RESUMO
Two structurally different forms of activated human Factor IX (Factor IXa alpha and IXa beta) have been previously reported to have essentially identical clotting activity in vitro. Although it has been shown that activated Factor IX Chapel Hill, an abnormal Factor IX isolated from the plasma of a patient with mild hemophilia B, and normal Factor IXa alpha are structurally very similar, the clotting activity of activated Factor IX Chapel Hill is much lower (approximately fivefold) than that of normal Factor IXa beta. In the present study we have prepared activated Factor IX by incubating human Factor IX with calcium and Russell's viper venom covalently bound to agarose. Fractionation of the activated Factor IX by high-performance liquid chromatography demonstrated the presence of both Factors IXa alpha and IXa beta. On the basis of active site concentration, determined by titration with antithrombin III, the clotting activities of activated Factor IX Chapel Hill and IXa alpha were similar, but both activities were less than 20% of the clotting activity of Factor IXa beta. Activated Factor IX activity was also measured in the absence of calcium, phospholipid, and Factor VIII, by determination of the rate of Factor X activation in the presence of polylysine. In the presence of polylysine, the rates of Factor X activation by activated Factor IX Chapel Hill, Factor IXa alpha, and Factor IXa beta were essentially identical. We conclude that the clotting activity of activated Factor IX Chapel Hill is reduced when compared with that of Factor IXa beta but essentially normal when compared with that of Factor IXa alpha.
Assuntos
Coagulação Sanguínea , Enzimas/fisiologia , Fator IX/fisiologia , Cálcio/farmacologia , Fator IXa , Fator X/metabolismo , Humanos , Polilisina/farmacologia , Sefarose/farmacologia , Venenos de Víboras/farmacologiaRESUMO
The regulation of human Factor IXa was studied in vitro in human and mouse plasma and in vivo in the mouse. In human plasma, approximately 60% of the 125I-Factor IXa was bound to antithrombin III (ATIII) by 2 h, with no binding to alpha 2-macroglobulin or alpha 1-proteinase inhibitor, as assessed by gel electrophoresis and IgG- antiproteinase inhibitor-Sepharose beads. In the presence of heparin, virtually 100% of the 125I-Factor IXa was bound to ATIII by 1 min. The distribution of 125I-Factor IXa in mouse plasma was similar. The clearance of 125I-Factor IXa was rapid (50% clearance in 2 min) and biphasic and was inhibited by large molar excesses of ATIII-thrombin and alpha 1-proteinase inhibitor-trypsin, but not alpha 2-macro-globulin-trypsin; it was also inhibited by large molar excesses of diisopropylphosphoryl - (DIP-) Factor Xa, DIP-thrombin, and Factor IX, but not by prothrombin or Factor X. The clearance of Factor IX was also rapid (50% clearance in 2.5 min) and was inhibited by a large molar excess of Factor IX, but not by large molar excesses of Factor X, prothrombin, DIP-Factor Xa, or DIP-thrombin. Electrophoresis and IgG- antiproteinase inhibitor-Sepharose bead studies confirmed that by 2 min after injection into the murine circulation, 60% of the 125I-Factor IXa was bound to ATIII. Organ distribution studies with 125I-Factor IXa demonstrated that most of the radioactivity was in the liver. These studies suggest that Factor IXa binds to at least two classes of binding sites on endothelial cells. One site apparently recognizes both Factors IX and IXa, but not Factor X, Factor Xa, prothrombin, or thrombin. The other site recognizes thrombin, Factor Xa, and Factor IXa, but not the zymogen forms of these clotting factors. After this binding, Factor IXa is bound to ATIII and the complex is cleared from the circulation by hepatocytes.
Assuntos
Fator IX/metabolismo , Inibidores de Proteases/sangue , Animais , Antitrombina III/metabolismo , Endotélio/fisiologia , Fator IXa , Fator X/metabolismo , Fator Xa , Humanos , Radioisótopos do Iodo , Isoflurofato/farmacologia , Cinética , Camundongos , Especificidade da Espécie , Trombina/metabolismo , Distribuição Tecidual , alfa 1-Antitripsina/metabolismo , alfa-Macroglobulinas/metabolismoRESUMO
The excretion of Piracetam was monitored by measuring the concentrations in maternal and fetal substrates during labor in nine volunteers. Piracetam was tolerated without side-effects and injected in the maternal cubital vein. Consecutively, maternal plasma and urine samples as well as amniotic fluid portions were collected during labor. at delivery, fetal blood from placenta and the first fetal urines were collected. Biostatistical methods showed that approximately 50% of Piracetam were eliminated 80 minutes after the injection of the drug. In amniotic fluid a continuous rise of Piracetam concentrations was monitored until delivery. In fetal plasma and urines the substance could be detected. The rapid excretion of Piracetam during labor was obviously typical for the situation sub partu; reasons are discussed.
Assuntos
Parto Obstétrico , Piracetam/análise , Pirrolidinonas/análise , Líquido Amniótico/análise , Feminino , Sangue Fetal/análise , Meia-Vida , Humanos , Recém-Nascido , Troca Materno-Fetal , Piracetam/sangue , Piracetam/urina , GravidezRESUMO
High-dosage medroxyprogesterone (Farlutal, 1 g/d orally) was administered to 42 female patients with progressive disseminated carcinomas of the breast after conventional cytostatic and hormonal treatment had failed. Besides evaluation of success of treatment the pharmacokinetics of medroxyprogesterone were investigated. A remission rate of 37% (total and partial remissions) indicated that high-dosage oral treatment with gestagens can be used as "failure-regime" in patients at the end of conventional treatment. The plasma level of the gestagen approached values corresponding to high-dosage intramuscular application.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Medroxiprogesterona/administração & dosagem , Administração Oral , Adulto , Idoso , Neoplasias da Mama/sangue , Avaliação de Medicamentos , Feminino , Humanos , Hidrocortisona/sangue , Cinética , Medroxiprogesterona/sangue , Pessoa de Meia-Idade , Metástase Neoplásica , Comprimidos , Fatores de TempoRESUMO
Activated human factor IX (factor IXa) was treated under mildly acidic conditions with a mixture of formaldehyde and morpholine. This reagent has been shown to react preferentially with gamma-carboxyglutamyl (Gla) residues and to convert these residues to gamma-methyleneglutamyl residues (Wright, S.F., Bourne, C.D., Hoke, R.A., Koehler, K.A., and Hiskey, R.G. (1984) Anal. Biochem. 139, 82-90). The modified enzyme was evaluated for coagulant activity and calcium-dependent fluorescence quenching. [14C]Formaldehyde was employed to allow quantitation of the modification and to facilitate localization of the modified residues in the primary structure of factor IXa. In the presence of the [14C]formaldehyde/morpholine reagent, factor IXa rapidly lost coagulant activity, which corresponded to incorporation of radiolabel. Examination of the relationship between protein modification (radiolabel incorporation) and the loss of coagulant activity suggested that modification of 1 mol of Gla/mol of factor IXa results in complete loss of factor IXa coagulant activity. Primary structure analysis of the radioactivity labeled factor IXa suggested that modification of any one of 11 Gla residues was responsible for the loss of coagulant activity. In the presence of calcium, modified factor IXa exhibited a smaller Gla-dependent decrease in protein fluorescence than native factor IXa, but the Gla-independent fluorescence change was the same for both proteins. It therefore appears that the Gla domain of factor IXa must be completely intact for the enzyme to undergo a functionally important calcium-dependent conformational change necessary for coagulant activity.