Vesna Subota

Effects of subacute oral warfarin administration on peripheral blood granulocytes in rats.

Warfarin affects mainly vitamin K dependent (VKD) processes, but the effects on some non-VKD-related activities such as tumor growth inhibition and mononuclear cell-mediated immune reactions were shown as well. In this study, the effect of subchronic (30 days) oral warfarin (0.35 mg/l and 3.5mg/l) intake on peripheral blood granulocytes in rats was investigated. Increase in prothrombin and partial thromboplastin time at high warfarin dose reflected its basic activity. Priming effect for respiratory burst was noted at both warfarin doses, while only high warfarin dose resulted in priming for adhesion, the rise in intracellular myeloperoxidase content/release and stimulation of nitric oxide production. Differential effects of high warfarin dose were noted on granulocyte cytokines IL-6 (lack of the effect), TNF-α (decreased release and mRNA expression) and IL-12 (increase in mRNA for IL-12 subunits p35 and p40). Changes in granulocytes seems not to rely on mitogen activated kinases p38 and ERK. Warfarin intake was associated with an increase in circulating IL-6, fibrinogen and haptoglobin and with changes in the activity of erythrocyte antioxidant enzymes superoxide dismutase and catalase. The effects of oral warfarin intake on peripheral blood granulocytes demonstrated in this study might be relevant for oral anticoagulant therapy strategies in humans.

Elevations in soluble CD40 ligand in patients with high platelet aggregability undergoing percutaneous coronary intervention.

High aggregatory responses despite antiplatelet treatment is associated with an increased risk of thrombotic complications following percutaneous coronary intervention (PCI). In the present study, we investigated the relationship between platelet aggregatory responses to ADP and the release of CD40L (sCD40L): an immunomodulatory compound involved in atherothrombosis – in patients undergoing PCI. ADP-induced platelet aggregation, sCD40L and soluble P-selectin (sP-selectin) were determined before and 24 h after PCI, in samples from 52 patients receiving aspirin and thienopyridines. Platelet aggregation to ADP above the median was defined as ‘high aggregation’, and aggregation below the median as ‘low aggregation’. Data below are medians and interquartile ranges. Patients with ‘high platelet aggregability’ had a significantly higher increase in both sCD40L (Δ-values: 0.78 (−0.19–3.18) vs. −0.65 (−2.10–0.00) ng/ml, P = 0.002) and sP-selectin (Δ-values: 8.0 (−2.00–16.00) vs. 4.50 (−13.00–0.50) ng/ml, P = 0.001) compared with patients with ‘low platelet aggregability’. In a multivariate linear regression analysis adjusted for clinical characteristics and type of preintervention therapy, the only independent predictors of sCD40L and sP-selectin were platelet aggregation to ADP before PCI (P < 0.001) and the combination of platelet aggregation to ADP before PCI and urgency of PCI (P < 0.001). Circulating CD40L is more markedly increased after PCI in patients with high ADP-induced platelet aggregation.

Oral warfarin affects peripheral blood leukocyte IL-6 and TNFα production in rats

Warfarin is a Vitamin K (VK) antagonist that affects Vitamin K-dependent (VKD) processes, including blood coagulation, as well as processes unrelated to hemostasis such as bone growth, calcification, and growth of some cell types. In addition, warfarin exerts influence on some non-VKD-related activities, including anti-tumor and immunomodulating activity. With respect to the latter, both immune stimulating and suppressive effects have been noted in different experimental systems. To explore the in vivo immunomodulatory potential of warfarin on one type of activity (i.e., cytokine production) in two different immune cell populations (i.e., mononuclear or polymorphonuclear cells), effects of subchronic oral warfarin intake in rats on pro-inflammatory cytokine (i.e., TNFα, IL-6) production by peripheral blood mononuclear and polymorphonuclear cells (granulocytes) was examined. Differential effects of warfarin intake on TNFα and IL-6 were noted, depending on the type of peripheral blood leukocytes and on the cytokine examined. Specifically, a lack of effect on TNFα and a priming of IL-6 production by mononuclear cells along with a decrease in TNFα and a lack of effect on IL-6 in polymorphonuclear cells were seen in warfarin-exposed hosts. The cell- and cytokine-dependent effects from subchronic oral warfarin intake on peripheral blood leukocytes demonstrated in this study could, possibly, differentially affect reactions mediated by these cells. Ultimately, the observed effects in rats might have implications for those humans who are on long-term/prolonged warfarin therapy.

