Bovine aortic endothelial cell transglutaminase. Enzyme characterization and regulation of activity.

Bovine aortic endothelial cells contain Ca2+-dependent tissue-type transglutaminase. Its activity in these cells was high, with apparent Km and Vmax. values with respect to putrescine of 0.203 mM and 18.5 nmol/min per mg of protein, and its activity was inhibited by the three competitive inhibitors dansylcadaverine, spermine and methylamine. The molecular mass of endothelial cell transglutaminase estimated by gel filtration chromatography was 88 kDa and it was immunoprecipitated by rabbit monospecific antiserum raised against rat liver transglutaminase. Its enzymic activity rose when the cell cultures reached confluence, and was further increased when their proliferation was arrested (synchronized at G0/G1 phase). Most of the enzymic activity was found in the 15,000 g soluble fraction, with only 4-22% of the activity found in the particulate fraction, depending on the state of cell proliferation. Examination of these cellular fractions by SDS/polyacrylamide-gel electrophoresis and immunoblotting revealed that at confluence endothelial cells have accumulated transglutaminase antigen in their 15,000 g particulate fraction. A series of experiments demonstrated the existence of a latent transglutaminase form in non-proliferating cells, and suggested that this might involve the formation of an inhibitory complex. Treatment of cell lysates and the 15,000 g particulate fraction with high salt concentration showed a significant increase in transglutaminase activity. Mixing experiments using the 100,000 g particulate fraction or purified rat liver transglutaminase on one hand and the cytosolic fraction on the other showed dose-dependent inhibition of the transglutaminase activity of the latter. It is concluded that endothelial cells contain a particulate fraction-residing inhibitor of transglutaminase which interacts via ionic interaction with the enzyme.

Investigations on plasma activity of low molecular weight heparin after intravenous and oral administrations.

This study investigated the absorption of low molecular weight heparin (LMWH) after oral administration in man. Six healthy subjects received 5,000 anti-Xa units of LMWH (Kabi 2165) by both i.v. and oral administration. The oral formulations were prepared in pH 4.0 and pH 7.0 buffered solutions. Multiple blood samples were collected after each dose for measurements of anti-Xa, anti-IIa and APTT plasma heparin activities. Pharmacokinetic parameters based on the anti-Xa activity measurements after the i.v. dose were as follows (mean +/- s.d.): t1/2, 1.82 +/- 0.23 h; V, 4.34 +/- 0.651; and CL, 28.0 +/- 6.2 ml min-1. Half-life values based on the anti-IIa and APTT activities were 1.63 +/- 0.43 and 1.09 +/- 0.51 h, respectively. Considerable prolongations in APTT were observed, with APTT at 30 min averaging 55.7 +/- 4.1 s (1.84 +/- 0.27 times baseline values). After oral administration, no measurable plasma heparin activities were observed with either LMWH preparation. The results of this investigation indicate that LMWH does not have detectable plasma activity after oral administration, and that after i.v. administration it has significant anti-IIa and APTT activities in addition to its anti-Xa activity.

Aspirin acetylates fibrinogen and enhances fibrinolysis. Fibrinolytic effect is independent of changes in plasminogen activator levels.

In addition to its antiplatelet effect, aspirin has been reported to have fibrinolytic and hypoprothrombinemic effects. The objective of this study was to investigate possible mechanisms underlying the enhanced fibrinolysis after aspirin. Five healthy subjects received 650 mg of aspirin every 12 hr for 5 days. Blood samples were collected before aspirin (control), and immediately before (0 hr) and 2 hr after (2 hr) the last dose for determinations of clot lysis time, time course of thrombin-induced fibrin aggregation, tissue plasminogen activator activity, intrinsic pathway fibrinolytic activity, plasminogen, fibrinogen, aspirin and salicylic acid, and the coagulation tests activated partial thromboplastin time, thrombin time and prothrombin time. Clot lysis time was shorter after aspirin, control: 9.1 +/- 12.4 min (mean +/- S.D.); 0 hr: 4.6 +/- 4.0 min; 2 hr: 5.7 +/- 6.2 min (P: .04) and the fibrin aggregation curves showed increased relative absorbance at 10 min, control: 8.4 +/- 2.2; 0 hr: 11.2 +/- 0.2; 2 hr: 13.3 +/- 5.4 (P: .02). Control values of tissue plasminogen activator (0.11 +/- 0.04 IU/ml), intrinsic pathway fibrinolytic activity (2.20 +/- 0.54 IU/ml), plasminogen (10.9 +/- 1.0 mg/dl), fibrinogen (288 +/- 37 mg/dl) and the coagulation tests were not different from those after aspirin. Aspirin concentration was below detection limits at 0 hr and 1.63 +/- 0.97 micrograms/ml at 2 hr, whereas salicylic acid concentration was 55.0 +/- 35.8 and 136 +/- 71.9 micrograms/ml at 0 and 2 hr, respectively. In vitro studies using fibrinogen-free plasma and added acetylated fibrinogen showed an inverse relationship between the extent of acetylation and clot lysis time.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of N-deacetylation and N-desulfation of heparin on its anticoagulant activity and in vivo disposition.

