RESUMO
Artificial recharge (AR) is gaining importance as a management tool in water stressed regions. The need to prove recovery performance requires new monitoring tools for AR systems. A novel combination of environmental isotope tracers (B, Li, O, H stable isotopes) was tested for the monitoring of AR of tertiary treated, desalinated domestic wastewater into a coastal dune aquifer in Flanders, Belgium. No significant isotope fractionation was observed for the treatment process, which includes low pH RO desalination. The wastewater, after infiltration through ponds and before recovery through pumping wells is characterized by low molar Cl/B ratios (3.3 to 5.2), compared to 130 to 1020 in the wider study area, delta(11)B values close to 0% per hundred, rather homogeneous delta(7)Li values (10.3 +/- 1.7% per hundred), and a 18O and 2H enrichment with respect to ambient groundwater due to evaporation in the infiltration ponds. This confers to the AR component a unique isotopic and geochemical fingerprint. Immediately downstream of the pumping wells and in the deeper part of the aquifer no evidence of AR wastewater could be found, indicating a high recovery efficiency. In the wider area and in the deeper part of the aquifer, isotopes evidence mixing of coastal rain and a fresh paleo-groundwater component with residual seawater as well as interaction with the aquifer material. Combining several isotope tracers provides independent constraints on groundwater flow and mixing proportions as a complement to hydrodynamic modeling and geochemical studies.
Assuntos
Meio Ambiente , Marcação por Isótopo/métodos , Osmose , Água do Mar , Eliminação de Resíduos Líquidos , Abastecimento de Água , Bélgica , Boro/química , Geografia , Hidrogênio/química , Isótopos , Lítio/química , Oxigênio/química , Solo , Água/química , Movimentos da Água , Purificação da ÁguaRESUMO
AMPA receptor-mediated excitotoxicity has been implicated in the selective motor neuron loss in amyotrophic lateral sclerosis. In some culture models, motor neurons have been shown to be selectively vulnerable to AMPA receptor agonists due to Ca(2+) influx through Ca(2+)-permeable AMPA receptors. Because the absence of GluR2 in AMPA receptors renders them highly permeable to Ca(2+) ions, it has been hypothesized that the selective vulnerability of motor neurons is due to their relative deficiency in GluR2. However, conflicting evidence exists about the in vitro and in vivo expression of GluR2 in motor neurons, both at the mRNA and at the protein level. In this study, we quantified electrophysiological properties of AMPA receptors, known to be dependent on the relative abundance of GluR2: sensitivity to external polyamines, rectification index, and relative Ca(2+) permeability. Cultured rat spinal cord motor neurons were compared with dorsal horn neurons (which are resistant to excitotoxicity) and with motor neurons that survived an excitotoxic insult. Motor neurons had a higher sensitivity to external polyamines, a lower rectification index, and a higher relative Ca(2+) permeability ratio than dorsal horn neurons. These findings confirm that motor neurons are relatively deficient in GluR2. The AMPA receptor properties correlated well with each other and with the selective vulnerability of motor neurons because motor neurons surviving an excitotoxic event had similar characteristics as dorsal horn neurons. These data indicate that the relative abundance of GluR2 in functional AMPA receptors may be a major determinant of the selective vulnerability of motor neurons to excitotoxicity in vitro.