Percutaneous Toxicity of Anticoagulant Warfarin in Rats

Percutaneous toxicity of anticoagulant rodenticides is usually manifested by coagulopathy and/or fatal outcome. There are, however, virtually no data on other biological effects of this class of pesticides that gain access into the organism via skin. In this study, percutaneous toxicity of epicutaneously applied warfarin was evaluated by measuring changes in peripheral blood granulocytes in rats. Application of 10 μg (0.05 mg/kg) or 100 μg (0.5 mg/kg) of warfarin (WF) for 3 consecutive days resulted in an increase in prothrombin time, documenting the access of warfarin to systemic circulation. Application of warfarin led to an increase in relative numbers of granulocytes at higher dose, whereas both doses resulted in increased metabolical viability, evaluated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay. Higher warfarin dose resulted in both granulocyte activation and priming (evaluated by cytochemical nitroblue tetrazolium, NBT, reduction assay of respiratory burst), whereas only a tendency toward activation was noted at lower WF dose. Soluble mediators from the circulation seem responsible for the observed effects, as exogenous plasma from WF-treated animals stimulated NBT reduction by isologous or naïve granulocytes. Data presented in this study are relevant for the recognition of biological effects, other than those affecting hemostasis, of anticoagulant rodenticides that gain access to systemic circulation through the skin.

Coexistence of hypofibrinogenemia and factor V Leiden mutation: is the balance shifted to thrombosis?

Congenital hypofibrinogenemia and afibrinogenemia are usually associated with an increased risk of bleeding, but occurrence of arterial or venous thrombosis has also been reported in individuals with fibrinogen deficiency. This study reports on a 25-year-old patient with hypofibrinogenemia (fibrinogen 0.6 g/l) and congenital thrombophilia due to heterozygous factor V Leiden mutation who developed spontaneous deep-vein thrombosis (DVT) in the right lower extremity. Regardless of hypofibrinogenemia, he was receiving anticoagulant therapy over 6 months, with no occurrence of bleeding. His father is also a heterozygous carrier of factor V Leiden, but with normal fibrinogen level and he remained asymptomatic despite having experienced surgery in the past. This case, as well as data from literature, suggests that risk of thrombosis in carriers of factor V Leiden mutation is not counterbalanced by moderate congenital hypofibrinogenemia, and that antithrombotic prophylaxis should not be omitted in high-risk situations for occurrence of thrombosis in patients with coinheritance of hypofibrinogenemia and factor V Leiden mutation.

