Disorders of thrombin interaction with the blood vessel wall and its inactivation by antithrombin III in experimental nephrotic syndrome

In the experiments on white rats was conducted a comparative study of 125I-alpha-thrombin clearance and its inactivation by antithrombin III in animals of the control group and rats with the experimental nephrotic syndrome (Heymann nephritis). It was determined that alterations of thrombin binding to the vascular wall in the nephrotic syndrome induced the prolongation of the labelled enzyme half-life in the blood stream. The formation of 125I-alpha-thrombin complexes with antithrombin III was delayed in the nephrotic syndrome, that suggests the violation of mechanisms of thrombin inactivation by antithrombin III. The distortions of endothelium-mediated thrombin elimination and inactivation in the nephrotic syndrome resulted in the enzyme interaction with fibrinogen, which threatened organism by thrombosis.     

Mechanisms of the development of acquired antithrombin III deficiency in the experimental nephrotic syndromes

In the experiments on white rats was studied the role of excessive thrombinogenesis in the development of acquired antithrombin III deficiency in the experimental nephrotic syndrome. It was determined that excess thrombin generation induced the marked acceleration of 125I-antithrombin III clearance from blood stream in consequence formation of thrombin antithrombin III complexes with the following limited proteolysis of the inhibitor by enzyme. These results give evidence that apart from proteinuria the excess thrombin generation accompanied by nephrotic syndrome play a part in the development of acquired deficiency of antithrombin III in this experimental pathology.     

Anticoagulant and antithrombotic effects of low molecular weight heparin

Low molecular weight heparin (Mr 8 kDa) was prepared from conventional heparin (Mr 18 kDa) by the chromatography on DEAE-sephadex with the recovery of 56%. Low molecular weight heparin had less affinity to antithrombin III than unfractionated heparin and had less anticoagulant and anti-IIa activities. The anti-Xa activity of low molecular weight heparin exceed by 17% the activity of conventional heparin. In the experiments on rats it was determined that the biological half-life of low molecular weight heparin exceed two-fold that of the unfractionated heparin. In the modified model of the arteriovenous shunt thrombosis in normal and nephrotic syndrome rats it was shown that the low molecular weight heparin was the most efficient antithrombotic remedy in normal and decreased level of antithrombin III in the organism.     

Antithrombin III activity in long-developing hypercoagulation in animals

Old rats aged 12-18 months and rats kept on an atherogenic diet for 3.5 months demonstrate high blood antithrombin III content at the initial period of the development of anticoagulation function suppression and of hypercoagulation. During long-developing hypercoagulation, the high content of antithrombin III might be regarded as compensatory reaction interfering with formation in the blood of thrombin microamounts. With hypercoagulation becoming more pronounced and with a further increase of blood thrombin concentration the content of antithrombin III progressively descends, which is accompanied by steady development of anticoagulation function suppression.     

A study of impaired fibrin polymerization in patients with the nephrotic syndrome.

Clotting studies have been performed on 64 consecutive patients with nephrotic syndrome. The thrombin time was prolonged in 59. Fibrin polymerization was studied in 42 of the 59 patients with a prolonged thrombin time and an abnormality was present in 22. There was a significant correlation between the prolongation of the thrombin time and impairment of polymerization (p = 0.018). No correlation was found between these two parameters and the patient's sex, age and drug therapy. Furthermore there was no correlation with the prothrombin time, APTT, fibrinogen, FDP, antithrombin III and platelet counts. There was however a significant negative correlation between the thrombin time and the serum albumin level (p less than 0.05). No abnormal bleeding was observed during or after renal biopsy in these patients. Renal biopsy may be performed safely despite the grossly prolonged thrombin time and abnormal fibrin polymerization in patients with the nephrotic syndrome.     

