RESUMO
Altered expression and functional responses to cardiac beta(3)-adrenergic receptors (ARs) may contribute to progressive cardiac dysfunction in heart failure (CHF). We compared myocyte beta(3)-AR mRNA and protein levels and myocyte contractile, [Ca(2+)](i) transient, and Ca(2+) current (I(Ca,L)) responses to BRL-37344 (BRL, 10(-8) mol/L), a selective beta(3)-AR agonist, in 9 instrumented dogs before and after pacing-induced CHF. Myocytes were isolated from left ventricular myocardium biopsy tissues. Using reverse transcription-polymerase chain reaction, we detected beta(3)-AR mRNA from myocyte total RNA in each animal. Using a cloned canine beta(3)-AR cDNA probe and myocyte poly A(+) RNA, we detected a single band about 3.4 kb in normal and CHF myocytes. beta(3)-AR protein was detected by Western blot. beta(3)-AR mRNA and protein levels were significantly greater in CHF myocytes than in normal myocytes. Importantly, these changes were associated with enhanced beta(3)-AR-mediated negative modulation on myocyte contractile response and [Ca(2+)](i) regulation. Compared with normal myocytes, CHF myocytes had much greater decreases in the velocity of shortening and relengthening with BRL accompanied by larger reductions in the peak systolic [Ca(2+)](i) transient and I(Ca,L). These responses were not modified by pretreating myocytes with metoprolol (a beta(1)-AR antagonist) or nadolol (a beta(1)- and beta(2)-AR antagonist), but were nearly prevented by bupranolol or L-748,337 (beta(3)-AR antagonists). We conclude that in dogs with pacing-induced CHF, beta(3)-AR gene expression and protein levels are upregulated, and the functional response to beta(3)-AR stimulation is increased. This may contribute to progression of cardiac dysfunction in CHF.
Assuntos
Insuficiência Cardíaca/fisiopatologia , Miocárdio/metabolismo , Receptores Adrenérgicos beta 3/genética , Receptores Adrenérgicos beta 3/metabolismo , Regulação para Cima , Antagonistas de Receptores Adrenérgicos beta 1 , Antagonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Western Blotting , Cálcio/metabolismo , Estimulação Cardíaca Artificial/efeitos adversos , Separação Celular , Modelos Animais de Doenças , Progressão da Doença , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Etanolaminas/farmacologia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/patologia , Contração Miocárdica/efeitos dos fármacos , Miocárdio/patologia , RNA Mensageiro/metabolismo , Receptores Adrenérgicos beta 3/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Platelet glycoprotein IIb/IIIa blockade with abciximab (ReoPro) improves the clinical outcomes of percutaneous coronary intervention. This registry was conducted to characterize the effects of repeated administration of abciximab during intervention. METHODS AND RESULTS: We recruited 500 consecutive patients at 22 centers in the United States who were receiving abciximab for at least a second time during percutaneous coronary intervention. Safety was measured as the incidence of hypersensitivity reactions, major bleeding, and thrombocytopenia. Efficacy was assessed as event-free clinical success. Human antichimeric antibody (HACA) responses were also characterized. There were no cases of hypersensitivity (95% upper confidence bound, 0.3%), major bleeding, or death. Clinical success was 94.4%. Thrombocytopenia occurred in 23 patients (4.6%; 95% CI, 2.8% to 6.4%), including 12 (2.4%; 95% CI, 1.1% to 3.7%) who developed profound thrombocytopenia (<20x10(9) cells/L). In 2 patients (0.4%), profound thrombocytopenia did not develop until after hospital discharge; in 4 (0.8%), profound thrombocytopenia recurred despite platelet transfusion. Before a first readministration, a positive HACA titer was present in 22 of 454 patients (4.8%); after a first readministration, an additional 82 of 432 (19.0%) became HACA-positive. HACA did not neutralize the in vitro inhibition of platelet aggregation by abciximab or correlate with clinical events. CONCLUSIONS: The results, including overall rates of thrombocytopenia, were consistent with randomized clinical trials of first abciximab treatment. However, there was a shift from mild to profound thrombocytopenia, and cases of delayed presentation and of recurrent thrombocytopenia were seen. These findings suggest that indications and guidelines for first-time use apply to retreatment, particularly the systematic monitoring for thrombocytopenia.
