Synthesis of factor II antigen by isolated perfused rat liver.

Rat Factor II (prothrombin), isolated and purified by chromatography on Blue Dextran-agarose, was used to raise an antiserum in rabbits. On the basis of single radial immunodiffusion measurements. Factor II synthesis by isolated perfused rat liver amounted to 0.54 mg/300 cm2 body surface area of the liver donor in 10 h. Corresponding measurements of Factor II coagulant activity revealed cumulative synthesis of 802 Iowa units. Coumadin added to the liver perfusate blocked production of Factor II coagulant activity, but did not change synthesis of the immunologically measured protein. In perfusions in which either heparin or citrate was used as anticoagulant, synthesis of albumin was not affected by the choice of anticoagulant but bile production and synthesis of Factor II were significantly less in citrate perfusions.

A plasma factor enhances activity of vitamin K-dependent coagulation proteins.

A plasma factor, "coagulopoietin", present in animals with depleted vitamin K-dependent coagulation factors, appears to enhance activity of these factors in normal animals. We have investigated the effects of "coagulopoietin" on synthesis of certain coagulation proteins by the isolated rat liver perfused for eight hours. Liver donor rats received plasma injections from vitamin K-deficient rats or from normal rats 24 hr before sacrifice. Coagulation activity of Factor VII and Factor II in liver perfusate samples was measured with a coagulation assay; Factor II synthesis was also measured by rocket immunoelectrophoresis and by activation with E. carinatus venom. Cumulative hepatic synthesis of Factor VII coagulation activity was increased by 43% when rat liver donors received vitamin K-deficient rat plasma compared to normal rat plasma. Cumulative synthesis of Factor II coagulation activity was increased by 51%, but synthesis of the protein measured immunologically or by activation with venom was not affected. The "coagulopoietin" factor in these studies appears to increase measurable coagulation factor activity without increasing total protein synthesis.

Testosterone effects on biosynthesis of coagulation proteins.

Studies were performed to assess the effects of testosterone on synthesis of selected coagulation proteins using the isolated rat liver perfused in vitro for 10 hours, as well as effects of testosterone on plasma levels of these same proteins. Pretreatment of castrated male rat liver donors for 14 days with pellets containing placebo or testosterone, 0.5 mg, 5.0 mg or 15.0 mg had no significant effects on cumulative biosynthesis of Factor II, Factor VII, antithrombin III, plasminogen or fibrinogen. Plasma concentrations of these proteins in liver donor animals were also unchanged by such hormonal manipulations. In contrast, biosynthesis of fibronectin was increased significantly by increasing doses of testosterone, and plasma concentrations of fibronectin in liver donor rats showed a similar effect.

The interaction of salicylate and vitamin K in synthesis of Factor II (prothrombin).

The effects of sodium salicylate on synthesis of a vitamin K-dependent plasma coagulation protein, Factor II (prothrombin) was studied using the isolated rat liver perfused for 10 hours in vitro. Cumulative synthesis of Factor II was measured by a standard coagulation assay, by activation with E. carinatus venom, and by rocket immunoelectrophoresis. When sodium salicylate 5 mg or 25 mg was added to the liver perfusate (volume 100 ml) at the outset of the perfusion, cumulative synthesis of both coagulation activity and immunoreactive protein was significantly less than that seen in control perfusions containing no salicylate. The inhibitory effect of salicylate was prevented by pretreatment of rat liver donors with supplemental vitamin K injected 24 hours before sacrifice. Although some interaction between salicylate and vitamin K was apparent from these experiments, the results from vitamin K-deficient rat liver donors were quite different from those containing salicylate. There was no assayable Factor II coagulation activity produced in 10 hours of perfusion of vitamin K-deficient rat livers, but cumulative synthesis of immunoreactive Factor II was quite comparable to that seen in control perfusions.

Measurements of rat plasma coagulation proteins during prolonged exposure to diethylstilbesterol.

The effects of diethylstilbesterol (DES) on concentration of selected plasma coagulation proteins in male rats was studied sequentially over a 28-day period. At the outset of the study, male rats underwent orchiectomy and implantation of a pellet containing DES 5.0 mg or a placebo pellet. At intervals of 7 days, 3 of the animals from each treatment group were sacrificed and blood samples were withdrawn for assay. Plasma concentrations of Factor II (prothrombin) and Factor VII, two vitamin K-dependent coagulation proteins, were significantly decreased in DES-treated animals (approximately 70% normal activity for both Factors) compared to placebo-treated animals. In contrast, Factor VIII activity was higher with DES treatment than with placebo. Plasma concentrations of both antithrombin III (AT III) and plasminogen were decreased in DES-treated animals compared to placebo by immunologic as well as activity assays. Albumin concentrations were not different between the two groups at any point of study, although both were increased at day 21 compared to beginning values. Fibronectin concentrations were slightly decreased in DES-treated animals compared to those which received placebo. The combination of orchiectomy and treatment with DES had profound metabolic effects on the animals; the DES treated animals had a mean body weight loss of 100 g after 28 days, while the placebo group gained an average of 78 g compared to a control group (no surgery) which had average weight gain of 85 g during the 28 day period of study.

