CYLD enhances severe listeriosis by impairing IL-6/STAT3-dependent fibrin production.

The facultative intracellular bacterium Listeria monocytogenes (Lm) may cause severe infection in humans and livestock. Control of acute listeriosis is primarily dependent on innate immune responses, which are strongly regulated by NF-κB, and tissue protective factors including fibrin. However, molecular pathways connecting NF-κB and fibrin production are poorly described. Here, we investigated whether the deubiquitinating enzyme CYLD, which is an inhibitor of NF-κB-dependent immune responses, regulated these protective host responses in murine listeriosis. Upon high dose systemic infection, all C57BL/6 Cyld(-/-) mice survived, whereas 100% of wildtype mice succumbed due to severe liver pathology with impaired pathogen control and hemorrhage within 6 days. Upon in vitro infection with Lm, CYLD reduced NF-κB-dependent production of reactive oxygen species, interleukin (IL)-6 secretion, and control of bacteria in macrophages. Furthermore, Western blot analyses showed that CYLD impaired STAT3-dependent fibrin production in cultivated hepatocytes. Immunoprecipitation experiments revealed that CYLD interacted with STAT3 in the cytoplasm and strongly reduced K63-ubiquitination of STAT3 in IL-6 stimulated hepatocytes. In addition, CYLD diminished IL-6-induced STAT3 activity by reducing nuclear accumulation of phosphorylated STAT3. In vivo, CYLD also reduced hepatic STAT3 K63-ubiquitination and activation, NF-κB activation, IL-6 and NOX2 mRNA production as well as fibrin production in murine listeriosis. In vivo neutralization of IL-6 by anti-IL-6 antibody, STAT3 by siRNA, and fibrin by warfarin treatment, respectively, demonstrated that IL-6-induced, STAT3-mediated fibrin production significantly contributed to protection in Cyld(-/-) mice. In addition, in vivo Cyld siRNA treatment increased STAT3 phosphorylation, fibrin production, pathogen control and survival of Lm-infected WT mice illustrating that therapeutic inhibition of CYLD augments the protective NF-κB/IL-6/STAT3 pathway and fibrin production.

Iron-induced fibrin in cardiovascular disease.

Accumulating evidence within the last two decades indicates the association between cardiovascular disease (CVD) and chronic inflammatory state. Under normal conditions fibrin clots are gradually degraded by the fibrinolytic enzyme system, so no permanent insoluble deposits remain in the circulation. However, fibrinolytic therapy in coronary and cerebral thrombosis is ineffective unless it is installed within 3-5 hours of the onset. We have shown that trivalent iron (FeIII) initiates a hydroxyl radical-catalyzed conversion of fibrinogen into a fibrin-like polymer (parafibrin) that is remarkably resistant to the proteolytic dissolution and thus promotes its intravascular deposition. Here we suggest that the persistent presence of proteolysis-resistant fibrin clots causes chronic inflammation. We study the effects of certain amphiphilic substances on the iron- and thrombin-induced fibrinogen polymerization visualized using scanning electron microscopy. We argue that the culprit is an excessive accumulation of free iron in blood, known to be associated with CVD. The only way to prevent iron overload is by supplementation with iron chelating agents. However, administration of free radical scavengers as effective protection against persistent presence of fibrin-like deposits should also be investigated to contribute to the prevention of cardiovascular and other degenerative diseases.

[Dependence of the anticoagulant activity of starch and inulin on their degree of sulfonation].

We have studied a relationship between the degree of sulfonation and anticoagulant activity of starch from Solanum tuberosum (molecular weight, 25000-30000 Da; sulfonation degree, 0.4-2.5) and inulin from Helianthus tuberosus (molecular weight, 7000-8000 Da; sulfonation degree, 0.6-1.6). Starch and inulin sulfates (i) increased the time of appearance of fibrin clots in plasma in coagulometric tests and (ii) reduced (via antithrombin) the rate of thrombin-induced hydrolysis of a chromogen substrate. The antithrombin (aIIa) activity of starch sulfates reached 16.8-70.0 IU/mg and the activity against factor Xa (aXa activity) was 2.3-16.6 IU/mg. The antithrombin activity of inulin sulfates was within 5.5-11.4 IU/mg and the activity against factor Xa (aXa activity) was within 0-1.4 IU/mg. An increase in the degree of sulfonation led to a growth in the anticoagulant activity of starch sulfates. The anticoagulant activity of starch sulfates and inulin sulfate with sulfonation degree 1.0 is mediated by antithrombin, which is the plasma inhibitor of serine proteases.