Assuntos
Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Neurotoxinas/farmacologia , Receptores de AMPA/fisiologia , Espermina/análogos & derivados , Animais , Cálcio/metabolismo , Morte Celular , Permeabilidade da Membrana Celular , Técnicas de Cocultura , Eletrofisiologia , Ácido Caínico/farmacologia , Neurônios Motores/fisiologia , Ratos , Ratos Wistar , Espermina/farmacologiaRESUMO
alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents, evoked with the agonist kainate, were studied with the gramicidin perforated-patch-clamp technique in cultured rat spinal cord motoneurons. Kainate-induced currents could be blocked by the AMPA receptor antagonist LY 300164 and displayed an apparent strong inward rectification. This inward rectification was not a genuine property of AMPA receptor currents but was a result of a concomitant decrease in outward current at potentials positive to -40.5 +/- 1.3 mV. The AMPA receptor current itself was nearly linear (rectification index 0.91). The kainate-inhibited outward current had a reversal potential close to the estimated K(+) equilibrium potential and was blocked by 30 mM tetraethylammonium. When voltage steps were applied, it was found that kainate inhibited both the delayed rectifier K(+) current K(V) and the transient outward K(+) current, K(A). The kainate-induced inhibition of K(+) currents was dependent on ion flux through the AMPA receptor, because no change in the membrane conductance was noticed in the presence of LY 300164. Removing extracellular Ca(2+) had no effect, whereas replacing extracellular Na(+) or clamping the membrane close to the estimated Na(+) equilibrium potential during kainate application attenuated the inhibition of the K(+) current. Sustained Na(+) influx induced by application of the Na(+) ionophore monensin could mimic the effect of kainate on K(+) conductance. These findings demonstrate that Na(+) influx through AMPA receptors results in blockade of voltage-gated K(+) channels.
Assuntos
Neurônios Motores/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Receptores de AMPA/metabolismo , Sódio/metabolismo , Medula Espinal/fisiologia , Animais , Benzodiazepinas/farmacologia , Cálcio/metabolismo , Técnicas de Cultura de Células , Eletrofisiologia , Imuno-Histoquímica , Ionóforos/farmacologia , Monensin/farmacologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Ratos , Ratos Wistar , Receptores de AMPA/antagonistas & inibidores , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neuron death. The exact mechanism responsible for this selectivity is not clear, although it is known that motor neurons are very sensitive to excitotoxicity. This high sensitivity is due to a high density of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors on their surface and to a limited Ca(2+) buffering capacity. Ca(2+) can enter the cell upon stimulation through voltage-operated Ca(2+) channels and through the Ca(2+)-permeable portion of AMPA receptors. How this Ca(2+) kills motor neurons is incompletely understood. In the present study, we report that kainate (KA)-induced motor neuron death is purely mediated through Ca(2+) entering motor neurons through Ca(2+)-permeable AMPA receptors and that voltage-operated Ca(2+) channels play no significant role. In contrast to what has been observed in other neuronal models or after N-methyl-D-aspartate stimulation, NO synthase inhibition and a number of antioxidants did not protect motor neurons from KA-induced death. Only PD150606, derived from alpha-mercaptoacrylic acid and considered as a selective calpain antagonist, inhibited dose-dependently the KA-induced motor neuron death. However, other calmodulin and calpain inhibitors were not effective. At least part of the inhibitory effect of PD150606 is due to an irreversible inhibition of the Ca(2+) influx through the Ca(2+)-permeable AMPA receptor. These results demonstrate the interesting property of PD150606 to interfere with excitotoxicity-dependent motor neuron death and show that PD150606 is not an exclusive calpain/calmodulin antagonist.
