Search

Antithrombin III (AT III), heparin cofactor has been known to be one of the important protease inhibitors on the regulatory mechanism of coagulation and fibrinolysis, but the site of synthesis of plasma AT HI has not yet been conclusively established, although some observations suggested that the major site of AT III production may be the liver and AT III levels in plasma is known to be decreased on the hepatic disorders. This paper reports the deposition of the AT III specific fluorescence in the hepatocytes of rat liver by immunofluorescent examination. Plasma AT III was purified by heparin sepharose affinity chromatography and gel filtrations. Rabbit was immunized with this purified AT III and Freund’s complete adjuvant and rabbit anti rat AT III serum was obtained. This antiserum was comfirmed to be monospecific to rat AT HI by means of immunoelectrophoresis. Freshly isolated rat liver was prepared as specimen and fixed in acid alcohol and processed to dehydration and embedding into paraffin in conventional way. Indirect immunofluorescent technique with FITC labeled anti rabbit gamma-globulin were utilized for immunofluorescent staining and histological observation was performed under Nikon FL fluorescent microscope. The localization of fluorescence was observed in the hepatocytes which scattered in the acinus structures. The fluorescence in the cytoplasma of these hepatocytes was homogeneously stained. As far as the fluorescence is concerned, the synthesis of AT III may be performed not synchronizing among hepatocytes. This immunofluorescent study supports the reported data of hepatic perfusion.

A radioimmunoassay method for antithrombin III (ATIII) was developed in order to detect the AT III levels correctly in plasma and tissues and the effect of heparin infusion was investigated on rat using this method and 125I labeled ATIII. Rat AT III was purified from rat defibrinated plasma by heparin sepharose affinity chromatography and gel filtrations. This purified AT III was used for the preparation of specific AT III antiserum. Labeling of AT III with 125I was performed according to the method by Hunter and Greenwood. Plasma level of AT III were significantly decreased in the treated group with heparin for 6 hours, although significant difference was not observed in AT III contents in various organs. The behavior of i.v. injected AT III laveled with 125I in the normal control and treated groups proved the difference on the half life of AT III. Control group gave 52 hours and it was shortened in the treated group. The percent radioactivity per ml plasma after 6 hours of heparin infusion was 1.16±0.51, and 2.01±0.38 in the control group, and significant difference was observed (p < 0.05). On the contrary the percent dose radioactivity per g tissue wet weight was significantly increased in the liver, lungs, and large intestine on the heparin treated group. The decreased amount of the intravenously injected laveled AT III appears to be trapped and metabolized in the various organs mainly in the liver during heparin infusion. The decrease of plasma AT III levels on the patients treated with heparin may be explained from these experimental results.

PGEl is one of prostaglandins which inhibit platelet functions and has vasodilating activity similar to PGI2. Newly developed PGEi analogue (OP-1206) was supplied for the clinical evaluations on oral administration. This research was performed to analyze the effect of orally administrated PGE1 analogue on platelet functions and to evaluate the usefulness on the thromboembolic disorders. Comparing with PGI2, this analogue demonstrated the similar inhibitory activity on the platelet aggregation in vitro study. Oral administration of OP-1206 on the patients with thromboembolic disorders showed the dose-dependent inhibition on platelet aggregation and adhesiveness. This activity continued for l80 min (max at 120 min). Daily oral administration (20μg and 30μg t.i.d.) was continued for two weeks and its effect on blood pressure, heart rate, ADP induced platelet aggregation, platelet adhesiveness and platelet c-AMP level were evaluated. Both of administration doses caused remarkable depression of platelet aggregation, increment of c-AMP level in the platelet and mild suppression on the platelet adhesiveness. Blood pressure was decreased, but heart rate remained unchanged. Clinical improvement of symptoms were observed in the patients with deep vein thrombosis or angina pectoris. These results emphasize the effectiveness and usefulness of this orally administrated PGE1 analogue against the prevention and treatment of thromboembolic disorders.