Biosynthesis of plasminogen by the perfused rat liver.

We investigated synthesis of plasminogen by the isolated rat liver perfused in vitro for 10 hours. Studies were performed under basal conditions as well as under conditions of hormone stimulation known to augment synthesis of acute-phase reactant proteins by the isolated perfused rat liver. In six control perfusions, mean cumulative synthesis of plasminogen after 10 hours of perfusion was 1.61 +/- 0.11 mg/300 cm2 body surface area of the rat liver donor, and in six "acute-phase" experiments, mean cumulative synthesis was 1.55 +/- 0.07 mg. When cycloheximide was added to the liver perfusate at the outset to inhibit protein synthesis, production of plasminogen was greatly reduced. Synthesis of fibrinogen in these same perfusions was characteristic of the known acute-phase reactant proteins. In control perfusions, 68.85 +/- 6.19 mg fibrinogen was produced in 10 hours compared with 106.20 +/- 6.93 mg in acute-phase perfusions. These studies indicate substantial synthesis of plasminogen by the isolated perfused rat liver, but suggest that synthesis of this protein is not affected by those conditions that enhance synthesis of known acute-phase reactant proteins.

Diethylstilbestrol selectively modulates plasma coagulation protein synthesis by the perfused rat liver.

The combined effects of orchiectomy and estrogen administration on synthesis of selected hepatic secretory proteins--antithrombin (AT) III, plasminogen, fibrinogen, factor II (prothrombin), factor VII, fibronectin, and albumin--were studied using the isolated rat liver perfused in vitro for ten hours. Male rat liver donors underwent orchiectomy under ether anesthesia and then received 5.0 mg of diethylstilbestrol (DES) by subcutaneous pellet implantation or a placebo pellet; 14 days later the livers were extracted and perfused in vitro for ten hours. In DES experiments, 1.0 mg of DES was also added directly to the liver perfusate at the outset. Pretreatment with DES resulted in significant increases in cumulative synthesis of factors II (65%) and VII (76%) and significant reduction in cumulative synthesis of both antithrombin III (20%; P = .03) and plasminogen (27%; P less than .01) compared to control perfusions, but synthesis of fibrinogen, fibronectin, and albumin was not significantly affected by addition of the hormone. Plasma samples collected from rat liver donors that had received DES showed similar effects on protein concentrations: significant decreases in concentration of plasminogen and antithrombin III were apparent with no significant changes in concentrations of fibrinogen, fibronectin, or albumin. In additional perfusions, "dose-response" experiments were conducted in which rat liver donors received a subcutaneous DES pellet of 0.5, 5.0, or 50 mg. Synthesis of plasminogen in this group of perfusions was progressively decreased as the concentration of DES administered to the rat liver donor increased. Synthesis of AT III was reduced to the same degree by 5.0 or 50 mg of DES, both being substantially lower than the 0.5-mg experiments. Concentrations of these two proteins in plasma samples from rat liver donors showed changes quite similar to those seen in perfusion experiments; however, plasma fibrinogen concentrations were not different among the three groups of rats.

Synthesis of fibronectin by the isolated perfused rat liver.

The major site(s) of synthesis of plasma fibronectin is unknown. Using the isolated perfused rat liver and anti-rat fibronectin antiserum, we have measured net hepatic synthesis of fibronectin during 10-hr perfusion periods. We calculated the circulating plasma fibronectin pool of a 195-g rat with body surface area of 300 sq cm to be 3.3 mg: net hepatic synthesis of 3.75 +/- 1.0 mg/300 sq cm body surface area of the rat liver donor was seen in liver perfusions of 10 hr. When the hormones cortisol and insulin were added to the liver perfusate, net synthesis of 5.05 +/- 0.82 mg/300 sq cm was seen. Synthesis of two known acute phase reactant proteins, fibrinogen and alpha-2 acute phase globulin, was also substantially greater in the presence of cortisol and insulin. Plasma fibronectin levels measured in intact rats that had abscess produced by subcutaneous turpentine injection were 0.57 +/- 0;05 mg/ml compared to 0.38 +/- 0.02 mg/ml in normal control animals. Our studies indicate that hepatic synthesis contributes substantially to the plasma fibronectin pool and show that such synthesis is enhanced by the hormones cortisol and insulin. In the intact rat, inflammation was associated with elevated levels of plasma fibronectin.

Heparin is not metabolized by the perfused rat liver.

The role of the liver in metabolism of heparin was studied using the isolated rat liver perfused in vitro for 10 hr. Porcine intestinal heparin (1000 u) was added to the recirculating liver perfusate, and serial heparin measurements were performed on the liver perfusate every 2 hr, as well as on bile samples secreted by the perfused liver. Heparin concentration remained at a constant level throughout the 10 hr of perfusion, and there was no detectable heparin secreted into bile samples. The findings suggest that hepatic metabolism/clearance plays a minimal role in heparin kinetics in plasma.