Assuntos
Acrilatos/farmacologia , Cálcio/metabolismo , Calpaína/metabolismo , Ácido Caínico/antagonistas & inibidores , Neurônios Motores/efeitos dos fármacos , Inibição Neural/efeitos dos fármacos , Animais , Cálcio/antagonistas & inibidores , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Calpaína/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Inibição Neural/fisiologia , Ratos , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/metabolismoRESUMO
The mechanism responsible for the selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is poorly understood. Several lines of evidence indicate that susceptibility of motor neurons to Ca(2+) overload induced by excitotoxic stimuli is involved. In this study, we investigated whether the high density of Ca(2+)-permeable AMPA receptors on motor neurons gives rise to higher Ca(2+) transients in motor neurons compared to dorsal horn neurons. Dorsal horn neurons were chosen as controls as these cells do not degenerate in ALS. In cultured spinal motor neurons, the rise of the cytosolic Ca(2+) concentration induced by kainic acid (KA) and mediated by the AMPA receptor was almost twice as high as in spinal neurons from the dorsal horn. Furthermore, we investigated whether increasing the motor neuron's cytosolic Ca(2+)-buffering capacity protects them from excitotoxic death. To obtain motor neurons with increased Ca(2+) buffering capacity, we generated transgenic mice overexpressing parvalbumin (PV). These mice have no apparent phenotype. PV overexpression was present in the central nervous system, kidney, thymus, and spleen. Motor neurons from these transgenic mice expressed PV in culture and were partially protected from KA-induced death as compared to those isolated from nontransgenic littermates. PV overexpression also attenuated KA-induced Ca(2+) transients, but not those induced by depolarization. We conclude that the high density of Ca(2+)-permeable AMPA receptors on the motor neuron's surface results in high Ca(2+) transients upon stimulation and that the low cytosolic Ca(2+)-buffering capacity of motor neurons may contribute to the selective vulnerability of these cells in ALS. Overexpression of a high-affinity Ca(2+) buffer such as PV protects the motor neuron from excitotoxicity and this protective effect depends upon the mode of Ca(2+) entry into the cell.
Assuntos
Agonistas de Aminoácidos Excitatórios/toxicidade , Neurônios Motores/efeitos dos fármacos , Neurotoxinas/toxicidade , Parvalbuminas/farmacologia , Esclerose Lateral Amiotrófica/etiologia , Animais , Western Blotting , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Células Cultivadas , Citoproteção/efeitos dos fármacos , Citoproteção/genética , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Ácido Caínico/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Especificidade de Órgãos , Parvalbuminas/genética , Parvalbuminas/metabolismo , Fenótipo , Células do Corno Posterior/citologia , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/metabolismo , Receptores de AMPA/metabolismoRESUMO
To obtain a sustainable water catchment in the dune area of the Flemish west coast, the integration of treated domestic wastewater in the existing potable water production process is planned. The hygienic hazards associated with the introduction of treated domestic wastewater into the water cycle are well recognised. Therefore, the concept of HACCP (Hazard Analysis and Critical Control Points) was used to guarantee hygienically safe drinking water production. Taking into account the literature data on the removal efficiencies of the proposed advanced treatment steps with regard to enteric viruses and protozoa and after setting high quality limits based on the recent progress in quantitative risk assessment, the critical control points (CCPs) and points of attention (POAs) were identified. Based on the HACCP analysis a specific monitoring strategy was developed which focused on the control of these CCPs and POAs.
Assuntos
Modelos Teóricos , Saúde Pública , Eliminação de Resíduos Líquidos , Abastecimento de Água , Conservação dos Recursos Naturais , Monitoramento Ambiental , Humanos , Medição de Risco , VírusRESUMO
To evaluate the role of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis (ALS), we compared the sensitivity of motor neurons and that of dorsal horn neurons to kainic acid (KA). Short exposure to KA resulted in the death of motor neurons, while dorsal horn neurons were unaffected. This selective motor neuron death was completely dependent on extracellular Ca(2+) and insensitive to inhibitors of voltage-operated Ca(2+) or Na(+) channels. It was also completely inhibited by the specific AMPA antagonist LY300164 and by Joro spider toxin (JSTx), a selective blocker of AMPA receptors that lack the edited GluR2 subunit. KA selectively killed those motor neurons that stained positive for the Co(2+) histochemical staining, a measure for the presence of Ca(2+)-permeable AMPA receptors. These results suggest that Ca(2+) entry via Ca(2+)-permeable AMPA receptors is responsible for the selective motor neuron death. As the Ca(2+) permeability of the AMPA receptor is regulated by its GluR2 subunit, we stained motor neurons for GluR2. Immunoreactivity was present in all motor neurons, albeit to a variable degree. However, double-staining experiments demonstrated that motor neurons clearly expressing GluR2, also expressed Ca(2+)-permeable AMPA receptors. This indicates that despite the abundant expression of GluR2, this subunit is excluded from a subset of AMPA receptors and that the activation of these receptors is responsible for the selective motor neuron death.
