet defects based on disorders of platelet adhesion, receptors, secretion and signal transduction. In some cases they are associated with thrombocytopenia, giant platelets and various comorbidities. This article gives an overview regarding diverse defects, their diagnosis and treatment options.
Assuntos
Transtornos Plaquetários/genética , Plaquetas/fisiologia , Genótipo , Transtornos Hemorrágicos/genética , Síndrome de Bernard-Soulier/sangue , Síndrome de Bernard-Soulier/diagnóstico , Síndrome de Bernard-Soulier/genética , Síndrome de Bernard-Soulier/terapia , Transtornos Plaquetários/sangue , Transtornos Plaquetários/diagnóstico , Transtornos Plaquetários/terapia , Plaquetas/patologia , Diagnóstico Diferencial , Transtornos Hemorrágicos/sangue , Transtornos Hemorrágicos/diagnóstico , Transtornos Hemorrágicos/terapia , Humanos , Programas de Rastreamento , Agregação Plaquetária/genética , Agregação Plaquetária/fisiologia , Testes de Função Plaquetária , Deficiência do Pool Plaquetário/sangue , Deficiência do Pool Plaquetário/diagnóstico , Deficiência do Pool Plaquetário/genética , Deficiência do Pool Plaquetário/terapia , Prostaglandina-Endoperóxido Sintases/genética , Receptores de Colágeno/genética , Receptores de Colágeno/fisiologia , Trombastenia/sangue , Trombastenia/diagnóstico , Trombastenia/genética , Trombastenia/terapia , Tromboxano-A Sintase/genéticaRESUMO
Thromboelastography methods have been used to predict or monitor treatment of haemophilia patients with recombinant activated factor VII (rFVIIa). However, neither of the two thromboelastographic methods (ROTEM and TEG) has as yet been validated. This multi-centre, randomised trial compared both methods in terms of intra- and inter- patient variability following in vivo and ex vivo rFVIIa administration to haemophilia A and B patients with and without inhibitors. Patients ((3)16 years old) received the same intravenous rFVIIa dose (45, 90 or 180 microg/kg) twice, 1-12 weeks apart. Blood samples were collected pre-dose and 15, 60, 120 and 240 minutes post-dose for ROTEM and TEG analysis. Pre-dose samples were also spiked ex vivo with rFVIIa (0.6, 1.2 or 2.4 microg/ml), to correspond to the three in vivo doses. Twenty-six haemophilia A and four haemophilia B patients were enrolled. A significant treatment effect was observed with in vivo rFVIIa (p<0.05) with more pronounced effects in inhibitor (n=14) versus non-inhibitor (n=16) patients. There was a strong positive correlation between ROTEM and TEG parameters. Intra- and inter-patient variation was large for all thromboelastography parameters at all time points and rFVIIa doses. Intra-patient variation was generally lower for non-inhibitor than inhibitor patients, and lower following ex vivo spiking versus in vivo rFVIIa administration. In conclusion, there was a clear effect of rFVIIa on all thromboelastography parameters, but the large intra- and inter-patient variability following in vivo rFVIIa administration renders the use of our method unsuitable for dose-response prediction for haemophilia patients in the clinical setting.
Assuntos
Fator VIIa/administração & dosagem , Hemofilia A/tratamento farmacológico , Tromboelastografia/métodos , Adolescente , Relação Dose-Resposta a Droga , Fator VIIa/farmacocinética , Fator VIIa/farmacologia , Humanos , Reprodutibilidade dos Testes , Tromboelastografia/normas , Resultado do TratamentoRESUMO
Anticoagulation is a standard treatment in patients with thrombosis and commonly used in surgical procedures for primary thrombosis prophylaxis. The occurrence of bleeding episodes is the predominant treatment complication. Although monitoring of hemostatic parameters reduces the risk of bleeding, bleedings occur in approximately 10% of patients. Besides standard life saving procedures it is crucial to ensure sufficient coagulation by administering factor concentrates (e.g. fresh frozen plasma, prothrombin complex, recombinant factor VIIa), platelet concentrates and fluid. Specific antidotes are not available for the majority of anticoagulant agents.