The cardioprotective effects of marrubiin, a diterpenoid found in Leonotis leonurus extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonotis leonurus L. (Lamiaceae) is used as a traditional medicine for a variety of ailments in South Africa. The diterpene marrubiin is the major product constituent in specimens of this plant occurring in South Africa. MATERIALS AND METHODS: Marrubiin isolated from South African specimens of L. leonurus in addition to an organic extract of L. leonurus were tested in vivo, ex vivo and in vitro for their anticoagulant, antiplatelet and anti-inflammatory activities. RESULTS: Marrubiin and the organic extract suppressed coagulation, platelet aggregation and inflammatory markers. For the coagulation markers it was found that the organic extract and marrubiin significantly prolonged activated partial thromboplastin time (APTT). Fibrin and D-dimer formation were drastically decreased. These findings were observed in an ex vivo model and an obese rat model. Chemokines enhance leukocyte recruitment to inflammatory sites. TNF-α and RANTES secretion were significantly reduced by the extract and marrubiin when determined in the obese rat model relative to the controls. Calcium mobilization and TXB(2) synthesis were suppressed by the extract and marrubiin. An in vitro model was used to elucidate the antiplatelet mechanism and it was found that the extract and marrubiin inhibited platelet aggregation by inhibiting the binding of fibrinogen to glycoprotein (GP) IIb/IIIa receptor in a concentration dependent manner. CONCLUSION: The findings reflect that marrubiin largely contributes to the extract's anticoagulant, antiplatelet and anti-inflammatory effects observed.

Changes in tissue factor and activated factor XII following an acute myocardial infarction were uninfluenced by high doses of n-3 polyunsaturated fatty acids.

Few data exist on the effects of n-3 polyunsaturated fatty acids (PUFAs) on the initiators and endstage products of coagulation following an acute myocardial infarction (MI). We assessed the long-term effects of n-3 PUFAs on postinfarct variations of tissue factor (TF), activated factor XII (FXIIa) and fibrin monomer (FM), and expected additional statin treatment to modify thrombogenicity. Acute MI patients (n = 300) were randomly allocated to a high dose of n-3 PUFAs or corn oil for at least one year. Plasma concentrations of TF, FXIIa and FM were unaffected by n-3 PUFAs as compared to corn oil, and were uninfluenced by additional statin treatment in subgroup analyses. TF decreased (p = 0.0001), while FXIIa increased during the first 6 weeks (p = 0.001). FM remained essentially unchanged during the entire observation period. In conclusion, TF, FXIIa and FM were unaffected by long-term treatment with high- dosed n-3 PUFAs and by additional statin treatment.

A screening procedure to evaluate the anticoagulant activity and the kinetic behaviour of direct thrombin inhibitors.

The development of a fibrin clot microassay to define both the kinetic behaviour and the anticoagulant activity of direct thrombin inhibitors targeting various domains of thrombin (catalytic site, anion binding exosite or both) is described. Since classical kinetics studies are difficult to perform in a fibrin-clot assay, methodological conditions were selected in order to obtain a linear relationship between fibrin formation and the thrombin concentration i.e. 0.67 nM thrombin, 6 microM fibrinogen, 5 minutes reaction. Under those conditions, the concentration of the complex thrombin-inhibitor can easily be calculated from a standard curve performed with increasing concentrations of thrombin and fitted versus the total inhibitor concentration using adapted equations. To detect the slow establishment of the thrombin inhibition, results obtained with a protocol in which the inhibitor is pre-incubated with thrombin before the addition of fibrinogen is compared to a protocol in which the inhibitor is pre-incubated with fibrinogen before thrombin is added. Our assay which is validated using different types of thrombin inhibitors (classical competitive: NAPAP and hirudin 55-65; tight binding: r-hirudin; slow tight binding: DUP-714), provides a rapid screening protocol allowing to evaluate the biochemical and anticoagulant properties of any direct thrombin inhibitor.

[Anticoagulants of the nondialyzed fractions of the ammonia extract of the herb Pulmonaria mollissima]. / Antikoagulianty nedializuiushchei fraktsii ammiachnogo ékstrakta travy medunitsy miagchaishei.

T-100 anticoagulants non-dialyzed through cellophane are isolated from ammonia extract of herb Pulmonaria mollissima. Their effect is mainly realized at the stage of coagulation conversions of fibrinogen, first of all at the stage of fibrin self-assembly. One of these anticoagulants is a peptide, another one is a glycopeptide which contains glucose residues like carbohydrate. Peptide components of the both anticoagulants are characterized by the high level of amino acids, their radicals are capable to ionization under physiological conditions. In contrast to animal-origin analogs, glycopeptide in nontoxic doses causes stable hypocoagulemia in animals. It is expedient to study Pulmonaria mollissima extracts as a source of direct anticoagulants.