Assuntos
Esclerose Lateral Amiotrófica/etiologia , Canais de Cálcio/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Receptores de AMPA/efeitos dos fármacos , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Benzodiazepinas/farmacologia , Canais de Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Neurônios Motores/metabolismo , Neurotoxinas/farmacologia , Nifedipino/farmacologia , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/metabolismo , Ratos , Ratos Wistar , Receptores de AMPA/metabolismo , Venenos de Aranha/farmacologiaRESUMO
Calin from the saliva of the medicinal leech, Hirudo medicinalis, is a potent inhibitor of collagen mediated platelet adhesion and activation. In addition to inhibition of the direct platelet-collagen interaction, we presently demonstrate that binding of von Willebrand to coated collagen can be prevented by Calin, both under static and flow conditions in agreement with the occurrence of binding of Calin to collagen, confirmed by Biospecific Interaction Analysis. To define whether Calin acted by inhibiting the platelet-collagen or the platelet-von Willebrand factor (vWF)-collagen-mediated thrombus formation, platelet adhesion to different types of collagens was studied in a parallel-plate flow chamber perfused with whole blood at different shear rates. Calin dose-dependently prevented platelet adhesion to the different collagens tested both at high- and low-shear stress. The concentration of Calin needed to cause 50% inhibition of platelet adhesion at high-shear stress was some fivefold lower than that needed for inhibition of vWF-binding under similar conditions, implying that at high-shear stress, the effect of Calin on the direct platelet-collagen interactions, suffices to prevent thrombus formation. Platelet adhesion to extracellular matrix (ECM) of cultured human umbilical vein endothelial cells was only partially prevented by Calin, and even less so at a high-shear rather than a low-shear rate, whereas the platelet binding to coated vWF and fibrinogen were minimally affected at both shear rates. Thus, Calin interferes with both the direct platelet-collagen interaction and the vWF-collagen binding. Both effects may contribute to the inhibition of platelet adhesion in flowing conditions, although the former seems to predominate.
Assuntos
Colágeno/metabolismo , Hormônios de Invertebrado/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Proteínas e Peptídeos Salivares/farmacologia , Fator de von Willebrand/metabolismo , Animais , Colágeno/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Cinética , Sanguessugas , Ligação Proteica , Estresse Mecânico , Fatores de Tempo , Fator de von Willebrand/efeitos dos fármacos , Fator de von Willebrand/isolamento & purificaçãoRESUMO
Interaction between exposed collagen and platelets and/or von Willebrand factor is believed to be one of the initiating events for thrombus formation at sites of damaged endothelium. Interference with this mechanism may provide an anti-thrombotic potential. Calin, a product from the saliva of the leech Hirudo medicinalis, was tested in vitro and for its in vivo activity in a thrombosis model in hamsters. Calin specifically and dose dependently (IC50:6.5 to 13 micrograms/mL) inhibited human platelet aggregation induced by collagen. In addition, specific platelet adhesion onto microtiter wells coated with collagen and detected with a monoclonal antiglycoprotein IIb/IIIa antibody-conjugated with horseradish peroxidase, could be completely prevented with Calin (IC50:22 micrograms/mL). A dose-response curve was constructed in groups of six hamsters in whom a standardized trauma was induced on the femoral vein. Thrombus formation was followed continuously using video recording and processing of the image obtained upon transillumination of the vessel. Intravenous Calin dose-dependently inhibited platelet-rich thrombus formation in this model with an ED50 of 0.07 mg/kg and complete inhibition with 0.2 mg/kg. No effects were seen on coagulation tests or bleeding times, whereas ex vivo aggregation induced by collagen was inhibited dose dependently. Local application of leech saliva, Calin, hirudin, or the combination of the latter two into the bleeding time wound of hamsters resulted in a mild prolongation of the bleeding time (twofold to threefold). A similar experiment in baboons did not cause any prolongation of the bleeding time. This is in sharp contrast with the long-lasting bleeding after a leech bite itself in both species. Calin from the leech Hirudo medicinalis is able, by binding to collagen, to effectively interfere with platelet-collagen interaction, which results in an antithrombotic effect observed in a platelet-rich thrombosis model in hamsters.
