Factor X activating enzyme from Russell''s viper venom: isolation and characterization

The protease from Russell's viper venom that activates factor X (Stuart factor), factor IX (Christmas factor), and protein C was purified by gel filtration on Sephadex G-150 and QAE-Sephadex A-50 column chromatography. The purified enzyme migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 79 000. A minimal molecular weight of 78 500 +/- 800 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. Upon reduction with 2-mercaptoethanol, a heavy chain (mol wt 59 000) and a light chain were observed. The light chain migrated as a single band (mol wt 19 000) in 7.5% polyacrylamide-sodium dodecyl sulfate gels but appeared as a doublet (mol wt 18 000 and 20 000) in 10% polyacrylamide-sodium dodecyl sulfate gels. The amino-terminal end of the heavy chain was heterogeneous and contained isoleucine, valine and serine. The amino-terminal sequence of the light chain was Val-Leu-Asp. The factor X activator contained 13% carbohydrate including 6.0% hexose, 1.7% N-acetyleneuraminic acid, and 5.3% galactosamine. Most of the carbohydrate was found to be present in the heavy chain, although some was also observed in both forms of the light chain. The factor X activator had no esterase activity toward benzoyl-Phe-Val-Arg-p-nitroanilide or benzoylarginine ethyl ester and was not inhibited by 0.05 M diisopropyl phosphorofluoridate. These data indicate that factor X activator from Russell's viper venom is a highly specific protease composed of one heavy chain and one light chain, and these chains are held together by a disulfide bond(s).     

Effects of wine on plasma fibrinolytic and coagulation systems

Energy expenditure was estimated using the doubly-labelled water (DLW) method in summer in five free-living adult, non-pregnant, non-lactating, red deer (Cervus elaphus) hinds (weight 107.3 (SE 0.9) kg; age 6 (SE 1) years) on lowland pasture under typical farming conditions. Climatic conditions were monitored throughout the experiment. Errors due to 2H losses in CH4 and faeces were calculated from previous estimates of stoichiometries. CH4 production, fractionated water loss, urinary N and O2 consumption were estimated using an iterative approach. The water flux (rH2O) in these animals consuming only fresh grass was 12 (SE 0.5) kg/d, the CO2 production (rCO2) was 1271 (SE 40) litres/d and the mean energy expenditure was 25 (SE 0.8) MJ/d. There were no significant differences in the isotope distribution spaces and flux rates, rH2O, rCO2 or energy expenditure using the multi-point or two-point approaches to calculation. The DLW-derived energy expenditure of 25 MJ/d is approximately 20% higher than the recommended intake of 21 MJ/d for adult hinds kept outdoors (Adam, 1986) and, at 757 kJ/kg0.75 per d, one third higher than the value of 570 kJ/kg0.75 per d for stags penned indoors (Key et al. 1984).     

Effects of eleutherococcus, elton, leuzea, and leveton on the blood coagulation system during training in athletes

Three hermetics are assessed: chemically hardened Delton, light-hardened Estiseal, and composite Evikrol. The study was carried out in 126 children aged 6 years. The decrease of the increment of dental caries depends on the retention of hermetics on the occlusion surface of the teeth, and the efficacy of caries prevention in permanent teeth is much higher if hermetic sealing of fissures and fossae is combined with local fluorine prophylaxis and oral hygiene. All types of hermetics can be used to prevent permanent teeth caries, but chemically hardened ones should be preferred.     

Absorption and elimination of viper venom after antivenom administration

The mechanisms by which antivenom neutralizes the venom are still poorly understood. In the present work, we studied the effects of antivenom, constituted with either F(ab')2 or Fab, on the processes of absorption and elimination of Vipera aspis venom in experimentally envenomed rabbits. We first concluded from this study that during the few hours after intramuscular injection, the venom rapidly disappeared from the site of injection but did not immediately reach the vascular system, suggesting that it is partly absorbed via the lymphatic circulation. Concerning the elimination process of the venom in the presence of antivenom, we observed that the elimination of F(ab')2/venom complexes is slower than that of free venom in the absence of antivenom but faster than that of free F(ab')2, suggesting that F(ab')2/venom complexes are eliminated by phagocytosis. The Fab/venom complexes, on the other hand, are eliminated more slowly than free Fab. These complexes are not eliminated through the renal route in agreement with their high molecular weight. In addition, we observed that the treatment of envenomed rabbits with antivenom made of Fab, but not F(ab')2, is responsible for an oliguria that could be responsible for clinical problems.     