Assuntos
Angioplastia Coronária com Balão , Anticorpos Monoclonais/administração & dosagem , Doença das Coronárias/terapia , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Sistema de Registros/estatística & dados numéricos , Trombocitopenia/diagnóstico , Grau de Desobstrução Vascular/efeitos dos fármacos , Abciximab , Angioplastia Coronária com Balão/efeitos adversos , Anticorpos/sangue , Anticorpos/farmacologia , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Aspirina/administração & dosagem , Doença das Coronárias/sangue , Intervalo Livre de Doença , Esquema de Medicação , Hemorragia/etiologia , Heparina/administração & dosagem , Humanos , Fragmentos Fab das Imunoglobulinas/efeitos adversos , Fragmentos Fab das Imunoglobulinas/imunologia , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/administração & dosagem , Inibidores da Agregação Plaquetária/efeitos adversos , Inibidores da Agregação Plaquetária/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Complicações Pós-Operatórias/prevenção & controle , Trombocitopenia/etiologia , Resultado do Tratamento , Estados UnidosRESUMO
BACKGROUND: The optimal level of platelet inhibition with a glycoprotein (GP) IIb/IIIa antagonist necessary to minimize thrombotic complications in patients undergoing a percutaneous coronary intervention (PCI) is currently unknown. METHODS AND RESULTS: Five hundred patients undergoing a PCI with the planned use of a GP IIb/IIIa inhibitor had platelet inhibition measured at 10 minutes, 1 hour, 8 hours, and 24 hours after the initiation of therapy with the Ultegra Rapid Platelet Function Assay (Accumetrics). Major adverse cardiac events (MACES: composite of death, myocardial infarction, and urgent target vessel revascularization) were prospectively monitored, and the incidence correlated with the measured level of platelet function inhibition at all time points. One quarter of all patients did not achieve >/=95% inhibition 10 minutes after the bolus and experienced a significantly higher incidence of MACEs (14.4% versus 6.4%, P=0.006). Patients whose platelet function was <70% inhibited at 8 hours after the start of therapy had a MACE rate of 25% versus 8.1% for those >/=70% inhibited (P=0.009). By multivariate analysis, platelet function inhibition >/=95% at 10 minutes after the start of therapy was associated with a significant decrease in the incidence of a MACE (odds ratio 0.46, 95% CI 0.22 to 0.96, P=0.04). CONCLUSIONS: Substantial variability in the level of platelet function inhibition is achieved with GP IIb/IIIa antagonist therapy among patients undergoing PCI. The level of platelet function inhibition as measured by a point-of-care assay is an independent predictor for the risk of MACEs after PCI.
Assuntos
Angioplastia Coronária com Balão , Cardiopatias/prevenção & controle , Inibidores da Agregação Plaquetária/uso terapêutico , Tirosina/análogos & derivados , Abciximab , Idoso , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/uso terapêutico , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Estudos de Coortes , Eptifibatida , Feminino , Cardiopatias/induzido quimicamente , Humanos , Fragmentos Fab das Imunoglobulinas/efeitos adversos , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Modelos Logísticos , Masculino , Análise Multivariada , Peptídeos/efeitos adversos , Peptídeos/uso terapêutico , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/efeitos adversos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Estudos Prospectivos , Fatores de Risco , Fatores de Tempo , Tirofibana , Tirosina/efeitos adversos , Tirosina/uso terapêuticoRESUMO
OBJECTIVES: This study determined the incidence of pseudothrombocytopenia during abciximab therapy administered for percutaneous coronary interventions and compared the clinical course of patients with pseudothrombocytopenia with the clinical courses of patients with thrombocytopenia and patients with normal platelet counts. BACKGROUND: Although pseudothrombocytopenia has been previously reported during therapy with abciximab, the incidence and significance of this occurrence are unknown. The failure to differentiate pseudothrombocytopenia from thrombocytopenia could lead to unnecessary interruption of abciximab infusions or to platelet transfusions. METHODS: The incidences of pseudothrombocytopenia and thrombocytopenia were determined in four large placebo-controlled abciximab trials: c7E3 Fab Antiplatelet Therapy in Unstable Refractory Angina (CAPTURE), Evaluation of 7E3 for the Prevention of Ischemic Complications (EPIC), Evaluation of Percutaneous Transluminal Coronary Angioplasty to Improve Long-term Outcome of c7E3 GpIIb/IIIa Receptor Blockade (EPILOG) and Evaluation of Platelet IIb/IIIa Inhibitor for Stenting (EPISTENT). The clinical features, bleeding complications and major clinical outcomes of patients with pseudothrombocytopenia and those with thrombocytopenia were compared with each other and with those of patients with normal platelet count. RESULTS: Pseudothrombocytopenia occurred in 2.1% (95% confidence intervals [CI]: 1.7%, 2.5%) of abciximab-treated patients and in 0.6% of placebo-treated patients (p < 0.001). Thrombocytopenia occurred in 3.7% (95% CI: 3.2%, 4.2%) of abciximab-treated patients and in 1.8% (95% CI: 1.3%, 2.3%) of placebo-treated patients (p < 0.001). Patients with thrombocytopenia had significantly higher rates of major bleeding, major decreases in hemoglobin and increased transfusion requirements of both blood and platelets compared with those without thrombocytopenia. By contrast, pseudothrombocytopenic patients did not differ from patients with normal platelet counts in any of the measures of blood loss or transfusion requirements. Thrombocytopenic patients, but not those with pseudothrombocytopenia, had increased rates of revascularization at 30 days and six months. As previously reported, there was also a higher rate of death and myocardial infarction in the thrombocytopenic patients. CONCLUSIONS: Pseudothrombocytopenia is the cause of more than one third (36.3%) of low platelet counts in patients undergoing coronary interventions who are treated with abciximab. This study demonstrates that pseudothrombocytopenia is a benign laboratory condition that does not increase bleeding, stroke, transfusion requirements or the need for repeat revascularization. It is important to recognize pseudothrombocytopenia so that the beneficial effects of abciximab are not lost by premature termination of therapy.