Renal leptin in experimental nephrotic syndrome

Leptin is a fat derived hormone involved in the regulation of metabolism and body composition. The kidney is the principle organ responsible for elimination of circulating leptin. Our aim is to evaluate if the nephrotic kidneys participate in the metabolism of leptin by comparing the serum leptin level in renal veins and in their renal arteries and to study the relationship between leptin and lipoprotein levels in healthy and nephrotic rats. Methods: Rats were divided into two equal groups: group 1 in which experimental nephrotic syndrome was produced by injecting them intraperitoneally with a supernatant of the homogenized mixture of their own kidney (obtained by previous unilateral nephrectomy) and complete Freund’s adjuvant. Another group constituted the control group. Leptin and lipid profile were estimated in blood samples of renal veins and renal arteries. There was a highly significant increase in leptin and lipid profile levels in the nephrotic rats compared with the normal group. There was a high significant decrease in leptin in the renal venous blood compared with its level in the renal arterial blood of normal and nephrotic rats. This work has stressed the involvement of kidney and the nephrotic renal tissue in the process of leptin metabolism and lipogenesis.     

A study of rabbit plasma progressive antithrombinic activity during acute phase inflammatory reaction and in experimental nephrotic syndrome.

1. The development of the progressive antithrombinic activity in rabbit plasma as a function of the level of the two alpha-macroglobulins in two experimental pathologies: acute phase inflammatory reaction and nephrotic syndrome, were studied. 2. In the first case, the antithrombinic activity is a result of the increased biosynthesis of plasmatic antithrombins: antithrombin III, alpha 1 antitrypsin and alpha macroglobulins. 3. In the nephrotic syndrome, this activity follows the increase in the alpha M levels, the other antithrombins having been massively eliminated from the circulation in the urine.     

Increased glomerular atrial natriuretic peptide receptor affinity in experimental nephrotic syndrome.

Atrial natriuretic peptide (ANP) is a cardiac hormone with natriuretic and diuretic effects. To better define the ANP hormonal system in the nephrotic syndrome, a condition associated with renal sodium retention, we undertook a study of glomerular ANP receptors in rats with puromycin aminonucleoside-induced nephrotic syndrome and in pair-fed controls. Nephrotic rats had significantly decreased serum albumin and total protein and significantly increased serum cholesterol, triglycerides and 24 hour urinary protein excretion. Plasma level of atrial natriuretic peptide was similar in both groups of rats. Competition binding inhibition studies in isolated glomeruli demonstrated one binding site in both groups of rats. The density of ANP binding sites in isolated glomeruli was similar in nephrotic and pair-fed rats while the binding affinity was increased significantly in the nephrotic rats. This is the first study to demonstrate alterations in renal ANP receptors in the nephrotic syndrome. Further studies will be necessary to determine whether alterations in glomerular ANP receptors contribute to renal sodium retention in the nephrotic syndrome.     

Inhibition of bovine factor IXa and factor Xabeta by antithrombin III.

Factor IXa and factor Xabeta are serine proteases which participate in the middle phase of blood coagulation. These two enzymes are inhibited by antithrombin III by the formation of an enzyme-inhibitor complex containing 1 mol of enzyme and 1 mol of antithrombin III. The complex was readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and loss of coagulant or esterase activity at increasing concentrations of inhibitor. The inactivation of factor IXa by antithrombin III was relatively slow, but the reaction was greatly accelerated by the addition of heparin.     

Lipid peroxidation in the kidneys of rats with nephritis induced by nephrotoxic serum and proteinuria induced by albumin overload]

Lipid peroxidation (LPO) stimulated by ascorbate was studied in renal cortex of 20 rats with nephrotoxic nephritis (NTN) and of 9 rats with proteinuria induced by a 3-day course of i. p. injections of the human serum albumin. At the early stages of NTN (0.5 h. and 3 h.) LPO activities were of the same values as in control rats. A small decrease in renal cortex LPO was found on the 4-th day of NTN when nephrotic syndrome has been developed. A significant reduction in LPO activity was observed on the 16-th day of NTN characterized by a more pronounced nephrotic syndrome. LPO activity in renal cortex of the rats with albumin overload proteinuria was also reduced. An inhibitory effect of proteinuria on LPO activity in kidney is discussed.     