An activator of blood coagulation factor X from the venom of Bungarus fasciatus

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mol. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.     

An investigation on the changes of coagulation and fibrinolytic system in intensive infection with multiple organs failure

An investigation was made for the significance of changes of coagulation and fibronolytic system in intensive infection with multiple system organ failure (MSOF). In 68 cases with various degrees of infection, hematological examinations, including estimation of PT, APTT, Fg, Fn and D-Dimer, activation of coagulation factor II, VII, X, XII (F II, F VII, F X, F XII), AT-III, PLG, alpha(2)-AP, t-PA and count of platelets were carried out. The results were as follows: In intensive infection with MSOF, PT and APTT increased significantly; activity of F II, F VII, F X and F XII decreased significantly platelet count and Fn decreased markedly; concentration of Fg and D-Dimer increased significantly; activity of PAI increased markedly; activity of t-PA and alpha(2)-AP decreased slightly. The incidence of MSOF not combined with DIC was 38.5%, but that combined with DIC was 79.7% (P < 0.01). It is suggested that DIC is the most important factor in the disorder of coagulation and fibrinolytic system. It play an important role in the pathogenesis and development of MSOF.     

Cerastocytin, a new thrombin-like platelet activator from the venom of the Tunisian viper Cerastes cerastes

Cerastocytin, a thrombin-like enzyme from the venom of the desert viper, Cerastes cerastes, has been purified to homogeneity by fast performance liquid chromatography (FPLC) on Mono-Q and Mono-S columns. It is a basic protein (isoelectric point higher than 9) made of a single polypeptide chain of 38 kDa. Its N-terminal polypeptide sequence shows strong similarities with other thrombin-like enzymes from snake venoms. Nanomolar concentrations of cerastocytin induce aggregation of blood platelets. This activity is inhibited by chlorpromazine, theophylline and mepacrine, as in the case of platelet aggregation stimulated by low doses of thrombin. Cerastocytin also possesses an amidolytic activity measured with the thrombin chromogenic substrate S-2238. The platelet aggregating activity and the amidolytic activity of cerastocytin were inhibited by PMSF, TPCK, TLCK and soybean trypsin inhibitors, suggesting that cerastocytin is a serine proteinase. On the other hand, both amidolytic activity and platelet aggregating activity of cerastocytin were unaffected by hirudin or by antithrombin III in the presence of heparin. High concentrations of cerastocytin (1-10 microM) also cleaved prothrombin and Factor X.     

Pathophysiological response of the blood coagulation system in acute glomerulonephritis

1. The effects of clonidine on blood pressure, cerebral norepinephrine content and vascular structures of the kidneys were investigated in 21 SHR. Although the body weight was not affected by long term clonidine treatment up to 36 weeks, the syatolic blood pressure was significantly reduced. The reduction of the blood pressure was already obvious after 1 week administration of clonidine but the effect was more prominent after long term treatment of 30 weeks or longer. 2. The cerebral norepinephrine content was significantly lower in SHR, regardless of with or without clonidine treatment, than in the control Wistar rats. Although the cerebral norepinephrine content was slightly increased following clonidine treatment SHR, the increase was not statistically significant. 3. Angiographic study of the kidneys revealed a poor opacification of the blood vessels and glomeruli in SHR compared with the control Wistar rats. There was no difference in the sizes of the arcuate and interlobular arteries in SHR and the control Wistar rats, although the medial muscular hypertrophy of the arteries was slightly more prominent in the SHR histologically. The more prominent in the SHR histologically. The angiographic and histologic findings of the renal arteries were not altered following long term clonidine treatment. A possibility was considered that the renal arterioles are mainly functionally affected in SHR.     