Assuntos
Anticorpos Monoclonais/efeitos adversos , Fragmentos Fab das Imunoglobulinas/efeitos adversos , Inibidores da Agregação Plaquetária/efeitos adversos , Trombocitopenia/induzido quimicamente , Abciximab , Idoso , Angina Instável/sangue , Angina Instável/tratamento farmacológico , Angina Instável/cirurgia , Anticorpos Monoclonais/administração & dosagem , Intervalos de Confiança , Ponte de Artéria Coronária , Diagnóstico Diferencial , Método Duplo-Cego , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Incidência , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Inibidores da Agregação Plaquetária/administração & dosagem , Contagem de Plaquetas/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Estudos Prospectivos , Trombocitopenia/sangue , Trombocitopenia/diagnósticoRESUMO
OBJECTIVES: The purpose of this study was to examine the incidence and clinical implications of thrombocytopenia that occurs in hospital after administration of thrombolytic therapy for acute myocardial infarction. BACKGROUND: The use of thrombolytic therapy in patients with acute myocardial infarction has improved mortaltiy rates, but hemorrhage remains a major complication. Because thrombocytopenia may be associated with hemorrhage after thrombolytic therapy, we examined the incidence and clinical implications of thrombocytopenia after administration of thrombolytic therapy for acute myocardial infarction. METHODS: The patient population comprised 1,001 patients enrolled in Phases 2, 3 and 5 of the Thrombolysis and Angioplasty in Myocardial Infarction (TAMI) trial and the urokinase trial. Patients received recombinant tissue-type plasminogen activator, urokinase or combination therapy in various dosing schemes. All patients received heparin, aspirin and a calcium-channel blocking agent. Thrombocytopenia occurring anytime after thrombolytic therapy was defined as a nadir platelet count either < 100,000/microliters or < 1/2 baseline. Blood loss was quantified by a bleeding index. Multiple logistic regression was used to evaluate the independent contribution of thrombocytopenia in a model predicting in-hospital mortality. RESULTS: Thrombocytopenia occurred in 16.4% of patients, with no difference among the thrombolytic regimens. Patients with thrombocytopenia had a lower median acute ejection fraction and a higher likelihood of three-vessel coronary artery disease than patients without thrombocytopenia. Patients with thrombocytopenia had more hemorrhage, a higher in-hospital mortality rate and a more complicated hospital course than patients without thrombocytopenia, even after consideration of other important variables, including age, acute ejection fraction, number of diseased vessels, bypass surgery and use of intraaortic balloon counterpulsation. CONCLUSIONS: Thrombocytopenia after thrombolytic therapy is a common event and is associated with excess hemorrhage and mortality. Platelet counts should be monitored daily after administration of thrombolytic therapy because the appearance of thrombocytopenia identifies a subset of patients at increased risk for hemorrhage and death.
