Prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin administration

The possibility of prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin devoid of proteolytic activity and capable of stimulating the reaction of anticoagulation system was studied. The injection of lethal thromboplastin dose was shown to produce a sharp increase in soluble fibrin blood content, total disappearance of fibrinolytic activity and intravascular blood coagulation. The animals died of thrombosis in 90% of cases. It was established that the injection of lethal thromboplastin dose 5 min after DIP-alpha-thrombin injection caused a 13% lethality from thrombosis. No reliable changes in fibrinolytic activity and soluble fibrin content were observed. A significant increase in thrombin and recalcification time was recorded. It is suggested that DIP-alpha-thrombin prevents intravascular blood coagulation induced by lethal thromboplastin dose due to mobilization of the reserve capacities of neuro-humoral anticoagulation system.     

Vascular smooth muscle cells synthesize,secrete and express coagulation factor V

Expression of cellular procoagulant activity may be one of the more important responses to vascular injury. Because factor V, a coagulation cofactor in the prothrombinase complex, catalyzes the conversion of prothrombin to thrombin, it may be a key to understanding this response. Therefore, we have investigated the synthesis, secretion and expression of factor V by vascular smooth muscle cells, which proliferate at sites of vascular injury. Cultured aortic vascular smooth muscle cells constitutively secreted Factor V activity, as determined by a functional assay. Labeled factor V was immunoprecipitated from conditioned medium of [³⁵S]methionine-labeled cells, indicating that the secreted factor V was synthesized by vascular smooth muscle cells. Treatment of vascular smooth muscle cells with tunicamycin prevented secretion of factor V, suggesting that its secretion was dependent on the presence of N-linked carbohydrate. Factor V activity was also expressed on the vascular smooth muscle cell surface, as indicated by the ability of cultured cells to promote factor X_a-catalyzed prothrombin activation. These data suggest that the proliferation of smooth muscle cells in response to vascular injury may be one mechanism that links vascular disease with thrombosis.     

A Simple Bioassay for Chemicals Active to the Photosynthetic or Respiratory Systems of Plants

After male rats of the Sprague Dawley strain, 5 weeks old, were fed a 20% casein diet with or without 0.5% nicotinamide for 13 days, 180 mg/kg body weight of alloxan was injected in- traperitoneally into the rats. The rats were kept for 18 days with the same diet. The level of blood glucose was increased 6-fold in the group on a 20% casein diet by the injection of alloxan, while there was only a 2-foid increase in the group on a nicotinamide-containing diet and the decreased body weight was also lower in the group on the nicotinamide diet than the group on the casein diet. The body weight was indirectly related to the concentration of blood glucose. A marked increase was observed in the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase upon the injection of alloxan with both diets; on the other hand, the activities of kynureninase and NAD⁺ synthetase were decreased by the injection of alloxan. The activity of kynurenine aminotransferase increased in the group on the 20% casein diet by the injection of alloxan, while in the group on the nicotinamide-containing diet its activity was not increased by the injection. These changes in the above enzyme activities mean that the conversion ratio from tryptophan to niacin is lower in the alloxan diabetic rat than normal rat. It was found that the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase were directly related to the concentration of blood glucose, and that the activities of kynureninase and NAD⁺ synthetase were inversely related. There was no difference in the activities of 3-hydroxyanthranilic acid oxygenase and nicotinamide mononucleotide adenylyltransferase upon the injection of alloxan with both diets.     

Effect of various levels of supplementation with sodium pivalate on tissue carnitine concentrations and urinary excretion of carnitine in the rat