Assuntos
Anticoagulantes/efeitos adversos , Hemorragia/etiologia , Hemorragia/prevenção & controle , HumanosRESUMO
FVIII therapy for haemophilia A is safe and effective, with the problem of individually sufficient efficacy unsettled. Routine one-stage clotting assays and tests employing chromogenic substrates poorly detect individual haemostatic effects of FVIII due to artificial test conditions. In particular, the use of cell-free and diluted plasma samples neglect the crucial role of platelets for thrombin and fibrin formation. To optimize FVIII substitution therapy, we measured in 40 patients with severe to mild haemophilia A before and after FVIII substitution the FVIII activity in cell-free plasma samples using a one-stage clotting assay as well a chromogenic substrate assay and compared the data with those obtained with cell-based coagulation tests, i.e. thrombin generation in platelet-rich plasma (PRP) and thromboelastography (TEG) in samples of citrated whole blood (WB). To determine the maximum ex vivo haemostatic effect we added 1 unit/ml of FVIII to samples of PRP and WB and measured the maximum thrombin generation in the thrombin generation test (TGT) and the maximum clot firmness (MCF) in TEG. After FVIII substitution we observed a nearly linear relation between the individual FVIII activities administered to the patients and the activities measured in the plasma samples. However, data obtained with TGT and TEG revealed a high inter-individual variation and a very poor correlation to the administered FVIII activity. Actually, it could be shown that FVIII substitution yielding in a FVIII plasma activity of about 30% is sufficient to get an ex vivo haemostatic effect of more that 90% as measured by maximum thrombin generation and MCF. FVIII substitution up to a plasma activity of more than 90% did not further enhance the haemostatic effect. Our data clearly demonstrate that the haemostatic effect of FVIII is not only dependent on the activity that is measured in plasma but also depends on the interplay between coagulation and blood cells, in particular with platelets. The use of cell-based coagulation tests such us TGT or TEG may help to optimize FVIII therapy by determining the individual FVIII dosage that produces a maximum haemostatic effect.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Fator VIII/farmacologia , Hemofilia A/tratamento farmacológico , Tromboelastografia/efeitos dos fármacos , Adulto , Biomarcadores/sangue , Testes de Coagulação Sanguínea/métodos , Determinação de Ponto Final , Humanos , Trombina/análise , Resultado do TratamentoRESUMO
Although the administration of recombinant coagulation factor VIIa (rFVIIa) is a well established treatment in haemophilia with inhibitory antibodies, monitoring the therapeutic efficacy is still a problem. This is because the complete haemostatic effect in vivo depends on negatively charged surfaces provided by exposed lipids from activated platelets which are not present in standard clinical assays using plasma. The thrombin generation assay, however, measures the endogenous thrombin potential (ETP) in platelet-rich plasma (PRP) and this assay might be useful for monitoring the haemostatic response to rFVIIa. We characterized the in vitro concentration-response relationship of rFVIIa and the thrombin generation parameters ETP, PEAK and TIME TO PEAK using platelet-rich plasma (PRP) and rFVIIa at concentrations between one and five times the therapeutic dose. We also studied the effect of inhibiting tissue-factor and the intrinsic coagulation pathway using excess TF-neutralizing antibodies and corn trypsin inhibitor (CTI), respectively. There was a sigmoid relationship between ETP and PEAK and the dose of rFVIIa. Increasing rFVIIa concentrations between 100 and 500 U/ml resulted in a progressive increase in ETP, whereas supratherapeutical concentrations led to a plateau phase. The plateau phase differed between patients, suggesting a biological variation in the maximum ETP. Neutralization of plasma TF with TF-Ab partially decreased FVIIa efficacy. The inhibitory effect of CTI on rFVIIa-induced thrombin generation via the intrinsic pathway was negligible. The thrombin generation assay using PRP is a useful test for determining the sufficient efficacy of rFVIIa in blood. Once a plateau level is reached, higher doses of rFVIIa have no additional effect on haemostatic efficacy. The variation in plateau levels between subjects indicates that there are inter-individual differences in the level of thrombin activity that can be generated. High doses of rFVIIa are effective even in the absence of TF.