Assuntos
Infarto do Miocárdio/tratamento farmacológico , Trombocitopenia/etiologia , Terapia Trombolítica/efeitos adversos , Idoso , Feminino , Hemorragia/etiologia , Mortalidade Hospitalar , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/mortalidade , Contagem de PlaquetasRESUMO
OBJECTIVES: This study sought to determine the frequency of thrombocytopenia and its relation with clinical outcomes in high risk patients undergoing percutaneous coronary revascularization who received either the platelet glycoprotein (GP) IIb/IIIa receptor antagonist abciximab (ReoPro, c7E3 Fab) or conventional therapy. BACKGROUND: The development of thrombocytopenia on exposure to GPIIb/IIIa antagonists threatens the utility and economic viability of this drug class for patients with vascular disease. METHODS: We analyzed data from the Evaluation of c7E3 for the Prevention of Ischemic Complications trial (EPIC), a 2,099-patient, randomized trial of placebo, abciximab bolus or abciximab bolus plus a 12-h infusion during high-risk coronary revascularization. RESULTS: Thrombocytopenia (nadir platelet count <100 x 10(9)/ liter) developed in 81 patients (3.9%) during their hospital stay, with 19 (0.9%) developing severe (<50 x 10(9)/liter) thrombocytopenia. Both thrombocytopenia and severe thrombocytopenia were more frequent in the bolus-plus-infusion arm (5.2% and 1.6%, respectively) than in the bolus-only and placebo arms combined (p = 0.020 and p = 0.025, respectively). Acute profound thrombocytopenia developed in two patients in the bolus-plus-infusion arm. Patients with thrombocytopenia experienced more unfavorable clinical outcomes than those who did not develop thrombocytopenia, regardless of treatment assignment, but those with thrombocytopenia who received abciximab had fewer worse outcomes at 30 days. Multivariable logistic modeling revealed a lower baseline platelet count, older age and lighter weight to be important predictors of thrombocytopenia. In a logistic regression model, bolus-plus-infusion treatment was a significant predictor of thrombocytopenia (p = 0.016) and remained so after adjustment for procedures and baseline risk factors (p = 0.0077). CONCLUSIONS: Thrombocytopenia was associated with adverse clinical outcomes and excessive bleeding, but patients receiving abciximab fared better than those receiving placebo.
Assuntos
Angioplastia Coronária com Balão/métodos , Anticorpos Monoclonais/efeitos adversos , Fragmentos Fab das Imunoglobulinas/efeitos adversos , Isquemia Miocárdica/prevenção & controle , Inibidores da Agregação Plaquetária/efeitos adversos , Trombocitopenia/etiologia , Abciximab , Fatores Etários , Idoso , Anticorpos Monoclonais/administração & dosagem , Peso Corporal , Feminino , Previsões , Hemorragia/etiologia , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Infusões Intravenosas , Injeções Intravenosas , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Placebos , Inibidores da Agregação Plaquetária/administração & dosagem , Contagem de Plaquetas , Fatores de Risco , Resultado do TratamentoRESUMO
Warfarin targets vitamin K 2,3-epoxide reductase (VKOR), the enzyme that produces reduced vitamin K, a required cofactor for g-carboxylation of vitamin K-dependent proteins. To identify VKOR, we used 4'-azido-warfarin-3H-alcohol as an affinity label. When added to a partially purified preparation of VKOR, two proteins were identified by mass spectrometry as calumenin and cytochrome B5. Rat calumenin was cloned and sequenced and the recombinant protein was produced. When added to an in vitro test system, the 47 kDa recombinant protein was found to inhibit VKOR activity and to protect the enzyme from warfarin inhibition. Calumenin was also shown to inhibit the overall activity of the complete vitamin K-dependent g-carboxylation system. The results were repeated in COS-1 cells overexpressing recombinant calumenin. By comparing calumenin mRNA levels in various tissues from normal rats and warfarin-resistant rats, only the livers from resistant rats were different from normal rats by showing increased levels. Partially purified VKOR from resistant and normal rat livers showed no differences in Km-values, specific activity, and sensitivity to warfarin. A novel model for genetic warfarin resistance in the rat is proposed, whereby the concentration of calumenin in liver determines resistance.
Assuntos
Anticoagulantes/farmacologia , Varfarina/farmacologia , Animais , Células COS , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/farmacologia , Relação Dose-Resposta a Droga , Resistência a Medicamentos/genética , Expressão Gênica , Fígado/metabolismo , Pulmão/metabolismo , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/metabolismo , Miocárdio/metabolismo , Marcadores de Fotoafinidade , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transfecção , Vitamina K Epóxido RedutasesRESUMO
The vitamin K-dependent protein, matrix Gla protein (MGP) is a binding protein for bone morphogenetic protein-2 (BMP-2). Here we present additional evidence that the Ca2+-induced conformer of the vitamin K-dependent Gla region in MGP is involved in BMP-2 binding. Recombinant BMP-2 binds to the Gla-containing region of MGP in the presence of Ca2+. Immunohistochemistry showed that calcified lesions in the aortic wall of aging rats contained elevated concentrations of MGP that was poorly gamma-carboxylated and did not bind BMP-2. In contrast, we were able to identify glandular structures in the mucosa of the rat nasal septum that gave bright fluorescent signals with both antigens; confocal microscopy confirmed their colocalization. These results demonstrate that the BMP-2/MGP complex exists in vivo, consistent with a role for MGP as a BMP-2 inhibitor. Age-related arterial calcification may be a consequence of under-gamma-carboxylation of MGP, allowing unopposed BMP-2 activity.