Assuntos
Colágeno , Veia Femoral , Hormônios de Invertebrado/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Proteínas e Peptídeos Salivares/farmacologia , Trombose/prevenção & controle , Animais , Mordeduras e Picadas , Colágeno/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta a Droga , Hirudinas/farmacologia , Humanos , Hormônios de Invertebrado/uso terapêutico , Sanguessugas , Masculino , Mesocricetus , Papio , Saliva/fisiologia , Proteínas e Peptídeos Salivares/uso terapêuticoRESUMO
Human B-cell lines were derived by limiting dilutions of Epstein-Barr virus (EBV) transformed peripheral B cells from a patient with an autoantibody against glycoprotein (GP)Ia/IIa, and manifesting defective collagen-induced platelet aggregation and a bleeding problem. Antibody-producing clones were selected for their reactivity with whole platelets or with affinity-purified GPIa/IIa by enzyme-linked immunosorbent assay (ELISA). One of these cell lines, selected for further evaluation, produced an IgM (E3G6) that interfered with platelet aggregation responses. Polymerase chain reaction (PCR) amplifications with two different sets of primers specific for human kappa-chains resulted in the rescue of a unique and identical sequence. The same was true for the mu-chain, from which it was concluded that the cell line was monoclonal. Further analysis showed that the kappa variable domain sequence is similar to the germline gene A30, to 2E7, an anti-GPIIb human autoantibody, and to HF2-1/17, a systemic lupus erythematosus (SLE)-associated broad-specificity human autoantibody. Thus, the specificity of our antibody, E3G6, appears to be determined by the mu-chain, the sequence of which is encoded by a VHIII gene segment strongly homologous to the germline gene DP-77, by a D gene that is not homologous to any of the germline D genes reported to date, and by JH4 gene segment that is germline. All four mutations versus DP-77 are in CDRs, and result in amino acid substitutions, which implies that E3G6 may have been derived from an antigen-driven response.
Assuntos
Autoanticorpos/biossíntese , Autoanticorpos/genética , Linfócitos B/imunologia , DNA Complementar/química , Imunoglobulina M/genética , Glicoproteínas da Membrana de Plaquetas/imunologia , Sequência de Aminoácidos , Especificidade de Anticorpos , Autoanticorpos/química , Autoanticorpos/farmacologia , Sequência de Bases , Linhagem Celular Transformada , Colágeno/farmacologia , Herpesvirus Humano 4 , Humanos , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/genética , Dados de Sequência Molecular , Agregação Plaquetária/efeitos dos fármacos , Reação em Cadeia da PolimeraseRESUMO
The effects of human recombinant plasminogen activator inhibitor (rPAI-1) on thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) were studied in a rabbit model of jugular vein thrombosis. Two functionally distinct rPAI-1 preparations were used in these experiments, including latent rPAI-1 (approximately 2 units of t-PA neutralizing activity per micrograms protein) and reactivated rPAI-1 (approximately 150 units/micrograms). Simultaneous intravenous infusion over 4 h of 1.7 mg/kg of reactivated rPAI-1 (inhibitory capacity approximately 0.5 mg/kg rt-PA) with 0.5 mg/kg of rt-PA completely prevented lysis of a jugular venous thrombus, whereas an equivalent amount of latent PAI-1 did not significantly influence clot lysis. These findings demonstrate that reactivated human rPAI-1 efficiently neutralizes thrombolysis with rt-PA in vivo. Since previous studies have suggested that elevated endogenous levels of PAI-1 do not attenuate the thrombolytic potency of rt-PA in the endotoxin-treated model, we compared the stability of complexes formed by 125I-rt-PA with reactivated human rPAI-1 and with rabbit PAI-1 in vitro. Our findings indicate that both forms of PAI-1 form SDS-stable complexes following incubation with 125I-rt-PA. Thus, it seems likely that elevated levels of active PAI-1 can negate the thrombolytic effects of rt-PA in vivo and argues against the possibility that t-PA can dissociate from PAI-1 and have its activity restored in the presence of a thrombus.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Inativadores de Plasminogênio/farmacologia , Terapia Trombolítica/métodos , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Animais , Radioisótopos do Iodo , Veias Jugulares , Inativadores de Plasminogênio/química , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Trombose/tratamento farmacológico , Ativador de Plasminogênio Tecidual/químicaRESUMO