In previous studies, sodium pivalate has been administered to rats in their drinking water (20 mmoles/L; equivalent to 0.3% of the diet) as a way to lower the concentration of carnitine in tissues and to produce a model of secondary carnitine deficiency. Although this level of supplementation results in a marked decrease in carnitine concentration in a variety of tissues, it does not produce the classical signs of carnitine deficiency (i.e., decreased fatty acid oxidation and ketogenesis). The present study was designed (1) to determine if increasing the level of pivalate supplementation (0.6, 1.0% of the diet) would further reduce the concentrations of total and free carnitine in rat tissues without altering growth or food intake, and (2) to examine the effect of length of feeding (4 vs. 8 weeks) on these variables. Male, Sprague-Dawley rats were randomly assigned to either a control (0.2% sodium bicarbonate) or experimental diet (0.3, 0.6, 1.0% sodium pivalate) for either four or eight weeks. Animals (n = 6/group) were housed in metabolic cages; food and water were provided ad libitum throughout the study. Supplementation with sodium pivalate did not alter water intake or urine output. Ingestion of a diet containing 1.0% pivalic acid decreased food intake (g/day; P < 0.05), final body weight (P < 0.007), and growth rate (P < 0.001) after four weeks. The concentration of total carnitine in plasma, heart, liver, muscle, and kidney was reduced in all experimental groups (P < 0.001), regardless of level of supplementation or length of feeding. The concentration of free carnitine in heart, muscle, and kidney was also reduced (P < 0.001) in rats treated with pivalate for either four or eight weeks. The concentration of free carnitine in liver was reduced in animals supplemented with pivalate for eight weeks (P < 0.05), but no effect was observed in livers from rats treated for four weeks. Excretion of total carnitine and short chain acylcarnitine in urine was increased in pivalate supplemented rats throughout the entire feeding period (P < 0.001). Free carnitine excretion was increased during Weeks 1 and 2 (P < 0.01), but began to decline during Week 3 in experimental groups. During Weeks 6 and 8, free carnitine excretion in pivalate supplemented rats was less than that of control animals (P < 0.01). In summary, no further reduction in tissue carnitine concentration was observed when rats were supplemented with sodium pivalate at levels greater than 0.3% of the diet. Food intake (g/day) and growth were decreased in rats fed a diet containing 1.0% sodium pivalate. These data indicate that maximal lowering of tissue carnitine concentrations is achieved by feeding diets containing 0.3% sodium pivalate or less.     

Increased risk of venous thrombosis by AB alleles of the ABO blood group and Factor V Leiden in a Brazilian population

Most cases of a predisposition to venous thrombosis are caused by resistance to activated protein C, associated in 95% of cases with the Factor V Leiden allele (FVL or R506Q). Several recent studies report a further increased risk of thrombosis by an association between the AB alleles of the ABO blood group and Factor V Leiden. The present study investigated this association with deep vein thrombosis (DVT) in individuals treated at the Hemocentro de Pernambuco in northeastern Brazil. A case-control comparison showed a significant risk of thrombosis in the presence of Factor V Leiden (OR = 10.1), which was approximately doubled when the AB alleles of the ABO blood group were present as well (OR = 22.3). These results confirm that the increased risk of deep vein thrombosis in the combined presence of AB alleles and Factor V Leiden is also applicable to the Brazilian population suggesting that ABO blood group typing should be routinely added to FVL in studies involving thrombosis.     

Effects of high-molecular-weight cryoprotectants on platelets and the coagulation system

The objective of this study is to examine the effects of the most widely used high-molecular-weight cryoprotectants on the coagulation system. Dextran, hydryoxyethyl starch (HES), polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), and albumin were added at different concentrations in the range between 0.01-1% (w/v) to solvent/detergent-treated plasma. Using a STA/STA Compact coagulation analyzer the following clotting tests were performed: prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Factor V, and Factor VIII percentage of activity. PVP and PEG caused a significant increase in APTT, a decrease in Factor VIII percentage of activity, and a slight decrease in TT, while PT and Factor V percentage of activity remained unchanged. Dextran, HES, and albumin did not effect the clotting tests. The effect of high-molecular-weight cryoprotectants on platelets was assessed by platelet-induced clot retraction (PICR) and aggregation with thrombin and agglutination with ristocetin. Platelet aggregation and agglutination were unaffected by all cryoprotectants tested; however, PICR was significantly reduced in the presence of PVP or PEG. Possible mechanisms by which PVP and PEG interfere with the coagulation system are discussed. We also raise issues concerning the development of one-step blood cryopreservation techniques which do not require cryoprotectant removal prior to transfusion.     