Assuntos
Plaquetas/fisiologia , Monitoramento de Medicamentos/métodos , Fator VII/farmacologia , Proteínas Recombinantes/farmacologia , Trombina/biossíntese , Adulto , Testes de Coagulação Sanguínea/métodos , Relação Dose-Resposta a Droga , Fator VIIa , Hemostasia/efeitos dos fármacos , Humanos , Masculino , Proteínas de Plantas , TromboplastinaRESUMO
BACKGROUND: The beneficial effect of moderate alcohol consumption in lowering the risk of cardiovascular disease has been shown in several epidemiologic studies. Such studies have also shown, however, that the protective effect of alcoholic beverages like wine and beer is not only due to the ethanol content but also to the presence of nonalcoholic constituents. The positive effect of alcoholic beverages has been attributed to changes in lipoprotein metabolism, but there is substantial evidence that effects on hemostasis play an important role. Whether the effects of alcoholic beverages on hemostasis are due exclusively to ethanol or are due, in part, to nonalcoholic components, is still under debate. METHODS: We have examined the hemostatic effects of 3 liters of beer, dealcoholized beer, and ethanol/water (v/v 4%), consumed over a period of 3 hr, in 12 young healthy volunteers. Platelet parameters CD62, PAC-1, and monocyte platelet aggregates were analyzed using flow cytometric measurements. The activity of factor VII was determined with a prothrombin time (PT) assay and plasminogen activator inhibitor activity using a chromogenic substrate. Thrombin generation was determined according to the method of Hemker. RESULTS: All three fluids administered, dealcoholized beer, beer, and ethanol, reduced the expression of activated fibrinogen receptor, the platelet activation marker CD62, and the formation of monocyte-platelet-aggregate. In addition, dealcoholized beer also showed significant inhibitory effects on thrombin generation, whereas beer and ethanol showed procoagulatory effects. CONCLUSIONS: This study has shown that the acute consumption of dealcoholized beer inhibits thrombogenic activity in young adults. This action could have a beneficial effect on the development of coronary artery disease. Thus, the consumption of dealcoholized beer could provide cardiovascular benefit without the negative effects of alcohol.
Assuntos
Consumo de Bebidas Alcoólicas/sangue , Cerveja , Etanol/administração & dosagem , Hemostasia/efeitos dos fármacos , Adulto , Cerveja/análise , Bebidas , Hemostasia/fisiologia , Humanos , Masculino , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Trombina/metabolismoRESUMO
Low molecular weight heparin (LMWH) is effective in the treatment of acute deep vein thrombosis (DVT) in adults. This has not been demonstrated for one LMWH alone. The relationship between venographic changes due to LMWH therapy and clinical outcome in the initial treatment period has not been reported. A pooled analysis of two clinical trials was performed. The trials compared a fixed-dose, body weight-independent, subcutaneous LMWH, certoparin (8000 antifactor Xa [aXa] U twice a day [b.i.d.]), with an adjusted-dose intravenous unfractionated heparin (UFH) with respect to venographic changes expressed as Marder score and occurrence of recurrent venous thromboembolism, major bleeding, and mortality. The Marder score was 23.2 +/- 8.4 in patients randomized to LMWH (n = 299 paired phlebograms) and 23.9 +/- 8.9 in patients allocated to UFH (n = 297 paired phlebograms) at entry (2p = 0.23) and 18.9 +/- 9.7 and 20.5 +/- 9.9 at the end of the initial therapy (2p = 0.04), respectively. The composite outcome of recurrent venous thromboembolism, major bleeding, and mortality occurred less frequently during treatment with LMWH (n = 393) than it did with UFH (n = 404, 1.3% versus 5.0%, risk reduction [RR] 0.26, 95% confidence interval [CI] 0.11 to 0.63, 2p = 0.004). Single events of recurrent thromboembolism (2p = 0.12), major bleeding (2p = 0.03), and mortality (2p = 0.12) were observed less frequently with LMWH. A trend toward a lack of regression of thrombus size was observed in recurrent venous thromboembolism (2p = 0.08). Body weight-independent LMWH significantly reduces thrombus size and the incidence of composite outcome during the initial treatment of acute proximal venous thrombosis compared with adjusted dose intravenous UFH. The data indicate a relation between an unimproved Marder score and a recurrent venous thromboembolism.