This investigation compares the anticoagulant activities and in vivo disposition characteristics of N-deacetylated heparin and N-desulfated heparin with those of standard heparin in an animal model. The N-deacetylated heparin retained 23% of the anticoagulant activity, 34% of the anti-Xa activity and 23% of the anti-IIa activity of standard heparin, whereas the N-desulfated heparin retained only 1.5% of the anticoagulant activity, 0.095% of the anti-Xa activity and 0.92% of the anti-IIa activity of the original heparin. After i.v. injections of 5 mg/kg of the modified heparins, both N-deacetylated and N-deacetylated heparin concentrations showed monoexponential declines with time. The elimination half-lives were similar, 11.7 +/- 3.8 and 13.5 +/- 5.2 min, respectively (N.S.). There were, however, significant differences in both total clearance (Cl) and apparent volume of distribution (Vd) for these heparins, both parameters being significantly larger for the N-deacetylated heparin (P = .0007). The Cl values were 4.15 +/- 1.11 and 0.58 +/- 0.26 ml/min/kg and the Vd values were 72.1 +/- 38.7 and 9.9 +/- 2.4 ml/kg for the N-deacetylated and N-desulfated heparins, respectively. We have reported previously that after a similar dose of standard heparin the elimination half-life was 69.0 +/- 13.1 min, Cl was 0.64 +/- 0.16 ml/min/kg and Vd was 62.6 +/- 16.7 ml/kg. These studies have thus demonstrated that selective N-deacetylation and N-desulfation of the glucosamine residues of heparin affect markedly both its anticoagulant activity and in vivo disposition characteristics. Although important for full anticoagulant activity of heparin, the N-acetyl groups are apparently not essential.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative estimates of beta-lactam residues in raw milk around a reference standard: collaborative study.

A collaborative study was conducted to determine the reliability of a Bacillus stearothermophilus disc assay method for differentiating various concentrations of penicillin in raw milk. Participating laboratories tested 10 different samples (including one negative) in blind duplicate. Triplicate standards were alternated with triplicate unknowns around the periphery of each of 5 different plates. Zone diameters were measured and the difference in zone size of pairs of adjacent standard and unknown samples were analyzed by a paired t-test. Penicillin concentrations 0.003 IU/mL different from the reference concentrations were consistently distinguishable at a 95% confidence level. Such discriminatory power was determined to be possible with as few as 3 plates (9 replicates) per unknown. The method has been adopted official first action.

Hydrogen shuttles in air versus water breathing fishes.

1. The malate-aspartate cycle was demonstrable in subcellular preparations of hearts from Arapaima, Lepidosiren, and Synbranchus (obligate air breathers), Hoplerythriunus (facultative air breather), and Osteoglossum and Hoplias (obligate water breathers). 2. Although no respiratory evidence for significant alpha-glycerophosphate cycle participation could be shown in the air breathers, this cycle was demonstrable in hearts of water breathers. 3. In agreement with the O2 uptake studies, it was possible to reconstruct the malate-aspartate, but not the alpha-glycerophosphate cycle, in isolated mitochondria from air breathers, while both shuttles could be reconstructed with heart mitochondria in the case of water breathing fishes.

Hydrogen shuttles in gills of water versus air breathing osteoglossids.

1. Using subcellular preparations of gills from Arapaima, an obligate air breather, and aruana, a related osteoglossid that is an obligate water breather, a comparison was made of the relative roles of the malate-aspartate cycle and the alpha-glycerophosphate (alpha-GP) cycle in transferring reducing equivalents from the cytosol to the mitochondria. 2. In aruana gill preparations, the alpha-GP cycle could be most clearly demonstrated by reconstructing it with purified isolated mitochondria, using the oxidation rate of exogenous NADH as a measure of the cycling activity. 3. Subcellular preparations of Arapaima gill, in contrast to the aruana gill, were not responsive to exogenous alpha-glycerophosphate, but a glutamate-malate stimulated O2 uptake was sensitive to aminooxyacetate, an aminotransferase inhibitor, a result that would be expected if the respiration were based on malate-aspartate cycling. 4. It was concluded that, compared to the alpha-glycerophosphate cycle, the malate-aspartate cycle was relatively more active in Arapaima gill than in aruana gill, and possible implications were discussed.