We studied a 5-year-old girl presenting with a markedly prolonged bleeding time. Her platelets were refractory to collagen stimulation, but the response to other agonists was normal. There were no coagulation abnormalities as measured by standard tests. Two-dimensional electrophoresis showed no abnormalities of the patient's platelet membrane glycoproteins. When the patient's plasma or purified plasma IgG was mixed with normal platelets, collagen-induced platelet aggregation was blocked. Western blotting showed the presence of an antibody in the patient's plasma directed against a protein of molecular weight 85 to 90 Kd under both reducing and nonreducing conditions. This protein comigrated with glycoprotein (GP) IV immunoprecipitated by OKM5 from 125I-labeled platelets. Immunoprecipitation of 125I-labeled normal platelet glycoproteins with the patient's IgGs also yielded an 85- to 90-Kd protein that migrated on the diagonal following nonreduced/reduced two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Despite similarities in electrophoretic behavior, the antigen was not demonstrated to be GPIV, since purified GPIV was not recognized by the antibody.
Assuntos
Anticorpos/análise , Colágeno/farmacologia , Hemorragia/etiologia , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/imunologia , Tempo de Sangramento , Pré-Escolar , Feminino , Humanos , Glicoproteínas da Membrana de Plaquetas/análiseRESUMO
BACKGROUND: Streptokinase (SK) is a bacteria-derived protein and one of the plasminogen activators that is currently available for therapeutic use. Exposure to SK induces synthesis of specific antibodies that may initiate platelet aggregation and paradoxical clot propagation during treatment. METHODS AND RESULTS: Using platelet-rich plasma (PRP), we found that SK (5,000 units/ml) but not urokinase (2,500 units/ml) or recombinant tissue-type plasminogen activator (2,500 units/ml) caused platelet aggregation in PRP from 14 of 100 normal volunteers. In 13 consecutive patients treated with SK for acute myocardial infarction, SK-mediated platelet aggregation was induced in five patients within 1 week after treatment. SK-mediated platelet aggregation was associated with significantly increased titers of both anti-SK antibodies and SK-neutralizing activity in plasma; it was partially inhibited by aspirin (1 mM) and by aprotinin (500 kallikrein inhibitor units/ml) and completely inhibited by tranexamic acid (1 mM) and by prostaglandin E1 (9 microM). Addition of SK (1,000 or 5,000 units/ml) induce a statistically significant dose-dependent thromboxane B2 release in mixtures of PRP with plasma from subjects with SK-induced aggregation but not in samples of PRP mixed with plasma from nonresponders; addition of recombinant tissue-type plasminogen activator (1 or 50 micrograms/ml) did not induce thromboxane B2 release. Mixing experiments with PRP and immunoglobulin G from reactive and nonreactive donors revealed that SK-induced aggregation requires the presence of anti-SK antibodies. When 125I-SK (50 nM) was used, platelets preincubated with plasminogen (0.5 microM) bound 9,500 +/- 600 (mean +/- SEM, n = 6) molecules SK/platelet, which increased to 25,000 +/- 3,100 molecules/platelet after thrombin stimulation. Tranexamic acid (1 mM) blocked specific binding of SK to resting platelets. CONCLUSIONS: These data demonstrate that SK-induced platelet aggregation is initiated by the binding of anti-SK antibodies to the SK-plasminogen complex located on the platelet surface. SK-induced platelet activation may limit the therapeutic effectiveness of the drug, and in view of the high prevalence of aggregation in a normal population, prospective evaluation of the effects of platelet aggregation during treatment with SK is warranted.