Reduced expressions of calmodulin genes and protein and reduced ability of calmodulin to activate plasma membrane Ca2+‐ATPase in the brain of protein undernourished rats: modulatory roles of selenium and zinc supplementation

The roles of protein undernutrition as well as selenium (Se) and zinc (Zn) supplementation on the ability of calmodulin (CaM) to activate erythrocyte ghost membrane (EGM) Ca²⁺‐ATPase and the calmodulin genes and protein expressions in rat's cortex and cerebellum were investigated. Rats on adequate protein diet and protein‐undernourished (PU) rats were fed with diet containing 16% and 5% casein, respectively, for a period of 10 weeks. The rats were then supplemented with Se and Zn at a concentration of 0.15 and 227 mg l⁻¹, respectively, in drinking water for 3 weeks. The results obtained from the study showed significant reductions in synaptosomal plasma membrane Ca²⁺‐ATPase (PMCA) activity, Ca²⁺/CaM activated EGM Ca²⁺ATPase activity and calmodulin genes and protein expressions in PU rats. Se or Zn supplementation improved the ability of Ca²⁺/CaM to activate EGM Ca²⁺‐ATPase and protein expressions. Se or Zn supplementation improved gene expression in the cerebellum but not in the cortex. Also, the activity of PMCA was significantly improved by Zn. In conclusion, it is postulated that Se and Zn might be beneficial antioxidants in protecting against neuronal dysfunction resulting from reduced level of calmodulin such as present in protein undernutrition. Copyright © 2016 John Wiley & Sons, Ltd.     

Congenital deficiency of factor VII in a canine family

Prolonged prothrombin time in the blood coagulation test was seen in some beagle dogs whose activated partial prothrombin times were distributed within the normal range. This phenomenon suggested possible abnormalities in coagulation factors II, V, VII, and/or X. Therefore, a revised cross-matching test was given and a determination of coagulation factors related to the extrinsic system was performed. We also determined whether or not factor VII inhibitor was present. The results were as follows: 1) In the revised cross-matching test, the prolonged prothrombin times were revised when normal canine serum was added to the plasma that showed prolongation of prothrombin time, but not when pooled normal canine plasma absorbed with BaSO4 was added to it. 2) The level of factor VII in the plasma with prolonged prothrombin time was 5 approximately 10% of the level in normal canine plasma. 3) Factor VII inhibitor was not detected in the plasma with prolonged prothrombin time or in normal plasma. Consequently, the prolongation of prothrombin time was attributed to a deficiency in factor VII. This abnormality was confirmed to be congenital.     

Aspirin-Induced Prolongation of the Ivy Bleeding Time—Its Diagnostic Usefulness

Ivy bleeding time values before and two hours after ingestion of 600 mg of aspirin (aspirin tolerance test) were studied in normal persons, in patients with a disorder of primary hemostasis and in patients with various coagulation factor deficiencies. Aspirin produced a significant prolongation of the bleeding time in patients with von Willebrand''s disease, uremia, and primary platelet disease, and in two patients with Factor XI deficiency. Dextropropoxyphene hydrochloride caused no prolongation of the bleeding time in normal persons.     

Effect of excess dietary histidine on rate of turnover of65Zn in brain of rat

The effect of the chronic administration of histidine on the brain zinc level was examined in growing, male Wistar rats. Using a purified diet, the minimum zinc requirement for normal growth and normal plasma and tissue zinc levels was found to be around 10 ppm. Given this zinc content; the diet was supplemented with 5% and 8% histidine, respectively, or with 10% glycine (as control). Brain zinc was analyzed by measuring the rate of turnover of⁶⁵Zn from 2–4 weeks after a single injection of the tracer. Feeding the diet supplemented with 5% histidine caused a small decrease in the plasma zinc concentration and a slight increase in the rate of turnover of⁶⁵Zn in the cerebrum and the cerebellum as compared to the control group. The animals fed the diet supplemented with 8% histidine became severely zinc deficient (as evidenced by a 50% reduction in the plasma zinc content), however, the rate of turnover of⁶⁵Zn in all brain regions examined was significantly decreased as compared to the control group. The results indicate that histidine has no specific complexing action on the brain zinc.     