Urinary Cystatin C as a Marker of Tubular Dysfunction

Urinary Cystatin C as a Marker of Tubular Dysfunction Cystatin C (CysC) is a nonglycosylated 13 KD protein that belongs to the type II cystatin gene family. It is a strong inhibitor of cysteine proteinases, freely filtered by the kidney glomerulus and reabsorbed by the tubulus, where it is almost totally catabolized. Remainder of the nonmetabolized CysC is eliminated in urine and may represent a useful marker of tubular dysfunction. The aim of the study was to confirm the clinical importance of the quantitative determination of CysC by an automated immunonephelometric method (DADE Behring). Two groups of patients were examined: one with glomerular (GD, n=36) and one with tubular dysfunction (TD, n=31), and compared with the control group (CG, n=31) of healthy males and females from laboratory personnel (n=11) and patients on routine systematic examination (n=20), from 25 to 58 years old. The patient groups were categorised according to the urine analysis of total proteins, creatinine and adequate proteins electrophoretic panel. CysC concentration in CG was in the range of 0.02-0.15 mg/L; 0.01-0.48 mg/L and 0.25-18 mg/L in GD and TD groups respectively. Values of means ± SD for patient groups (GD=0.11 ± 0.14; TD=3.92 ± 3.75 mg/L) showed statistical significance (p<0.001) in the TD group in relation to GD and CG groups. It confirms that quantitative determination of CysC in one urine portion, with a fast laboratory method, might be a useful marker of tubular dysfunction, especially in emergency situations, taking into account that there is no interference of circadian variation on its concentration. Cistatin C U Urinu Kao Pokazatelj Tubulskog Oštećenja Cistatin C (CysC) je neglikozirajući protein molekulske mase od 13 KD koji pripada tipu II genske familije cistatina. Ima funkciju inhibitora cistein proteinaze koji se potpuno filtrira u glomerulima a zatim reapsorbuje i kataboliše u proksimalnim tubulima. Preostali i nerazgrađen CysC biva eliminisan urinom pa zato može da bude koristan marker tubulskih oštećenja. Cilj ispitivanja bio je da se utvrdi klinički značaj kvantitativnog određivanja CysC automatizovanom imunonefelometrijskom metodom (DADE Behring). Ispitane su dve grupe bolesnika: sa tubulskom (TI) i glomerulskom (GI) insuficijencijom, i upoređene sa kontrolnom grupom (KG) koju su činile zdrave osobe oba pola (n=31), laboratorijski personal (n=11) i pacijenti na sistematskom pregledu (n = 20), starosne dobi od 25 do 58 godina. Kategorizacija grupa sa GI (n=36) i TI (n=31) ustanovljena je na osnovu koncentracije ukupnih proteina, kreatinina i adekvatnih elektroforetskih denzitometrijskih varijanti karakterističnih za obe grupe. Koncentracija CysC u KG bila je u opsegu 0,02-0,15 mg/L, u grupi sa GI 0,01-0,48 mg/L, a u grupi sa TI 0,25-18 mg/L. Rezultati vrednosti (medijana±SD) za grupu sa GI (0,11 ± 0,14 mg/L) i grupu sa TI (3,92 ± 3,75 mg/L) pokazali su statistički značajnu razliku (p< 0,0001) između grupe sa TI i grupa sa GI i KG. Pokazano je da određivanje CysC u jednom uzorku urina brzim laboratorijskim postupkom može biti koristan test za diferencijalnu dijagnostiku tubulskih u odnosu na glomerulska oštećenja, posebno u urgentnim stanjima, s obzirom na činjenicu da na njegovu koncentraciju nema uticaja cirkadijalnih ritmova.

Transdermal toxicity of topically applied anticoagulant rodenticide warfarin in rats.

Occupational/accidental exposure data have showed hemorrhage as a result of transdermal exposure to warfarin, however, other effects are not known. In the present study, the impact of epicutaneous application of 10 μg or 100 μg of warfarin (three times, once a day) on peripheral blood polymorphonuclear (PMN) and mononuclear cells (PBMC) was examined in rats. Both doses resulted in prolongation of prothrombin time and changes in hematologic parameters. Increases in PMN intracellular myeloperoxidase (MPO) activity were seen at higher warfarin dose and both doses resulted in higher percentages of granular CD11b(+) cells. In contrast, a decrease in PMN TNF and IL-6 production (ELISA) and gene expression (RT-PCR) was observed. Epicutaneous application of warfarin resulted in decreased numbers of PBMC, higher numbers of mononuclear CD11b(+) cells, but without effect on PMBC cytokine production. The data obtained showed differential effects of transdermal exposure to warfarin depending on leukocyte type and activity.