Factor IX of the blood coagulation system: a review

Factor IX is a factor of the blood coagulation system. Its activation occurs on the surface of phospholipid membranes. It can be activated by the factor VIIa-TF (tissue factor)-Ca2+ complex via an extrinsic pathway and by factor XIa in the presence of Ca2+ via the intrinsic pathway of blood coagulation system activation. The activated factor IXa is a serine proteinase. The main function of the activated factor IXa in complex with factor VIIIa and phospholipids in presence of Ca2+ consists of the activation of factor X. Factor IX is synthesized in the liver and is subject to a number of posttranslational modifications including gamma-carboxylation, beta-hydroxylation, and glycosylation. It forms a subgroup of vitamin K-dependent plasma proteins including factors VII and X and protein C characterized by identical domain structures having high levels of homology. Factor IX consists of an NH2-terminal Gla domain, two epidermal growth factor (EGF)-like domains, and a C-terminal domain containing Ser in its active site. Factor IX deficiency in human plasma results in the disease known as hemophilia B.     

Use of snake venom fractions in the coagulation laboratory

The G1 cyclin, cyclin D1, has been implicated in the development of human and mouse tumors. Here we describe immunohistochemical analyses of cyclin D1 for a large panel of mouse B cell tumors. In addition, we characterize cyclin D1 expression in a series of cultured cell lines that represent transformed B cells at different stages of development. Immunohistochemical analysis showed that for low-grade lymphomas, cyclin D1 was expressed by 83% of centroblastic centrocytic (CBCC) and 14% of small lymphocytic lymphomas (SLL). For high-grade tumors, 28% of B lymphoblastic and 23% of centroblastic tumors expressed cyclin D1, while all immunoblastic lymphomas were negative. Studies of RNA and protein prepared from cultured B lineage tumors showed that cyclin D1 was expressed by all pre-B and most B cell tumors but not by cell lines representative of late B cell differentiation or by plasma cells. Expression of cyclin D1 in the lymphomas was not associated with alterations in the genomic structure of the Fis-1 (Bcl-1) common proviral integration site or cyclin D1 itself or with cell growth activity as assessed by expression of proliferating cell nuclear antigen (PCNA).     

Effects of divalent cations on calcium levels, aggregation and morphology of human blood platelets in vitro

Some effects of cadmium, nickel, strontium and manganese ions upon human platelets in vitro are reported. Both Mn and Cd induced aggregation, Mn being the most effective. Ni and Sr did not have this effect. Scanning electron microscopic observation showed that Mn and Cd, but not Ni and Sr induced shape changes in the platelets. X-ray microanalysis of pyroantimonate fixed platelets revealed that calcium was displaced from both membrane and cytoplasmic regions by all four cations. The magnitude of displacement occurred in the sequence Mn > Cd > Ni approximately or equal to Sr. Manganese was detected both in the cytoplasm and membrane region of those platelets treated with this ion. It is suggested that the cations are influencing directly certain macromolecular ligands whose condition controls platelet surface properties.     

Effects of mazindol and d-fenfluramine of 5-hydroxytryptamine uptake, storage and metabolism in blood platelets

Mazindol induced a dose-related inhibition of the uptake of labelled 5-hydroxytryptamine (5-HT) by guinea pig blood platelets. It was more potent than d-fenfluramine. Mazindol and d-fenfluramine decreased 5-hydroxy-indoleacetic acid formation in intact platelets but not in sonicated ones. The inhibitory effects of both drugs appeared to the competitive in nature and were markedly reduced in platelets suspended in plasma instead of in Tyrode solution. Mazindol neither decreased the stored endogenous 5-HT nor caused efflux of the labelled amine from preloaded platelets, whereas d-fenfluramine induced a significant release of the amine. It is concluded that mazindol, like d-fenfluramine, competes with 5-HT for the same transport mechanisms at the cytoplasmic membrane level but this effect is not accompanied, as is the case with d-fenfluramine, by a concomitant release of the amine.     

Effect of para-aminobenzoic acid on the fibrinolytic activity of blood

In an infant with transient neonatal diabetes mellitus, control of the blood glucose concentration was attained with ultralente insulin treatment, without any episodes of hypoglycemia. We recommend subcutaneous injection of ultralente insulin, rather than lente or isophane (NPH) insulin, to avoid hypoglycemia during the treatment of transient neonatal diabetes mellitus.     

Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases

It has been well documented that drug-associated cues are important for the development and expression of drug tolerance. The Pavlovian conditioning analysis of tolerance emphasizes the importance of a drug-associated cues to tolerance by equating a drug administration with a learning trial. According to this analysis, tolerance should be subject to external inhibition, the disruption of a conditional response by a novel stimulus. We previously reported that tolerance to the ataxic effect of ethanol was attenuated by a novel strobe/noise presentation (31). In this article we report evidence of a compensatory CR in rats tolerant to the ataxic effect of ethanol as tested on the tilting plane. Both the compensatory CR and tolerance were disrupted by the presentation of a novel strobe/noise stimulus providing converging evidence that the attenuation of tolerance by a novel stimulus results from external inhibition of Pavlovian conditioning. The disruption of ethanol tolerance and the conditional response mediating tolerance was also apparent when the novel omission of the strobe/noise stimulus was used as the external inhibitor in rats made tolerant to ethanol with the stimulus on. Finally, we have shown that the disruptive effect of a novel stimulus on ethanol tolerance is decreased when there is a 10-day delay between the final tolerance development session and testing, demonstrating that the interval between training and testing is important when assessing associative tolerance.     

Effects of interleukin 6 administration on platelets and haemopoietic progenitor cells in peripheral blood

Platelet numbers and circulating haemopoietic progenitor cells were examined in 12 patients with advanced malignancies who were receiving recombinant human interleukin-6 (rhIL-6) as part of an investigation of its thrombopoietic effects. Patients received recombinant glycosylated IL-6 by daily subcutaneous injection for 7 consecutive days in doses of 1, 3 or 10 micrograms/kg/day. Platelet numbers increased reaching a peak on days 12-15 with a mean on day 15 of 198.1% of pre-treatment values. This was accompanied by a significant fall in the mean platelet volume (mean decrease of 10.6%, P = 0.0044). No significant correlation was seen between the IL-6 dose and the change in platelet number. No significant differences were observed between pre- and post-treatment levels of circulating erythroid burst-forming units (E-BFU) and granulocyte macrophage colony-forming units (GM-CFU) but a small significant increase was seen in circulating primitive progenitor cells measured in a plastic-adherent (P delta) assay (P = 0.025). As positive controls, a group of patients treated with cyclophosphamide/G-CSF showed significant increases in GM-CFU (P = 0.018), E-BFU (P = 0.018) and P delta progenitors (P = 0.028). These data suggest that the thrombopoietic effects of IL-6 are mediated at a relatively late stage via effects on megakaryocyte differentiation, with a relatively small effect on circulating haemopoietic progenitors.     

Neutralizing capacity of ++antiophidic sera against the venom of Bothrops moojeni (lance-headed viper)

Human oncostatin M (OM) is a M(r) 28,000 glycoprotein that has been shown to regulate cell proliferation and differentiation. The biological activities of OM can be mediated by two different heterodimeric receptor complexes, the leukemia inhibitory factor (LIF)/OM shared receptor and the OM-specific receptor. In this study, we have examined the growth-regulatory effect of OM on 10 breast cancer cell lines derived from human tumors. The cellular proliferation of seven of these breast cancer cell lines was inhibited by OM. The three cell lines that did not respond to OM treatment lacked the expression of OM receptors. The growth-inhibitory activity of OM is examined further in the H3922 breast cancer cell line, which expresses the high-affinity OM receptor at a relatively higher level. We found that the cellular proliferation of H3922 cells was induced strongly by extrogenous epidermal growth factor (EGF), EGF-like factor, and basic fibroblast growth factor. The proliferative activities of these growth factors can be abolished totally by cotreatment of H3922 cells with OM. Treatment of H3922 cells with OM for 24 h did not block EGF binding or the induction of EGF receptor tyrosine phosphorylation. This finding suggests that OM interferes with the mitogenic signal at steps distal to the EGF receptor. Examination of proto-oncogene expression demonstrated that OM down-regulates the c-myc gene in H3922 cells. The biological effects reported herein are not shared by the OM-related cytokines interleukin 6 or LIF, as demonstrated by the inability of these proteins to inhibit cell growth or modulate c-myc gene expression in breast cancer cells. Additionally, the high-affinity binding of labeled OM cannot be displaced by LIF. Together, these data suggest that OM is a growth inhibitor for breast cancer cells. The inhibitory activity is mediated predominantly through the OM-specific receptor, and activation of this receptor abrogates growth factor stimulation and down-regulates the c-myc proto-oncogene.