Activation of coagulation factor V by calcium-dependent proteinase

Factor V is a key coagulation cofactor, regulating the rate of Factor Xa-catalyzed prothrombin conversion. Activation of Factor V markedly accelerates coagulation. This study describes a new class of Factor V activators, sulfhydryl proteinases. Of the enzymes studied, calcium-dependent proteinase was the most effective activator. Activation of Factor V by this enzyme was associated with cleavage of 125I-labeled Factor V to peptides distinct from those generated by previously described activators. Calcium-dependent proteinase-activated Factor Va peptides with molecular weights of 114,000 and 93,000 bound both to Factor Xa and to cultured endothelial cells. Calcium-dependent proteinase was identified in vascular endothelial cells, a tissue that also synthesizes Factor V. These findings suggest a previously unknown mechanism for cellular regulation of coagulation.     

Blood coagulation studies in normothermic,hibernating, and aroused Spermophilus franklini

The blood coagulation system of Spermophilus franklini was evaluated from normothermic, hibernating, and aroused individuals. Clotting time, thrombin time, prothrombin time, and partial thromboplastin time were measured to test the state of coagulability. The concentrations of the formed elements and the titers of five plasma factors were also determined.During hibernation, clotting time significantly increased above normothermic levels. Arousal resulted in clotting time returning toward normothermic values. Both thrombin time and partial thromboplastin time significantly increased above normothermic levels in blood from hibernators. The two tests exhibited normothermic levels in arousing individuals. Prothrombin time did not increase in blood from hibernating animals.Erythrocytes, leukocytes, and platelets were found to be significantly reduced in number in hibernating animals. Leukocyte and platelet numbers returned to normothermic levels during arousal.Factor VII, Factor X, prothrombin, and heparin concentrations did not significantly change from normothermic levels in hibernating individuals. Factor V, however, displayed a 45% decrease in concentration in hibernating individuals, with arousal resulting in near-normothermic levels. Aroused individuals displayed a doubling of prothrombin concentrations relative to normothermic individuals.     

Effects of skim milk powder intake and treadmill training exercise on renal,bone and metabolic parameters in aged obese rats

Purpose

we aim to examine whether adding exercise has impact on obesity prevention and bone metabolism in senior rats, to which dietary obesity was induced through skim milk intake.

Methods

We used 47, 14-week old Sprague -Dawley (SD) female rats (CLEA Japan, Inc.). The Rats were separated into four random groups: 1) a Non-Ex group with a normal diet (n = 12), 2) an Ex group with a normal diet (n = 12), 3) a Non-Ex group with a skim milk diet (n = 11), and 4) an Ex group with a skim milk diet (n = 12). As the exercise for each Ex group, rats ran on a treadmill starting at 27-week old (TREADMILL CONTROL LE8710 and TREADMILL CONTROL LE8700, Harvard Bioscience). Training protocol stipulated a frequency of five times a week for 12 weeks.

Results

The leptin concentration differed with dietary content: compared to the Ex group with a skim milk diet, Non-Ex and Ex groups with a normal diet showed significantly higher values (p < 0.05). The Ex group had significantly lower values in both the normal diet and skim milk diet groups with or without exercise (p < 0.05). Compared to the Non-Ex group with a normal diet, BS/BV (mm²/mm³), BV/TV (%), Tb.Th (μm), TBPf (/mm) and Tb.N (/mm) had significantly lower in the Ex group, the Ex and Non-Ex groups with a whey protein diet, and the Ex group with a skim milk protein diet (p < 0.05).

Conclusion

These findings suggest that senior female rats fed SMP would have higher bone structural and strength parameters than rats fed a normal diet.     