Assuntos
Doenças da Aorta/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Calcinose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas da Matriz Extracelular , Fator de Crescimento Transformador beta , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/química , Proteínas Morfogenéticas Ósseas/genética , Cálcio/química , Proteínas de Ligação ao Cálcio/farmacologia , Bovinos , Feminino , Imunofluorescência/métodos , Hormônio do Crescimento/química , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Humanos , Imuno-Histoquímica , Microscopia Confocal , Dados de Sequência Molecular , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Mucosa Nasal/ultraestrutura , Septo Nasal/anormalidades , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes , Vitamina K/farmacologia , Proteína de Matriz GlaRESUMO
The PAI-1 binding site for VN was studied using two independent methods. PAI-1 was cleaved by Staph V8 protease, producing 8 fragments, only 2 of which bound to [125I]-VN. These fragments were predicted to overlap between residues 91-130. Since PAI-2 has structural homology to PAI-1, but does not bind to vitronectin, chimeras of PAI-1 and PAI-2 were constructed. Four chimeras, containing PAI-1 residues 1-70, 1-105, 1-114, and 1-167 were constructed and expressed in vitro. PAI-1, PAI-2, and all of the chimeras retained inhibitory activity for t-PA, but only the chimera containing PAI-1 residues 1-167 formed a complex with VN. Together, these results predict that the VN binding site of PAI-1 is between residues 115-130.
Assuntos
Inibidor 1 de Ativador de Plasminogênio/química , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Vitronectina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA Complementar/genética , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/genética , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/metabolismoRESUMO
Tyrosine sulfation is a widespread modification of secreted proteins including several coagulation proteins synthesized in the liver. Since factor V, a tyrosine-sulfated protein, is also synthesized in megakaryocytes, we determined whether platelets posses tyrosylprotein sulfotransferase (TPST) activity. Using the synthetic substrate EAY, substantial TPST activity (0.405 +/- 0.049 pmol EAY-SO4 formed min-1 mg-1) was detected in platelet homogenates. This activity could not be accounted for by contaminating leukocytes, erythrocytes or plasma. The Km of platelet TPST for EAY was 3.7 microM and Vmax 0.09 pmol/min. For the cofactor 3' phosphoadenosine 5' phosphosulfate (PAPS) the Km was 1.7 microM and Vmax 0.11 pmol/min. The PAPS analogue 3',5'-adenosine diphosphate inhibited platelet TPST with an IC50 of 15.4 microM. These findings suggest that tyrosine sulfation of factor V will occur in megakaryocytes. Platelets may be a useful source for further study of TPST.
Assuntos
Plaquetas/enzimologia , Sulfotransferases/sangue , Animais , Bovinos , Humanos , Microssomos Hepáticos/enzimologia , Peptídeos/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Matrix GLA protein (MGP) is an inhibitor of calcification in the arterial wall and its activity is dependent upon vitamin K-dependent gamma-carboxylation. This modification is carried out by a warfarin sensitive enzyme system that converts specific Glu residues to gamma-carboxyglutamic acid (GLA) residues. Recent studies have demonstrated that the gamma-carboxylation system in the arterial wall, in contrast to that in the liver, is unable to use vitamin K as an antidote to warfarin. By use of immunohistochemistry we demonstrate that MGP is expressed in the arterial wall and immunocytochemistry localized the MGP precursors to the endoplasmic reticulum in vascular smooth muscle cells. Resting smooth vascular muscle cells in the aortic wall and proliferating cells from explants of the aorta have all the enzymes needed for gamma-carboxylation of MGP. However, when compared to the liver system, expression of the enzymes of the gamma-carboxylation system in vascular smooth muscle cells is different. Of particular interest is the finding that the specific activity of the warfarin sensitive enzyme vitamin K epoxide reductase is 3-fold higher in vascular smooth muscle cells than in liver. DT-diaphorase, which catalyses the antidotal pathway for vitamin K reduction in liver, is 100-fold less active in resting vascular smooth muscle cells than in liver. Data obtained from an in vitro gamma-carboxylation system suggest that the antidotal pathway catalyzed by DT-diaphorase in the vessel wall is unable to provide the carboxylase with enough reduced vitamin K to trigger gamma-carboxylation of MGP. This finding provides an explanation to the inability of vitamin K to work as an antidote to warfarin intoxication of the arterial wall. Therefore the vitamin K dependent gamma-carboxylation system in the arterial wall share a common feature with the system in bone cells by being unable to utilize vitamin K as an antidote.