Relations between parameters of lipid metabolism and hemostasis in vascular diseases

In 98 patients with arterial insufficiency of arteriosclerotic and diabetic genesis correlation analyses were carried out between total cholesterol, HDL cholesterol, triglycerides, creatinine, blood pressure, platelet counts, platelet function and antithrombin III. Besides the known relations between the parameters of lipid metabolism and blood pressure, correlations were found and analysed between antithrombin III, platelet function, renal function and between platelet counts and HDL cholesterol. The importance of the findings for the diagnosis of vascular insufficiency and the relation to normal values are discussed.     

Soy protein diet ameliorates renal nitrotyrosine formation and chronic nephropathy induced by puromycin aminonucleoside

It has been shown that reactive oxygen species are involved in chronic puromycin aminonucleoside (PAN) induced nephrotic syndrome (NS) and that a 20% soy protein diet reduces renal damage in this experimental model. The purpose of the present work was to investigate if a 20% soy protein diet is able to modulate kidney nitrotyrosine formation and the activity of renal antioxidant enzymes (catalase, glutathione peroxidase, Cu,Zn- or Mn-superoxide dismutase) which could explain, at least in part, the protective effect of the soy protein diet in rats with chronic NS induced by PAN. Four groups of rats were studied: (1) Control rats fed 20% casein diet, (2) Nephrotic rats fed 20% casein diet, (3) Control rats fed 20% soy protein diet, and (4) Nephrotic rats fed 20% soy protein diet. Chronic NS was induced by repeated injections of PAN and rats were sacrificed at week nine. The soy protein diet ameliorated proteinuria, hypercholesterolemia, and the increase in serum creatinine and blood urea nitrogen observed in nephrotic rats fed 20% casein diet. Kidney nitrotyrosine formation increased in nephrotic rats fed 20% casein diet and this increase was ameliorated in nephrotic rats fed 20% soy protein diet. However, the soy protein diet was unable to modulate the antioxidant enzymes activities in control and nephrotic rats fed 20% soy protein diet. Food intake was similar in the two diet groups. The protective effect of a 20% soy protein diet on renal damage in chronic nephropathy induced by PAN was associated with the amelioration in the renal nitrotyrosine formation but not with the modulation of antioxidant enzymes.     

Effect of Ultrasound on Structure and Functional Properties of Antithrombin III and Proteins of PPSB Complex

A combination of gel-permeation HPLC, affinity chromatography on heparin-Sepharose, gel electrophoresis, and estimation of inhibitory activity showed that effect of low-frequency ultrasound (26 W/cm(2), 37 degrees C, pH 7.4) on homogeneous antithrombin III was accompanied by formation of aggregates and a latent form of serpin. Heparin and pentosan polysulfate stabilized antithrombin III; this resulted in decrease in ultrasonic-induced formation of the aggregate and latent forms. The influence of ultrasound was not accompanied by significant changes in the contents of non-activated blood coagulation factors in the PPSB complex.     

The physiological inhibitors of blood coagulation. 1. Antithrombin III (AT III)

In a brief survey, recent data on the molecular structure and functions of antithrombin III as well as on the similarity of identity with antithrombin III of newborns and mammals are referred to. Moreover, the survival time in circulation and its broad inhibiting spectrum comprising not only all activating factors of coagulation, but also other proteases, is discussed. The accelerating effect of heparin on response and possible mechanisms of this acceleration are discussed. The various procedures of determination are briefly dealt with. Furthermore, the distinct tendency towards thromboembolic complications occurring in patients with only a slight reduction of antithrombin III level is dealt with, as it was described in inherited and acquired deficiency of antithrombin III. Finally, the possibilities of therapy for this deficiency are discussed and is underlined on the key position of antithrombin III for maintaining the balance of coagulation.     

