Vasiliki D. Papakonstantinou

Re(I) tricarbonyl complex of 1,10-phenanthroline-5,6-dione: DNA binding, cytotoxicity, anti-inflammatory and anti-coagulant effects towards platelet activating factor

The complex fac-[Re(CO)3(phendione)Cl] (1) (where phendione=1,10-phenanthroline-5,6-dione) has been synthesized and fully characterized by UV-visible, FTIR, and NMR techniques. The DNA binding properties of 1 are investigated by UV-spectrophotometric (melting curves), covalent binding assay, CV (cyclic voltammetry), circular dichroism (CD) and viscosity measurements. Experimental data indicate that 1 fits into the major groove without disrupting the helical structure of the B-DNA in contrast to the free phendione which intercalates within the base pairs of DNA. Upon irradiation, complex 1 promotes the cleavage of plasmid pBR322 DNA from supercoiled form I to nicked form II via a proton coupled electron transfer mechanism. This comes as a result of experimental data in anaerobic/aerobic conditions and in the presence of DMSO. The biological activities of 1 and its precursors [Re(CO)5Cl] and phendione are tested towards a series of cancerous cell lines as glioblastoma (T98G), prostate cancer (PC3) and breast cancer (MCF-7) as well as platelet activating factor (PAF)-aggregation. Moreover, all the aforementioned compounds are tested for their ability to modulate PAF-basic metabolic enzyme activities in preparations of rabbit leukolytes. The in vitro experiments indicate that phendione has a better antitumor effect than cisplatin whereas [Re(CO)5Cl] is a better PAF inhibitor than both the phendione ligand and 1. Moreover, for the first time it is indicated that [Re(CO)5Cl], with a IC50 of 17nM is comparable to the widely used PAF receptor antagonists, BN52021 and WEB2170 with IC50 of 30 and 20nM, respectively, whereas 1 affects PAF-catabolism.

Effects of Highly Active Antiretroviral Therapy on Platelet Activating Factor Metabolism in Naïve HIV-Infected Patients: II) Study of the Abacavir/Lamivudine/Efavirenz Haart Regimen

Human Immunodeficiency Virus (HIV)-infected patients are at increased risk for cardiovascular diseases partly due to chronic inflammation. Some antiretroviral drugs and Highly Active Anti-Retroviral Therapy (HAART) regimens seem to be related and amplify this increased risk, especially the ones containing abacavir. Platelet-Activating-Factor (PAF) is a potent inflammatory mediator that is implicated in both cardiovascular diseases and HIV-related manifestations. Our objective is to study the in vivo effect of the abacavir/lamivudine/efavirenz first-line HAART regimen on PAF metabolism in HIV-infected patients. The specific activities of PAF basic biosynthetic enzymes in leukocytes and platelets, PAF-cholinephosphotransferase (PAF-CPT) and lyso-PAF-acetyltransferase (Lyso-PAF-AT), but also those of PAF-basic catabolic enzymes, PAF acetylhydrolase (PAF-AH) in leukocytes and platelets and Lipoprotein-associated-Phospholipase-A2 (LpPLA2) in plasma, were measured in blood samples of 10 asymptomatic naïve male HIV-infected patients just before and after 1, 3 and 6 months of treatment. CD4 cell counts, viral load and several biochemical markers were also measured in the same blood samples of these patients. The repeated ANOVA measures and the Pearson r criterion were used for studying statistical differences and correlations - partial correlations respectively. Even though viral load was decreased and CD4 cell counts were beneficially increased after treatment with the abacavir/lamivudine/efavirenz regimen, the main enzyme of the remodelling PAF-synthesis that is implicated in pro-atherogenic inflammatory procedures, Lyso-PAF-AT activity, was increased at 3 months of treatment in both leukocytes and platelets, while the main enzyme of PAF-degradation, PAF-AH, was increased as a response only in leukocytes at the 3rd month. Although the abacavir/lamivudine/efavirenz HAART regimen exhibits very efficient antiretroviral activities, on the other hand it induces an in vivo transient increase in the inflammation-related remodeling PAF-biosynthetic pathway. This finding supports the hypothesis of inflammation-mediated increased cardiovascular risk in HIV-infected patients during the first months of abacavir-containing HAART.