Assuntos
Aorta/patologia , Aorta/fisiologia , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas da Matriz Extracelular/biossíntese , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiologia , Animais , Osso e Ossos/fisiologia , Ácidos Carboxílicos , Células Cultivadas , Masculino , Ratos , Ratos Sprague-Dawley , Proteína de Matriz GlaRESUMO
Vitronectin (VN) stabilizes plasminogen activator inhibitor type 1 (PAI-1) activity and prevents the fibrin(ogen)-induced acceleration of plasminogen activation by t-PA. These antifibrinolytic activities as well as other functions are mediated by the glycosaminoglycan (GAG) binding domain of VN. Since the GAG binding region is rich in arginyl and lysyl residues, it is a potential target for enzymes such as plasmin. In this paper, the dose and time-dependent proteolysis of VN by plasmin is demonstrated. The addition of urokinase or streptokinase (200 units/ml) to plasma also produced proteolysis of VN. With minimal proteolysis, the 75 kDa band was degraded to a 62-65 kDa form of VN. This minimal proteolysis destroyed the binding of [3H]-heparin to VN and reversed the neutralization of heparin by VN. Thus, the plasmin-mediated proteolysis of the GAG binding activity of VN could destroy the antifibrinolytic activity of VN during physiologic conditions and during thrombolytic therapy. Furthermore, other functions of VN in complement and coagulation systems that are mediated by the GAG binding domain may be destroyed by plasmin proteolysis.
Assuntos
Proteínas Sanguíneas/metabolismo , Fibrinolisina/metabolismo , Glicoproteínas/metabolismo , Heparina/sangue , Eletroforese em Gel de Poliacrilamida , Humanos , Immunoblotting , Ligação Proteica , Ensaio Radioligante , VitronectinaRESUMO
Matrix Gla protein (MGP) is an inhibitor of calcification of the arterial wall but the mechanism of inhibition has not been resolved. Since chondrogenesis has been identified in calcified arteries from MPG null mice, we hypothesized that locally produced MGP might inhibit calcification by neutralizing the known effect of bone morphogenetic proteins (BMPs) as promotors of chondrogenesis and bone formation. As the first step to test this hypothesis, we demonstrate that MGP is a binding protein for 125I-BMP-2. Optimal binding is dependent on metals which suggests that the metal binding Gla region in MGP is involved. MGP is shown to undergo a Ca++ induced conformational change despite the presence of the gamma-carboxylase binding site being part of the mature protein sequence. The data propose that MGP matures earlier in the secretory pathway than other vitamin K-dependent proteins. Antibodies were used in an attempt to identify MGP in bovine serum. Conformational specific MGP antibodies were shown to also recognize the Gla region in prothrombin and factor X but did not identify MGP in serum. This finding is supported by electrophoresis data which demonstrate the absence of MGP among Ba-citrate absorbed vitamin K-dependent serum proteins. We conclude that MGP does not exist in normal bovine serum.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular , Fator de Crescimento Transformador beta , Animais , Anticorpos/metabolismo , Proteína Morfogenética Óssea 2 , Cálcio/metabolismo , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/imunologia , Bovinos , Sequência Conservada , Epitopos/metabolismo , Fator X/imunologia , Humanos , Plasma/química , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Protrombina/imunologia , Proteína de Matriz GlaRESUMO
Baseline plasminogen activator inhibitor (PAI) levels were examined for their influence on the responses to thrombolysis with recombinant tissue plasminogen activator (rt-PA) administered for acute myocardial infarction during the Thrombolysis and Myocardial Infarction (TAMI)-I study. Baseline PAI activity was 19 +/- 21 IU/ml (normal less than 5 IU/ml) and baseline PAI-1 antigen 54 +/- 53 ng/ml (normal 27 +/- 16 ng/ml), confirming previous findings of elevated PAI levels during acute myocardial infarction. Among clinical outcomes, lower PAI-1 antigen levels correlated weakly with greater patency at the 90 min angiogram. Thus, high baseline plasma PAI-1 levels may be detrimental to reperfusion with t-PA. There was no correlation with other major in-hospital clinical outcomes including reocclusion at the 7-10 day angiogram, survival to discharge, or bleeding. During the follow up period of 2.0 +/- 0.4 years, no relationship between baseline PAI levels and post-discharge reinfarction was observed.