Assuntos
Heparina de Baixo Peso Molecular/administração & dosagem , Trombose Venosa/tratamento farmacológico , Idoso , Relação Dose-Resposta a Droga , Feminino , Hemorragia/induzido quimicamente , Heparina de Baixo Peso Molecular/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Flebografia , Recidiva , Taxa de Sobrevida , Resultado do Tratamento , Trombose Venosa/complicações , Trombose Venosa/mortalidadeRESUMO
AIMS: The objective of our study was to define the interaction between either unfractionated heparin (UFH) or a low molecular weight heparin, reviparin (REV), and the pharmacodynamic profile of the GPIIb/IIIa-antagonists abciximab (ABC) or tirofiban (T). METHODS: Two studies each containing 18 healthy subjects were performed, and all were pretreated with aspirin (ASA) for 3 days. Volunteers then received UFH (5000 IU bolus/infusion 7 IU kg(-1) h(-1) for 7 h, n = 6), REV (4200-anti-Xa-IU s.c., n = 6) or placebo (n = 6). One hour later, ABC (study I) or T (study II) were given by i.v. infusion for 6 h. The pharmacodynamic effects measured were bleeding time (BT), fibrinogen-binding at the GPIIb/IIIa-receptor (FIB), expression of the platelet secretion marker CD62, and ADP (20 microM)- and collagen (5 microg ml(-1))-induced platelet aggregation. RESULTS: After treatment with both GPIIb/IIIa-antagonists, prolongation of BT occurred to a similar magnitude (approximately 25-30 min) and was not affected by UFH or REV-comedication. ABC or T with ASA alone resulted in nearly the same magnitude of reduction in FIB and platelet aggregation. After coadministration with UFH, FIB was significantly higher (thus less inhibited) than after after T + ASA alone (19 +/- 16% vs 55 +/- 36%) or ABC + ASA alone (8 +/- 9% vs 32 +/- 11%). This attenuation of FIB was not seen with REV. Inhibition of ADP-and collagen-induced aggregation tended to be attenuated by treatment with UFH (e.g. ADP-induced aggregation at 0.25 h after ABC + ASA alone =13 +/- 4%; after coadministration with UFH = 40 +/- 26%). No such changes were noted with REV. Minor reductions in CD62-expression were seen in subjects given ABC or T alone, but expression was not affected by UFH or REV. CONCLUSIONS: Co-medication with UFH attenuated platelet inhibition during treatment with GPIIb/IIIa-antagonists, but these effects were not seen with the low molecular weight heparin reviparin. The results show that administration of reviparin together with abciximab or tirofiban did not adversely affect the pharmacodynamic profile of these GPIIb/IIIa-antagonists.
Assuntos
Anticoagulantes/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Abciximab , Adolescente , Adulto , Anticorpos Monoclonais/farmacologia , Ligação Competitiva/efeitos dos fármacos , Tempo de Sangramento , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Interações Medicamentosas , Inibidores do Fator Xa , Fibrinogênio/metabolismo , Citometria de Fluxo , Heparina/farmacologia , Heparina de Baixo Peso Molecular/farmacologia , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Masculino , Tempo de Tromboplastina Parcial , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Selectinas/efeitos dos fármacos , Selectinas/metabolismo , Tirofibana , Resultado do Tratamento , Tirosina/análogos & derivados , Tirosina/farmacologiaRESUMO
Vascular endothelial growth factor (VEGF) has been implicated in the reendothelialization of the vascular wall after balloon injury. This study investigated whether thrombin, which is formed during activation of the coagulation cascade at sites of vascular injury, upregulates VEGF expression in vascular smooth muscle cells (VSMCs). VEGF expression was assessed in native and cultured VSMCs by Northern blot analysis and reverse transcription-polymerase chain reaction and the release of VEGF protein by immunoassay. alpha-Thrombin time- and concentration-dependently increased VEGF mRNA levels, mainly that mRNA coding for the soluble splice variant VEGF(164/165), and stimulated the release of VEGF protein. These effects required the proteolytic activity of thrombin and were mimicked by a thrombin receptor activating-peptide. Upregulation of VEGF expression was also induced by conditioned medium from alpha-thrombin-stimulated VSMCs. Both the early and the delayed alpha-thrombin-induced VEGF expressions were attenuated by antioxidants and by diphenyleneiodonium. alpha-Thrombin-induced VEGF release was significantly reduced by a platelet-derived growth factor (PDGF)-, a transforming growth factor (TGF)-beta-, and a basic fibroblast growth factor (bFGF)-neutralizing antibody. Thrombin caused a redox-sensitive upregulation of expression of VEGF in VSMCs through a direct and an indirect effect, which was dependent on the endogenous formation of PDGF, TGF-beta, and bFGF. Upregulation of VEGF expression may represent an important mechanism by which the coagulation cascade contributes to the regeneration of the endothelial lining at sites of balloon injury.