Assuntos
Plaquetas/efeitos dos fármacos , Agregação Plaquetária , Estreptoquinase/farmacologia , Adulto , Anticorpos/análise , Plaquetas/metabolismo , Coleta de Amostras Sanguíneas , Feminino , Humanos , Imunoglobulina G/farmacologia , Masculino , Estreptoquinase/imunologia , Estreptoquinase/metabolismoRESUMO
The pharmacokinetics of human recombinant plasminogen activator inhibitor-1 (rPAI-1) was studied in rabbits. Latent rPAI-1 (0-2 units of tissue-type plasminogen activator neutralizing activity per microgram protein); reactivated rPAI-1 (approximately 150 units/micrograms); and chloramine T-oxidized, nonreactivatable rPAI-1 (approximately 0.7 units/microgram) were studied. The pharmacokinetic parameters for the disposition of rPAI-1 antigen after an intravenous bolus injection of 1.0 or 2.5 mg/kg rPAI-1 were very similar for all three forms: the initial volume of distribution was approximately 60 ml/kg, the initial half-life in plasma was 6 minutes, and the plasma clearance was approximately 4 ml/kg/min. The disposition of PAI activity after injection of reactivated rPAI-1 was similar to that of rPAI-1 antigen. Injection of latent rPAI-1 was associated with a nearly threefold increase in the specific activity of circulating PAI-1 from 2 units/micrograms to 5.0 +/- 1.1 units/micrograms (p less than 0.01) within 1 minute, followed by a cumulative 25-fold increase in specific activity over 1 hour (p = 0.01). In contrast, the specific activity of oxidized or reactivated preparations of rPAI-1 did not increase in the first several minutes after injection. These findings support the existence of a fast-acting but low-capacity mechanism for the reactivation of rPAI-1 in vivo.
Assuntos
Reativadores Enzimáticos , Inativadores de Plasminogênio/farmacocinética , Análise de Variância , Animais , Ensaio de Imunoadsorção Enzimática , Meia-Vida , Inativadores de Plasminogênio/administração & dosagem , Inativadores de Plasminogênio/sangue , CoelhosRESUMO
Cell surface binding sites for the constituent proteins of the fibrinolytic system may play a role in the localization and regulation of fibrinolysis. In the present study, specific binding of recombinant human tissue-type plasminogen activator (rt-PA) to human blood platelets was identified and characterized. 125I-labeled rt-PA was found to bind specifically, saturably, and reversibly to the surface of gel-filtered platelets, reaching equilibrium within 5 min at 22 degrees C. Scatchard analysis revealed a single class of binding sites. Unstimulated platelets bound 120,000 +/- 24,000 (mean +/- S.D.) molecules/platelet with an apparent Kd of 340 +/- 25 nM, whereas thrombin-stimulated platelets bound 290,000 +/- 32,000 molecules/platelet with an apparent Kd of 800 +/- 60 nM. Binding of 0.1 microM 125I-rt-PA was greater than 90% reversible by a 50-fold excess of unlabeled rt-PA. Binding was not inhibited by fibrinogen or single chain urokinase-type plasminogen activator, but plasminogen partially competed for binding of 125I-rt-PA to platelets (up to 40% displacement). These findings indicate that the platelet surface possesses a large number of specific, low affinity binding sites for t-PA and provide further evidence for the role of platelets in localization and regulation of fibrinolysis.