Assuntos
Infarto do Miocárdio/tratamento farmacológico , Inativadores de Plasminogênio/sangue , Ativador de Plasminogênio Tecidual/uso terapêutico , Angioplastia Coronária com Balão , Testes de Coagulação Sanguínea , Terapia Combinada , Angiografia Coronária , Seguimentos , Hemorragia/induzido quimicamente , Humanos , Infarto do Miocárdio/sangue , Prognóstico , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Ativador de Plasminogênio Tecidual/efeitos adversosRESUMO
Understanding the clinical risks of intravenous thrombolytic therapy is critical to appropriate patient selection. The major risks can be classified into 5 major categories: intracranial hemorrhage, systemic hemorrhage, immunologic complications, hypotension, and myocardial rupture. Although theoretical concern exists about thromboembolic complications, they rarely occur. Although cardiac rhythm disturbances are somewhat more likely to occur at the time of reperfusion, the clinical significance of "reperfusion arrhythmias" is minimal. Intracranial hemorrhage, the most devastating complication, occurs in 0.2-1% of patients treated with thrombolytic therapy. Factors associated with incremental risk are now being identified from large clinical trials. Systemic hemorrhage is uncommon in patients without major vascular punctures and seldom leads to serious adverse outcomes. Immunologic complications--including anaphylaxis, which is rare, and immune complex disease, which is more common--occur only with streptokinase or agents with a streptokinase moiety, including anistreplase (anisoylated plasminogen--streptokinase activator complex, APSAC). Hypotension, which can be managed easily in most patients, is also observed much more frequently with streptokinase and anistreplase. Myocardial rupture is increasingly being recognized as a possible complication of late thrombolysis. A proper perspective on clinical risk can only be gained in the context of potential benefit of therapy. In many cases individual patients considered to be at highest risk for complications also stand to gain the most from treatment. Many of the questions raised by currently available data about bleeding risk are being addressed in the ongoing Global Utilization of t-PA and Streptokinase (GUSTO) Trial. A paradigm for considering this decision making problem is presented.
Assuntos
Hemorragia/induzido quimicamente , Terapia Trombolítica/efeitos adversos , Anistreplase/efeitos adversos , Humanos , Infarto do Miocárdio/tratamento farmacológico , Fatores de Risco , Estreptoquinase/efeitos adversosRESUMO
Clinical depression has been identified as an independent risk factor for increased mortality in patients with coronary artery disease. Enhanced platelet activity has been suggested as the mechanism responsible for this adverse association. Selective serotonin reuptake inhibitors (SSRIs) are known to inhibit platelets in patients undergoing coronary stenting. We sought to determine whether concomitant therapy with SSRIs would yield additional anti-platelet benefit in patients with congestive heart failure (CHF) already treated with antecedent aspirin. A total of 88 patients with left ventricular ejection fraction (LVEF) <40% or CHF symptoms in the setting of preserved systolic function and NYHA Class II-IV were analyzed. Of these, 23 patients (26%) were chronic SSRI users (SSRI+), and 65 patients were free from SSRI therapy (SSRI-). All patients received aspirin (325 mg) for at least 1 month prior to platelet studies. Platelets were assessed by aggregometry, flow cytometry and a rapid analyzer. The SSRI+ group exhibited a substantial decrease in platelet activity when compared with SSRI- patients, as manifested by a significant reduction in ADP- (P=0.001), and collagen-induced (P=0.02) aggregation, and the expression of PECAM-1 (P=0.03), GPIb (P=0.03), GP IIb/IIIa antigen (P=0.02) and GP IIb/IIIa activity with PAC-1 antibody (P=0.04) and P-selectin (P=0.02). Therapy with SSRIs also resulted in the reduced formation of platelet-leukocyte microparticles (P=0.01). Epinephrine-induced aggregation in plasma, collagen-induced whole blood aggregation, closure time and expression of vitronectin receptor, CD63, CD107a, CD107b and CD151 did not differ between groups. In patients with CHF already on aspirin, SSRI therapy was associated with further inhibition of platelet function. This observation may help to explain some of the clinical benefits associated with SSRI therapy. Further clinical trials may help to elucidate the potential outcome benefits of SSRIs in other potential thrombotic circumstances.
Assuntos
Aspirina/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Inibidores da Agregação Plaquetária/uso terapêutico , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Idoso , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Função Plaquetária , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
In this study, we examined the effects of age and growth hormone replacement on both coronary blood flow and capillary density. Blood flow was measured by using [(14)C]-iodoantipyrine in three groups of anesthetized Brown Norway x Fischer 344 rats: young vehicle-treated animals (6 months; n = 13), old vehicle treated animals (30 months; n = 9), and old animals treated with bovine growth hormone (200 microg/kg) twice a day for 30 days (30 months; n = 7). Capillary density was measured by color segmentation analysis of sections stained for platelet endothelial cell adhesion molecule-1. In all regions examined, coronary blood flow decreased with age, and growth hormone administration resulted in an increase in flow compared to vehicle-treated animals. Capillary density decreased with age in the apex and the left ventricular middle segment. In response to growth hormone administration, capillary density increased significantly in the apex but not in other regions of the heart. Our results demonstrate that growth hormone enhances regional myocardial blood flow in the aged heart and suggest that part of this effect could be due to an increase in capillary density.