Assuntos
Fatores de Crescimento Endotelial/biossíntese , Linfocinas/biossíntese , Músculo Liso Vascular/metabolismo , Espécies Reativas de Oxigênio/fisiologia , Trombina/farmacologia , Acetilcisteína/farmacologia , Angioplastia com Balão/efeitos adversos , Animais , Antioxidantes/farmacologia , Arteriopatias Oclusivas/etiologia , Ácido Ascórbico/farmacologia , Células Cultivadas , Fatores de Crescimento Endotelial/genética , Fator 2 de Crescimento de Fibroblastos/biossíntese , Humanos , Cinética , Linfocinas/genética , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/biossíntese , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Ativação Transcricional , Fator de Crescimento Transformador beta/biossíntese , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
AIMS: The aim of this study was to assess the inter- and intra-laboratory variation of the concentration-response to the GPIIb/IIIa-antagonists abciximab and eptifibatide on platelet aggregometry and to compare results with flow cytometric tests as well as the rapid platelet function analyser (RPFA). METHODS: In five different laboratory sites, blood from three to five healthy donors was spiked with abciximab or eptifibatide, followed by the assessment of: (1) aggregometry (anticoagulant: sodium citrate 3.18% or hirudin 5 microg/ml); (2) flow cytometry (fibrinogen binding or PAC1-expression), or (3) RPFA. Dose-response curves were established on the basis of a sigmoidal Imax)-model [I=(Imax)*Cg)/(IC50g + Cg)]. RESULTS: For citrated blood, aggregation induced by 20 microM ADP was blocked up to 100% by both GPIIb/IIIa-antagonists, IC50 values varied between 0.11-0.22 microg/ml for eptifibatide and 1.25-2.3 microg/ml for abciximab. I(max) of the response to 5 microg/ml collagen ranged from 46% to 100%, and IC50 values varied between 0.28-0.34 microg/ml for eptifibatide and 2.3-3.8 microg/ml for abciximab. In hirudinized blood, IC50 values for eptifibatide were 1.5- to 3-fold higher than those obtained with citrated plasma. Inhibition of PAC1-expression by abciximab (IC50) 0.84 microg/ml) showed results similar those of the RPFA (approx. 1.0 microg/ml); larger differences between PAC1 and RPFA results were observed for eptifibatide. Based on aggregometry, eptifibatide concentrations for 80% inhibition varied from 0.27 to 0.55 microg/ml, and were considerably less when the RPFA was taken as basis (0.15 or 0.22 microg/ml). A similar pattern was observed for abciximab. CONCLUSIONS: We found quite a low inter- and intra-laboratory variation in the in vitro pharmacodynamic characterization of GPIIb/IIIa-antagonists by aggregometry, making results of these tests obtained from different laboratories during clinical trials at least comparable. The RPFA exhibits a higher sensitivity to inhibitory GPIIb/IIIa-effects, in keeping with the "real" inhibition of the activated receptor (PAC1) as assessed with more elaborate flow cytometry.
Assuntos
Técnicas de Laboratório Clínico/normas , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Abciximab , Anticorpos Monoclonais/farmacologia , Técnicas de Laboratório Clínico/estatística & dados numéricos , Relação Dose-Resposta a Droga , Eptifibatida , Fibrinogênio , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Microesferas , Variações Dependentes do Observador , Peptídeos/farmacologia , Testes de Função Plaquetária/instrumentação , Testes de Função Plaquetária/normas , Testes de Função Plaquetária/estatística & dados numéricos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologiaRESUMO
Myelodysplastic syndromes (MDS) are characterized by a haematopoetic insufficiency that can lead to acute leukemia. A multistep pathogenesis caused by a clonal stem cell defect affecting several differentiation pathways has been proposed for MDS. Contrary to the better characterized alteration of lymphoid and myeloid differentiation, defects in thrombocytopoesis in MDS remain less clear. In the present study, we analyzed the expression of platelet glycoprotein (GP) Ia/IIa, IIb/IIIa, Ib/IX, and IV in 21 MDS patients (12 RA, 2 RARS, 4 RAEB, 1 RAEB-T, 2 CMML) and healthy controls by flowcytometric analysis and quantitation of platelet GP RNA using fluorescence-based PCR. We observed a reduced cell surface expression of GPIb (p<0.01) and GPIIb/IIIa (p<0.01), while GPIa/IIa and GPIV expression was only marginally different between patients and controls. In contrast, there was a two-fold increase of platelet GPIb and GPIIb RNA and a three-fold increase of GPIV RNA among MDS patients. Increased levels of platelet GPIb and GPIIb RNA were significantly more prominent among patients with RAEB(-T)/CMML (p<0. 05) in comparison to patients with RA/RARS. In conclusion, we demonstrate alterations in the cell surface expression and RNA content of platelet GPs in MDS patients. These data are consistent with dysmegakaryocytopoiesis and a defect in thrombocytopoiesis among MDS patients resulting from the clonal stem cell defect in MDS.