Pharmacological evidence for involvement of the sympathetic nervous system in the increase in renin secretion produced by a low sodium diet in rats

To determine the degree to which increased sympathetic activity contributes to the increase in renin secretion produced by a low sodium diet, the beta-adrenergic blocking drug propranolol or saline vehicle was injected through indwelling jugular cannulas in rats fed a normal diet and rats fed a low sodium diet for 9 days. Plasma renin activity (PRA) and plasma renin concentration (PRC) were elevated by the low sodium diet, and these values were reduced 42-45% by propranolol, although they were still higher than in the normal diet controls. Plasma corticosterone was moderately elevated in cannulated rats on regular diet, compared to decapitated controls, but corticosterone did not differ between cannulated and decapitated rats on low salt diet; propranolol reduced plasma corticosterone. However, PRA and PRC were comparable in cannulated rats and decapitated controls on both the normal and the low sodium diets, and propranolol did not produce a significant reduction in PRA and PRC in rats fed the normal diet. This indicates that the effects of propranolol on PRA and PRC in the low sodium rats were not simply due to reduction of a stress-induced increase in renin secretion. The results indicate that increased sympathetic activity makes a substantial contribution to the increase in renin secretion produced by 9 days of dietary sodium restriction.     

Biochemical changes in cuprizone-induced spongiform encephalopathy

Cuprizone (biscyclohexanone oxaldihydrazone) which is known to produce a status spongiosus and demyelination in the CNS was administered in the diet of weanling male mice at a concentration of 0.4% by weight for a period of six weeks before returning animals to a normal diet. Changes in body weight but not brain weight were reversible. Based on the decline in CNP'ase activity and the concentration of galactocerebroside, the loss of myelin was around 70% in those sections of the cerebrum with a high content of white matter while the cerebellum was less affected. The activity of oligodendroglial HFA-ceramide galactosyl transferase was also reduced. These biochemical parameters of myelination were increased after withdrawal of Cuprizone. Remyelination in the cerebrum but not the cerebellum was incomplete. The activity of plasmalogenase hydrolysing the alkenyl group of alkenyl, acyl-phospholipids increased 2-fold in those sections in which myelin loss was most severe. The increase preceded the greatest loss of myelin components (3 to 6 weeks on Cuprizone). The origin of the increased phospholipase activity in demyelinating tissue is discussed. Following myelination, there was a deficit in plasmalogenase activity particularly in the frontal cortex of the cerebrum, where the plasmalogen concentrations was higher than in controls.     

The influence of Annexin32, a new Ca2+-dependent phospholipid-binding protein, on coagulation time and thrombosis

The pharmacodynamics of Annexin32, a new Ca2+-dependent phospholipid-binding protein, was studied by measuring coagulation time in rabbits and venous thrombosis in rabbits and rats. Rabbits and rats were given Annexin32 by intravenous administration. Then Kaolin partial thromboplastin time (KPTT), thrombosis in vitro and in vivo were assayed. The results showed that KPTT of rabbits was prolonged (p < 0.01), and the length and weight of thrombus in vitro were reduced (p < 0.01) after administration of Annexin32 at 1 mg/kg. It also inhibited thrombosis in vivo and reduced the weight of venous thrombus significantly in rats (p < 0.01). All these results suggested that Annexin32 possesses the characteristic of antithrombotic effect and fewer side effects on coagulation time.     

Increase in DPP-IV in the intestine, liver and kidney of the rat treated with high fat diet and streptozotocin

High fat diet or insulin deficiency is commonly seen in Type II diabetes, while the mechanism remains unclear. To test our hypothesis that DPP-IV contributes to Type II diabetes, we examined the expression and activity of DPP-IV in rats (n=8 to each group) treated for 12 weeks with 3 separate diets: a) normal control; b) a high fat diet; and c) a high fat diet plus streptozotocin, a chemical for induction of insulin-deficient diabetes. Compared to rats on the normal diet, the rats with a high fat diet significantly increased DPP-IV's expression and activity about 142-152% in the intestine (P<0.05) and 153-240% in kidneys (P<0.05), but there was no change in the liver. Administration of streptozotocin to the rats treated with the high fat diet showed an insufficient insulin secretion and higher blood glucose in response to glucose/insulin tolerance test, and an increase in expression of DPP-IV and activity by 188-242% in the intestine (P<0.01); 191-225% in liver (P<0.01); and 211-321% in the kidneys (P<0.01). Immunohistochemistry studies confirmed the above results, showing increased DPP-IV immunostaining localized primarily in intestinal epithelium, hepatocytes and renal tubular cells. This study, for the first time reports an increase in DPP-IV associated with a high fat diet, as well as in the combination of a high fat diet with an insulin deficiency. Since both high fat diet and insulin deficiency are closely linked with etiology of Type II diabetes, the evidence in this study suggests a role of DPP-IV in development of Type II diabetes.     