Protective effects of mesenchymal stromal cells on adriamycin-induced minimal change nephrotic syndrome in rats and possible mechanisms

Background aimsMinimal change nephrotic syndrome is the most frequent cause of nephrotic syndrome in childhood. Current treatment regimes, which include glucocorticoid hormones and immunosuppressive therapy, are effective and have fast response. However, because of the side effects, long treatment course, poor patient compliance and relapse, novel approaches for the disease are highly desired.MethodsThe adriamycin-induced nephrotic rat model was established. Rats were allocated to a model group, a prednisone group or mesenchymal stromal cell (MSC) group. Clinical parameters in each treatment group were determined at 2 weeks, 4 weeks and 8 weeks. The messenger RNA (mRNA) levels of synaptopodin, p21 and monocyte chemoattractant protein-1 were determined through the use of quantitative real-time–polymerase chain reaction. Protein levels were determined by means of Western blot or enzyme-linked immunosorbent assay. Podocytes were isolated and apoptotic rate after adriamycin with or without MSC treatment was analyzed by means of flow cytometry.ResultsMSC intervention improved renal function as assessed by urinary protein, blood creatinine and triglyceride levels. MSC intervention reduced adriamycin-induced renal tissue damage visualized by immunohistochemistry and light and electron microscopic analysis and reduced adriamycin-induced podocyte apoptosis. After MSC intervention, mRNA and protein levels of synaptopodin and p21 in renal cortex were significantly increased. MSCs also restored synaptopodin mRNA and protein expression in isolated podocytes. In addition, monocyte chemoattractant protein-1 mRNA in renal cortex and protein level in serum of the MSC treatment group were significantly decreased compared with that in the adriamycin-induced nephropathy model group.ConclusionsOur data indicate that MSCs could protect rats from adriamycin-induced minimal change nephrotic syndrome, and the protective effects of MSCs are mediated through multiple actions.     

Effects of atorvastatin and aspirin combined therapy on inflammatory responses in patients undergoing coronary artery bypass grafting

This study was conducted to compare the effects of atorvastatin plus aspirin combined therapy on inflammatory responses, endothelial cell function, and blood coagulation system in patients undergoing coronary artery bypass grafting (CABG) to aspirin monotherapy. The patients were randomized into atorvastatin plus aspirin combined therapy group and aspirin monotherapy group. Reduced total cholesterol in the combined therapy group was found in a short term of medication for 14 days. On postoperative day (POD)-14, inhibitory effects of the combined therapy on whole blood aggregation as well as platelet activation assessed by flow cytometry were stronger than those of the monotherapy. Furthermore, cytokine, cytokine receptors, c-reactive protein and α1-acid glycoprotein in the combined therapy group were down-regulated on POD-14. At the same time, circulating levels of thromboxane A₂, vascular endothelial growth factor and thrombin–antithrombin III complex as well as P-selectin, L-selectin and intercellular adhesion molecule-1 were down-regulated, while E-selectin and transforming growth factor-β1 was up-regulated. Atorvastatin plus aspirin combined therapy may improve inflammatory responses, accelerated platelet function, vascular endothelial cell function, blood coagulation system at the early stage such as 14th day after CABG. In conclusion, atorvastatin and aspirin combined therapy may bring beneficial effects to the patient after CABG.     

Increased cyclosporine bioavailability induced by experimental nephrotic syndrome in rats

Components of whole blood and plasma are highly altered during the presentation of nephrotic syndrome. The present study was aimed to explore the influence of nephrotic syndrome on the pharmacokinetics of cyclosporine (CsA) (10 mg/kg) administered i.v. to control or puromycin-induced nephrotic rats (P-NS). We found an increase in CsA bioavailability in the nephrotic group compared with controls. The area under the curve of blood CsA versus time (AUCiv) increased from 27.7 +/- 5.3 to 60.6 +/- 13.8 mug.h.mL-1 in control and P-NS rats, respectively. The AUCiv augmentation was positively correlated with cholesterol levels. On the other hand, the total body clearance was significantly lower (0.38 +/- 0.06 vs. 0.17 +/- 0.03 L.(kg body mass)-1.h-1) and the volume of distribution at steady state (3.70 +/- 0.52 vs. 2.85 +/- 0.32 L/kg) was significantly smaller in nephrotic rats as compared with control. These pharmacokinetic changes lead to a longer terminal half-life of CsA in P-NS rats (11.8 +/- 1.6 vs. 6.9 +/- 0.91 h). We conclude that the physiopathologic changes induced by the nephrotic syndrome in P-NS animals result in a significant increase in CsA blood exposure by both the decrease in drug distribution and the reduction in elimination rate of CsA.     