Assuntos
Síndromes Mielodisplásicas/sangue , Glicoproteínas da Membrana de Plaquetas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD36/genética , Antígenos CD36/metabolismo , Estudos de Casos e Controles , Feminino , Hematopoese , Humanos , Integrina alfa2 , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/classificação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , RNA Mensageiro/metabolismoRESUMO
Vascular injury after balloon angioplasty results in the rapid activation of platelets leading to the release of growth factors and vasoactive substances. In addition, up-regulation of tissue factor (TF) and an increased production of reactive oxygen species (ROS) have been detected at sites of vascular injury. We investigated whether platelet-derived products (PDP) released from activated human platelets increase ROS production, resulting in the induction of TF expression in vascular smooth muscle cells (SMC). PDP induced a time- and concentration-dependent increase in ROS generation in cultured SMC that was mediated mainly by PDGF-AB and TGF-beta1 and impaired by the flavin inhibitor diphenylene iodonium. Increased ROS formation was associated with enhanced mRNA levels of the small NAD(P)H oxidase subunit p22phox or its smooth muscle isoform. Transient transfection with a p22phox antisense vector decreased PDP-induced ROS generation. PDP up-regulated TF mRNA expression, which was redox sensitive and reduced by transfection of the p22phox antisense vector. In addition, PDP-stimulated reporter gene activity of two TF promoter constructs was decreased by coexpression of the p22phox antisense vector. These results indicate that activated platelets up-regulate TF expression and that this response involves ROS generation and a p22phox-containing NAD(P)H oxidase in SMC.
Assuntos
Plaquetas/metabolismo , Proteínas de Membrana Transportadoras , Músculo Liso Vascular/metabolismo , NADPH Desidrogenase/metabolismo , Estresse Oxidativo , Fosfoproteínas/metabolismo , Ativação Plaquetária/fisiologia , Tromboplastina/metabolismo , Animais , Células Cultivadas , Grupo dos Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , NADPH Desidrogenase/genética , NADPH Oxidases , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosfoproteínas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tromboplastina/genética , Fatores de Tempo , Transfecção , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVES: To assess the interaction between aspirin and clopidogrel in healthy male volunteers and the interaction of the glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitors abciximab and SR121566A with blood from those pretreated subjects (ex vivo-in vitro). METHODS: Aspirin (300 mg/day), clopidogrel (75 mg/day), or the combination of both drugs were administered orally for 8 days. Group 1 (n = 5) started with aspirin and group 2 (n = 5) with clopidogrel. From day 4 to day 8, subjects of both groups received the combined treatment. Blood from these subjects was spiked with abciximab (0.5 and 1.5 microg x mL(-1)) and SR121566A (31 and 62 ng x mL(-1)). RESULTS: In vivo, average bleeding times were 6.8 minutes at baseline, 20.3 minutes for clopidogrel alone (P < .01), 10.9 minutes for aspirin alone (difference not significant), and 24.0 minutes (P < .01) for the combined treatment. Fibrinogen binding to the platelet GPIIb/IIIa receptor was reduced for aspirin to 69% (difference not significant), to 63% for clopidogrel (difference not significant), and to 63% for the clopidogrel plus aspirin combination (P < .01). CD62 expression as a marker of platelet granular secretion was reduced to 66% by clopidogrel (P < .01) and to 41% by the combination of clopidogrel and aspirin; aspirin alone had no effect. In vitro, with pretreatment with aspirin and clopidogrel, inhibitory effects of the GPIIb/IIIa inhibitors on fibrinogen binding were additive to changes observed with aspirin or clopidogrel alone. No effect on CD62 expression was observed with either GPIIb/IIIa inhibitor. Aspirin and clopidogrel reinforced effects of the GPIIb/IIIa inhibitors on adenosine diphosphate (5 micromol/L)-induced aggregation in an additive manner, a supra-additive effect was observed with collagen (2 microg x mL(-1))-induced aggregation. CONCLUSION: The augmentation of the antiaggregatory effects of GPIIb/IIIa inhibitors by aspirin and clopidogrel and the lack of antisecretory effects of GPIIb/IIIa inhibitors may favor their combination with clopidogrel.