Blood coagulation and fibrinolysis of rats fed fish oil: reduced coagulation factors especially involved in intrinsic pathway and increased activity of plasminogen activator inhibitor

Differences in the coagulation and fibrinolytic system of rats fed a fish oil based diet (fish oil diet) and fed a soybean oil based diet (control diet) were determined. Concentrations of plasma lipids were depressed in rats fed the fish oil diet. Prothrombin time (PT) and activated partial thromboplastin time (APTT) of rats fed the fish oil diet were longer than for the rats fed the control diet. Fish oil intake lowered the activities of most of the blood coagulation factors, and strongly depressed the factors involved in the intrinsic pathway. Fish oil also affected the fibrinolysis of rats. Plasminogen activator inhibitor (PAI) activity was elevated in rats fed the fish oil diet. In this study, both blood coagulation and fibrinolysis were down-regulated by feeding the fish oil diet.     

Prevalence of H1299R polymorphism in the Factor V gene among the Taif-Saudi Arabia population using polymerase chain reaction-reverse hybridization technique

Cardiovascular disease (CVD) remains a major health hazard worldwide. Single nucleotide polymorphisms (SNPs) represent a part of risk factors that contributes to cardiovascular disease. SNP in the coagulation factor V genes have been shown to play a role in the development of cardiovascular disease. Coagulation Factor V is an enzyme cofactor of the coagulation system and contributes to a normal haemostatic balance. The His1299Arg polymorphism in the Factor V gene has been identified and linked to hereditary thrombophilia. The aim of the present study is to determine the prevalence of HR2 haplotype and allele frequency of His1299Arg polymorphism in the Factor V gene among randomly selected healthy individuals from Taif population which belonging to western region of Saudi Arabia. Genotyping of this SNP was carried out via CVD StripAssay, which based on a polymerase chain reaction-reverse hybridization technique. Two hundred healthy unrelated individuals were included in the study. Seventeen out of the studied population (8.5%) had the HR2 haplotype; 14 (7%) were heterozygous (R1/R2), and three (1.5%) were homozygous (R2/R2), with an allelic frequency of 0.05. This is the first report for a Saudi Arabian population that estimates the prevalence of HR2 haplotype and its allele frequencies. In conclusion, the His1299Arg mutant was noticeable within population of western Saudi Arabia. Further larger studies are needed to (1) estimate the prevalence of this mutant among individuals belonging to different KSA locations (2) assess the relative contribution of this mutational event separately and in combination with other thrombophilic polymorphisms in the etiology of cardiovascular disease in KSA.     

Cyclic peptide analogs of 558–565 epitope of A2 subunit of Factor VIII prolong aPTT. Toward a novel synthesis of anticoagulants

Novel anticoagulant therapies target specific clotting factors in blood coagulation cascade. Inhibition of the blood coagulation through Factor VIII–Factor IX interaction represents an attractive approach for the treatment and prevention of diseases caused by thrombosis. Our research efforts are continued by the synthesis and biological evaluation of cyclic, head to tail peptides, analogs of the 558–565 sequence of the A2 subunit of FVIII, aiming at the efficient inhibition of Factor VIIIa–Factor IXa interaction. The analogs were synthesized on solid phase using the acid labile 2-chlorotrityl chloride resin, while their anticoagulant activities were examined in vitro by monitoring activated partial thromboplastin time and the inhibition of Factor VIII activity. The results reveal that these peptides provide bases for the development of new anticoagulant agents.