The heparin binding site of human antithrombin III. Selective chemical modification at Lys114, Lys125, and Lys287 impairs its heparin cofactor activity

Heparin binds to human antithrombin III and accelerates its inhibitory activity in the blood coagulation system. Previous reports (Rosenberg, R. D., and Damus, P. S. (1973) J. Biol. Chem. 248, 6490-6505; Pecon, J. M., and Blackburn, M. N. (1984) J. Biol. Chem. 259, 935-938) have shown that selective chemical modification of a limited number of lysine residues in antithrombin III causes drastic loss of its heparin cofactor activity. We have performed chemical modification of antithrombin III with trinitrobenzene sulfonic acid in order to determine the location of these lysine residues. When antithrombin III was treated with 100 M excess of trinitrobenzene sulfonic acid for 10 min, about 3.2 mol of amino group per mol of antithrombin III were modified. The heparin cofactor activity dropped to about 25%, whereas the progressive inhibitory activity (in the absence of heparin) remained essentially intact (about 95%). The modified amino groups were identified to be Lys114 (75%), Lys125 (94%), and Lys287 (96%). These results were obtained by comparing and analyzing the cyanogen bromide fragments derived from native antithrombin III and the 10-min modified antithrombin III. When antithrombin III was pretreated with heparin, followed by trinitrobenzene sulfonic acid modification, the extent of modification at Lys114 and Lys125 decreased from 75% and 94% to 20% and 40%, respectively, whereas the modification at Lys287 remained nearly quantitative (greater than 95%). Based on these results, we conclude that Lys114 and Lys125 are essential for the heparin cofactor activity of human antithrombin III.     

Coagulation factors and fibrinolytic system in subretinal liquid in patients with rheumatogenous retinal detachment

The work deals with estimation of some factors of blood coagulation and fibrinolytic systems, which include antithrombin III, factor X, prothrombin, plasminogen, protein C concentrations in the subretinal fluid of the patients with rhegmatogenous retinal detachment retina. The tendency to increase of the blood coagulation and fibrinolysis factor levels, except protein C, was revealed in the patients with complicated forms of the disease. The investigations mentioned above are capable of serving as a diagnostical and forecasting test characterizing the rhegmatogenous retinal detachment retina and surgical treatment proceeding.     

Thrombin generation and inactivation in the presence of antithrombin III and heparin

We have determined the rate constants of inactivation of factor Xa and thrombin by antithrombin III/heparin during the process of prothrombin activation. The second-order rate constant of inhibition of factor Xa alone by antithrombin III as determined by using the synthetic peptide substrate S-2337 was found to be 1.1 X 10(6) M-1 min-1. Factor Xa in prothrombin activation mixtures that contained prothrombin, and either saturating amounts of factor Va or phospholipid (20 mol % dioleoylphosphatidylserine/80 mol % dioleoylphosphatidylcholine, 10 microM), was inhibited by antithrombin III with a second-order rate constant that was essentially the same: 1.2 X 10(6) M-1 min-1. When both factor Va and phospholipid were present during prothrombin activation, factor Xa inhibition by antithrombin III was reduced about 10-fold, with a second-order rate constant of 1.3 X 10(5) M-1 min-1. Factor Xa in the prothrombin activation mixture that contained both factor Va and phospholipid was even more protected from inhibition by the antithrombin III-heparin complex. The first-order rate constants of these reactions at 200 nM antithrombin III and normalized to heparin at 1 microgram/mL were 0.33 and 9.5 min-1 in the presence and absence of factor Va and phospholipid, respectively. When the prothrombin concentration was varied widely around the Km for prothrombin, this had no effect on the first-order rate constants of inhibition. It is our conclusion that factor Xa when acting in prothrombinase on prothrombin is profoundly protected from inhibition by antithrombin III in the absence as well as in the presence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)