Effects of low and high dose oral contraceptives on blood coagulation and thrombogenesis induced by vascular subendothelium exposed to flowing human blood.

We investigated the effect of oral contraceptives with low and high estrogen concentration on blood coagulation and thrombogenesis, induced by vascular subendothelium of rabbit aorta exposed to flowing human blood. Twenty healthy women intending to take oral contraceptives were studied [1] before drug ingestion (control), and subsequently during the intake of oral contraceptives with [2] low estrogen content (20 micrograms ethinyl estradiol and 150 micrograms desogestrel per day) and [3] high estrogen content (50 micrograms ethinyl estradiol and 125 micrograms desogestrel per day). All experiments were performed between day 17 and 21 of the menstrual cycle and drug effects were studied during the third tablet cycle. Deposition of fibrin, platelets and platelet thrombi on vascular subendothelium was tested at a defined blood flow and wall shear rate (10 ml/min, 650 s-1) and was quantified by morphometrical techniques. Treatment with the low and high dose contraceptive increased the plasma levels of ethinyl estradiol (728 +/- 139 and 1438 +/- 212 vs. 0 fmol/l [low and high dose vs. control], means +/- SEM, P less than 0.001) and fibrinogen (2.3 +/- 0.1 and 2.6 +/- 0.1 vs. 2.0 +/- 0.1 g/l, P less than 0.05); and decreased antithrombin III activity (95 +/- 3 and 92 +/- 3 vs. 101 +/- 3 %, P less than 0.05). Fibrin deposition on vascular subendothelium was enhanced by the high dose contraceptive only (47 +/- 4 vs. 35 +/- 4 % coverage of the subendothelial surface with fibrin, high dose vs. control, P less than 0.05). The subendothelial deposition of platelets and platelet thrombi was not changed by contraceptive treatment. These results indicate that treatment with high dose contraceptives leads to an increase of fibrin-subendothelial interactions, whereas low dose contraceptives do not significantly alter the blood-subendothelium interactions. observed in this ex vivo model of thrombogenesis.

Current issues in thrombosis prevention with antiplatelet drugs.

In this review, the major current problems related to the pharmacology and clinical use of antiplatelet drugs are discussed in relation to the physiopathology of the platelet-vessel wall interaction and arterial thrombus formation. Although platelet adhesion to injured vessels is a crucial step in thrombogenesis, none of the currently used antiaggregating drugs prevents this phenomenon. Why the normal endothelium does not react with platelets is not known. Thus we are unable to pharmacologically restore endothelial 'non-thrombogenicity' when lost by single or repeated injury. In contrast, more information is available on the mechanisms controlling and amplifying platelet activation by physiological stimuli (such as collagen and thrombin), and on their pharmacological modulation. The 3 main amplification loops involve arachidonic acid metabolism, ADP release and possibly the availability of a phospholipid platelet activating factor. These pathways are in turn activated by the phosphatidylinositol cycle. The most widely used antiaggregating drug is aspirin. It prevents the formation of arachidonic acid metabolites both in platelets and in vascular cells. The use of low-dose aspirin, thromboxane-synthase inhibitors, thromboxane receptor antagonists, epoprostenol (prostacyclin) and its stable analogues, and ticlopidine all appear to be promising pharmacological approaches, but none has so far been tested in clinical trials for thrombosis prevention. On the other hand, aspirin (in relatively large doses of 300 to 1500 mg daily), sulphinpyrazone and dipyridamole have been tested alone or in combination in the secondary prevention of thromboembolic complications. Aspirin has significantly reduced both the occurrence of myocardial infarction and mortality rate in patients with unstable angina and/or previous myocardial infarction; it has also proved beneficial in cerebrovascular disease. The beneficial effect of aspirin was dose-independent. In some of these trials aspirin was combined with either dipyridamole or sulphinpyrazone. When used alone, the latter compound has reduced sudden death or thromboembolic complications in patients with myocardial infarction. It remains to be established whether antiplatelet therapy may prevent or stop the progression of atherosclerosis.

Are lobster fibrinogen and cold-insoluble globulin related molecules?

Several of the molecular properties of lobster fibrinogen and CIg have been compared. There are some significant similarities between these two proteins; however, to date, no direct experiments have demonstrated molecular homologies. Studies involving cyanogen bromide mapping, additional immunologic probing, and modifications of cell morphology effected by lobster fibrinogen are currently in progress. It is anticipated that these and other experimental approaches will provide direct evidence to answer the question, posed by the title of this paper.