Assuntos
Plaquetas/metabolismo , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tecidual/sangue , Sítios de Ligação , Membrana Celular/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Humanos , Técnicas In Vitro , Cinética , Agregação Plaquetária , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/sangue , Trombina/fisiologiaRESUMO
F(ab')2 fragments of a murine monoclonal anti-platelet GPIIb/IIIa antibody (7E3) are a potent platelet aggregation inhibitor, which in a canine coronary artery thrombosis model accelerate lysis with recombinant tissue-type plasminogen activator (rt-PA) and prevent reocclusion (7). In the present study, we have investigated the potential value of platelet aggregation inhibition as adjunctive therapy to lysis of venous thrombi, by measuring the thrombolytic potency of 7E3-F(ab')2 and rt-PA used alone or in combination, in dogs with a 125I-fibrin labeled femoral vein thrombus. The dose-response of thrombolysis with rt-PA infused over 4 hours was linear: doses of 0.075 mg/kg, 0.15 mg/kg and 0.3 mg/kg produced 37 +/- 3, 57 +/- 11 and 83 +/- 4% lysis respectively, against a background value of 20 +/- 2%. With F(ab')2 fragments of 7E3 given as a bolus of 1.2 mg/kg, which saturated 70% of the platelet GPIIb/IIIa receptors and prolonged the bleeding to more than 30 min, lysis was not significantly increased over background. Combination of 0.3 or 0.6 mg/kg of 7E3-F(ab')2 with either 0.03 or 0.06 mg/kg of rt-PA did not produce more lysis than obtained with a comparable dose of rt-PA alone. No significant changes in plasma fibrinogen or alpha 2-antiplasmin were observed with either agent alone or with the combination. It is concluded that extensive inhibition of platelet aggregation does not potentiate the thrombolytic effect of rt-PA in this venous thrombosis model.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Fibrinólise/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/imunologia , Tromboflebite/terapia , Ativador de Plasminogênio Tecidual/uso terapêutico , Animais , Terapia Combinada , Modelos Animais de Doenças , Cães , Veia Femoral , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Tromboflebite/sangueAssuntos
Oxirredutases/antagonistas & inibidores , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Prostaglandina/efeitos dos fármacos , Trombose/tratamento farmacológico , Tromboxano-A Sintase/antagonistas & inibidores , Humanos , Técnicas In Vitro , Agregação Plaquetária/efeitos dos fármacos , Receptores de TromboxanosRESUMO
We have developed an experimental model for the study of local prostaglandin production by platelets and the vessel wall following stimulation 'in vivo'. A nylon thread was inserted into the external jugular vein of rabbits; its presence did not induce an occluding thrombus. Thromboxane (TXB2) values in the blood, sampled through the facial vein, immediately distal to the stimulus, rose and remained high for at least 4 hr, while 6-keto prostaglandin (PG) F1 alpha levels, after a first increase, gradually returned to normal ('exhaustion' of the endothelial cells?). No changes were observed in the contralateral jugular vein without thread. After infusion via the femoral vein of 10 mg/kg dazoxiben, a thromboxane synthetase inhibitor, local TXB2 production was completely abolished, whereas 6-keto PGF1 alpha formation no longer returned to basal values, but tended to increase. This leads to the conclusion that upon inhibition of TXB2 formation endoperoxide metabolism is reoriented 'in vivo' towards prostacyclin, and this mainly at the site where platelets are activated. Injection of 100 mg/kg lysine acetylsalicylic acid resulted in complete inhibition of TXB2 and 6-keto PGF1 alpha formation, the latter, however, slowly recovering with time. The administration of nafazatrom to the animals did not influence the local TXB2 changes, but partially prevented the decline of 6-keto PGF1 alpha with time. The antithrombotic properties of this drug thus could be related to protection of the endothelial cells from 'exhaustion'.