Subclinical Atherosclerosis in Patients with Rheumatoid Arthritis and Low Cardiovascular Risk: The Role of von Willebrand Factor Activity

Background To evaluate association between von Willebrand factor (vWF) activity, inflammation markers, disease activity, and subclinical atherosclerosis in patients with rheumatoid arthritis (RA) and low cardiovascular risk. Methods Above mentioned parameters were determined in blood samples of 74 non-diabetic, normotensive, female subjects, with no dyslipidemia(42 patients, 32 matched healthy controls, age 45.3±10.0 vs. 45.2±9.8 years). Intima-media thickness (IMT) was measured bilaterally, at common carotid, bifurcation, and internal carotid arteries. Subclinical atherosclerosis was defined as IMT>IMTmean+2SD in controlsat each carotid level and atherosclerotic plaque as IMT>1.5 mm. Majority of RA patients were on methotrexate (83.3%), none on steroids >10 mg/day or biologic drugs. All findings were analysed in the entire study population and in RA group separately. Results RA patients with subclinical atherosclerosis had higher vWF activity than those without (133.5±69.3% vs. 95.3±36.8%, p<0.05). Predictive value of vWF activity for subclinical atherosclerosis was confirmed by logistic regression. vWF activity correlated significantly with erythrocyte sedimentation rate, fibrinogen, modified disease activity scores (mDAS28–ESR, mDAS28–CRP), modified Health Assessment Questionnaire (p<0.01 for all), duration of smoking, number of cigarettes/day, rheumatoid factor concentration (p<0.05 for all), and anti-CCP antibodies (p<0.01). In the entire study population, vWF activity was higher in participants with subclinical atherosclerosis (130±68% vs. 97±38%, p<0.05) or atherosclerotic plaques (123±57% vs. 99±45%, p<0.05) than in those without. Duration of smoking was significantly associated with vWF activity (β 0.026, p = 0.039). Conclusions We demonstrated association of vWF activity and subclinical atherosclerosis in low-risk RA patients as well as its correlation with inflammation markers, all parameters of disease activity, and seropositivity. Therefore, vWF might be a valuable marker of early atherosclerosis in RA patients.

Effects of warfarin on biological processes other than haemostasis: A review

Warfarin is the worlds most widely used anticoagulant drug. Its anticoagulant activity is based on the inhibition of the vitamin K-dependent (VKD) step in the complete synthesis of a number of blood coagulation factors that are required for normal blood coagulation. Warfarin also affects synthesis of VKD proteins not related to haemostasis including those involved in bone growth and vascular calcification. Antithrombotic activity of warfarin is considered responsible for some aspects of its anti-tumour activity of warfarin. Some aspects of activities against tumours seem not to be related to haemostasis and included effects of warfarin on non-haemostatic VKD proteins as well as those not related to VKD proteins. Inflammatory/immunomodulatory effects of warfarin indicate much broader potential of action of this drug both in physiological and pathological processes. This review provides an overview of the published data dealing with the effects of warfarin on biological processes other than haemostasis.

Oral Warfarin Affects Some Aspects of Systemic Immunomodulation with Topical Dinitrochlorobenzene (DNCB) in Rats

Abstract Purpose: The efficacy of topical dinitrochlorobenzene (DNCB) in the treatment of some skin dermatoses is based both on local and systemic effects. It is not known, however, whether it can be applied to patients receiving some other therapy associated with systemic immunomodulation. The aim of the present paper using a rat model was to examine whether oral warfarin (WF) intake, as shown by others and by us, had an immunomodulatory potential to interfere with effects of topical DNCB as systemic immunotherapy. Materials and methods: Rats received 3.5 mg/l of WF sodium in drinking water for 30 days and were thereafter skin-sensitized with 0.4% DNCB. Changes in the oxidative activity (myeloperoxidase/MPO, reduction of nitroblue tetrazolium/NBT and nitric oxide/NO production) as well as tumor necrosis factor (TNF) production by peripheral blood polymorphonuclear cells (PMN) were measured and compared with PMN from sensitized unexposed to WF rats. Results: WF intake enhanced some aspects of PMN activity (intracellular MPO activity and unstimulated NO production) as well as their responsiveness to exogenous stimulation (NBT reduction and TNF production from sensitized animals). However, WF also decreased PMN responsiveness of NO production to stimulation. WF affected NO and TNF production solely by PMN, as no effect on these activities of peripheral blood mononuclear cells was seen. Conclusion: Having in mind that polymorphonuclear leukocytes are the most abundant cell type in peripheral blood in humans, increase of basic aspects of PMN activity described in the present paper might be relevant for consideration of using WF as therapeutic modality in patients topically treated with DNCB.