Assuntos
Anticorpos Monoclonais/farmacologia , Aspirina/farmacologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/farmacologia , Ticlopidina/análogos & derivados , Abciximab , Administração Oral , Adulto , Análise de Variância , Anticorpos Monoclonais/administração & dosagem , Aspirina/administração & dosagem , Benzilaminas , Tempo de Sangramento , Clopidogrel , Esquema de Medicação , Interações Medicamentosas , Fibrinogênio/efeitos dos fármacos , Fibrinogênio/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Masculino , Nefelometria e Turbidimetria , Selectina-P/efeitos dos fármacos , Selectina-P/metabolismo , Piperidinas , Inibidores da Agregação Plaquetária/administração & dosagem , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/administração & dosagem , Valores de Referência , Tiazóis , Ticlopidina/administração & dosagem , Ticlopidina/farmacologiaRESUMO
The aim of this study was to compare fibrinogen binding, inhibition of platelet aggregation and secretory potential of the MAb abciximab (0.5-5 microg/mL) and the peptidomimetic compound SR121566A (15-250 ng/mL) in vitro in whole blood. Fibrinogen binding was followed by flow cytometry; platelet function was evaluated by light transmittance and by impedance aggregometry. Secretory functions of platelets were evaluated using ATP as marker for early secretion by dense granulae and P-selectin (CD62) for alpha-granular secretion as well as CD63 for lysosomal degranulation. Results showed that fibrinogen binding induced by 5 microM TRAP was maximally inhibited greater than 80% at 3 microg/mL abciximab or at 250 ng/mL SR121566A. At these concentrations of antagonists, platelet aggregation induced by 5 microM ADP or 2 microg/mL collagen was inhibited completely. Expression of CD62 was reduced 34% with abciximab or 15% with SR121566A; CD63 expression was reduced 22% with both agents. With both agents, the EC50 for inhibition of CD62 and CD63 expressions was in similar magnitudes than the EC50 for fibrinogen binding inhibition. With 3 microg/mL abciximab, ATP secretion was maximally reduced to 50% of the control, whereas SR121566A at 250 ng/mL had no inhibitory effect on this parameter. A slight increase in ATP secretion was seen with 0.5 microg/mL abciximab and with SR121566A in concentrations of less than 45 ng/mL. The data suggest a discoupling between the anti-aggregatory and the antisecretory effects of IIb/IIIa antagonists. Because it is not established to what extend CD62 or CD63 expression can be reduced by any means, the reduction by 20-30% obtained by 3 microg/mL abciximab or 250 ng/mL SR121566A might already be the maximum possible inhibition by these agents.
Assuntos
Anticorpos Monoclonais/imunologia , Fibrinogênio/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Adulto , Antígenos CD/metabolismo , Benzilaminas , Sítios de Ligação , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Feminino , Humanos , Técnicas In Vitro , Masculino , Oligopeptídeos/farmacologia , Selectina-P/metabolismo , Piperidinas , Inibidores da Agregação Plaquetária/sangue , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Tetraspanina 30 , TiazóisRESUMO
Low molecular weight heparins (LMWHs) are effective agents in both venous and arterial thrombosis. Extensive preclinical studies in various animal models show that there are substantial differences in antithrombotic efficacy between LMWHs, and that the relative efficacy of individual agents depends on the thrombogenic stimulus used. Relative efficacy cannot be predicted on the basis of molecular weight, nor on the basis of anti-factor Xa or anti-factor IIa potency. This suggests that other mechanisms of action, e.g., the release of tissue factor pathway inhibitor (which differs among the LMWHs), are important. Data obtained with a specific LMWH cannot be extrapolated to other compounds within the same class and their substitution in the clinical setting is, therefore, inappropriate.