Assuntos
Envelhecimento/fisiologia , Capilares/patologia , Permeabilidade Capilar/fisiologia , Circulação Coronária/fisiologia , Hormônio do Crescimento/fisiologia , Análise de Variância , Animais , Permeabilidade Capilar/efeitos dos fármacos , Circulação Coronária/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hormônio do Crescimento/farmacologia , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/análise , Masculino , Modelos Animais , Análise Multivariada , Probabilidade , Radioimunoensaio , Ratos , Ratos Endogâmicos F344 , Valores de ReferênciaRESUMO
In recent years, major progress has been made in the design and synthesis of fibrinogen antagonists, which are peptidomimetic Arg-Gly-Asp (RGD) analogs. These RGD analogs are very promising antiplatelet agents. However, the clinical development of orally active RGD analogs has been hindered by the low oral bioavailability of many such RGD analogs. Aimed at enhancing their oral bioavailability, we have synthesized several coumarin-based cyclic prodrugs of RGD analogs, which have the two most polar functional groups, a carboxyl and an amino group, masked as an ester and an amide, respectively. As expected, these cyclic prodrugs have higher membrane interaction potentials as estimated by determining their partitioning between aqueous buffer and an immobilized artificial membrane than the corresponding RGD analogs. Consequently, these cyclic prodrugs are 5-6-fold more able to permeate monolayers of Caco-2 cells, an in vitro cell culture model of the intestinal mucosa barrier. Preliminary studies using dog also indicate the promising potential of using this coumarin-based prodrug strategy to improve the oral bioavailability of such RGD analogs.
Assuntos
Anticoagulantes/administração & dosagem , Cumarínicos/administração & dosagem , Mucosa Bucal/metabolismo , Oligopeptídeos/farmacocinética , Pró-Fármacos , Receptores Imunológicos/metabolismo , Animais , Anticoagulantes/farmacocinética , Anticoagulantes/farmacologia , Biotransformação , Linhagem Celular , Fenômenos Químicos , Físico-Química , Cumarínicos/farmacocinética , Cumarínicos/farmacologia , Cães , Meia-Vida , Humanos , Mucosa Bucal/citologia , Inibidores da Agregação Plaquetária/farmacocinética , Inibidores da Agregação Plaquetária/farmacologia , Tirofibana , Tirosina/análogos & derivados , Tirosina/farmacocinética , Tirosina/farmacologiaRESUMO
Monitoring coagulation parameters during thrombolytic therapy could be useful for prediction and treatment of haemorrhagic episodes. Technology based on dry reagent chemistry has been developed that allows rapid (less than 10 min) assays on small samples of whole blood. The assay principle is based on the restriction of motion of paramagnetic particles during fibrin polymerization, and subsequent liberation of particle motion during fibrinolysis. This technology was used to monitor prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen levels and fibrinolysis profiles during thrombolytic therapy with tissue plasminogen activator for acute myocardial infarction. The PT and aPTT obtained with the COAG-1 correlated well with conventional assays (r = 0.93 and 0.92 for PT and aPTT, respectively; p = 0.0001). Fibrinogen estimates, obtained by COAG-2 also correlated well with modified Clauss assays (r = 0.86, p = 0.0001). The rapid determination of the aPTT may improve management of adjunctive anticoagulant therapy following thrombolysis. The fibrinolysis profile may be useful during thrombolytic therapy to verify that a lytic state has been achieved, to monitor the lytic state throughout therapy, and to verify that the lytic state normalizes once therapy has been completed.
Assuntos
Coagulação Sanguínea/fisiologia , Fibrinólise/fisiologia , Monitorização Fisiológica/métodos , Terapia Trombolítica , Testes de Coagulação Sanguínea , Fibrinogênio/metabolismo , Hemorragia/induzido quimicamente , Hemorragia/diagnóstico , Hemorragia/tratamento farmacológico , Humanos , Tempo de Tromboplastina Parcial , Projetos Piloto , Tempo de Protrombina , Terapia Trombolítica/efeitos adversos , Fatores de TempoRESUMO
Necrobiosis lipoidica (NL) is a granulomatous disease that usually presents on the lower extremities of patients with diabetes mellitus. We present a rare case of NL köbnerizing in a surgical scar on the upper extremity of a patient with diabetes. NL is generally refractory to treatment.