In vivo effect of two first-line ART regimens on inflammatory mediators in male HIV patients

BackgroundPersistent immune activation and inflammation are lying behind HIV-infection even in the setting of ART mediated viral suppression. The purpose of this study is to define the in vivo effect of two first-line ART regimens on certain inflammatory mediators in male HIV patients.MethodsMale, naive, HIV-infected volunteers were assigned either to tenofovir-DF/emtricitabine/efavirenz (Group_T) or abacavir/lamivudine/efavirenz (Group_A). Platelet Activating Factor (PAF) levels and metabolic enzymes together with HIV-implicated cytokines (IL-1beta, IL-6, IL-8, IL-10, IL-12p70, TNFa) and VEGF were determined for a 12-month period. Differences within each group were determined by non-parametric Friedman and Wilcoxon test, while the differences between the groups were checked by ANOVA repeated measures.ResultsBoth ART regimens present pronounced effect on inflammatory mediators, resulting in decreased PAF levels and Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity for tenofovir-containing regimen and same as baseline PAF levels with a peak though at the 3rd month as well as elevated Lp-PLA2 activity for abacavir-containing regimen.ConclusionsStudies regarding the effect of first-line ART regimens on inflammation may be beneficial in preventing chronic morbidities during HIV-treatment. From this point of view, the present study suggests an anti-inflammatory effect of tenofovir-containing ART, while the temporary increase of PAF levels in abacavir-containing ART may be the link between the reported cardiovascular risk and abacavir administration.

A Review on Platelet Activating Factor Inhibitors: Could a New Class of Potent Metal-Based Anti-Inflammatory Drugs Induce Anticancer Properties?

In this minireview, we refer to recent results as far as the Platelet Activating Factor (PAF) inhibitors are concerned. At first, results of organic compounds (natural and synthetic ones and specific and nonspecific) as inhibitors of PAF are reported. Emphasis is given on recent results about a new class of the so-called metal-based inhibitors of PAF. A small library of 30 metal complexes has been thus created; their anti-inflammatory activity has been further evaluated owing to their inhibitory effect against PAF in washed rabbit platelets (WRPs). In addition, emphasis has also been placed on the identification of preliminary structure-activity relationships for the different classes of metal-based inhibitors.

In vitro effects of vitamin supplements on platelet-activating factor and its metabolism in age-related macular degeneration

Abstract Objective: The purpose of our study was to investigate for the first time a series of vitamin supplements used for age-related macular degeneration (AMD) as potential inhibitors of platelet-activating factor (PAF). Materials and methods: Various vitamin supplements were tested in washed rabbit platelets (WRPs), in order to investigate the interaction between vitamin supplements (InShape, Nutrof, Ocuvite, Vitalux) and inhibition of PAF-induced platelet aggregation. Additionally, we examined their ability to affect PAF-metabolism, through their in vitro effect on PAF basic metabolic enzymes (PAF-CPT, lyso PAF-AT, and PAF-AH). Results: Nutrof exhibited the strongest anti-PAF activity, while Vitalux was the most potent anti-inflammatory factor. Conclusion: This is the first study to bring in surface potent anti-inflammatory and anti-angiogenic activities of some vitamin supplements used against AMD, through their in vitro anti-PAF effects in WRPs and the rabbit plasma and leukocyte PAF metabolism, suggesting a promising role of vitamin supplements and especially resveratrol, concerning its potent anti-angiogenic activity in AMD.

Platelet activating factor levels and metabolism in tangier disease: a case study

BackgroundTangier disease (TD) is a phenotypic expression of rare familial syndrome with mutations in the ABCA1 transporter. The risk of coronary artery disease in patients with TD is variable. On the other hand the pivotal role of Platelet-Activating Factor (PAF) mediator in atheromatosis was found. Plasma lipoproteins are transporters of the PAF acetylhydrolase (PAF-AH) in cells and known as lipoprotein-phospholipase A2 (Lp-PLA2) in plasma and regulators of PAF levels in blood. In addition, PAF can be biosynthesized from the remodeling and the de novo pathways in which Lyso-platelet activating factor-acetyltransferase (Lyso-PAF-AT) and platelet activating factor-cholinephosphotransferase (PAF-CPT) are the regulatory enzymes. The aim of this study is to investigate in a TD patient with a unique mutation (C2033A), the concentration of PAF in blood, the Equivalent Concentration for 50% aggregation (EC50) values of platelet rich plasma (PRP) toward PAF, adenosine diphosphate (ADP) and thrombin, and the activities of PAF metabolic enzymes Lp-PLA2, PAF-AH, Lyso-PAF-AT and PAF-CPT.MethodsThe EC50 value of PRP was measured by an aggregometer. The determination of the specific activity of PAF-CPT and Lyso-PAF-AT was made after in vitro enzymatic assay, chromatographic separation and measurement of the produced PAF in a biological assay with washed rabbit platelets. The determination of PAF-AH and Lp-PLA2 was made after an in vitro enzymatic assay from the decay of radioactive PAF.ResultsThe TD patient had lower bound-PAF values in blood, decreased specific activity of PAF-CPT and Lyso-PAF-AT, increased specific activity of PAF-AH in platelets and leukocytes and Lp-PLA2 activity in plasma compared to healthy women. The EC50 of PAF and Thrombin were higher compared to healthy women.ConclusionThe increased Lp-PLA2 activity, as well as, the decreased activities of PAF-CPT and Lyso-PAF-AT, explain the decreased bound-PAF level in TD patient and the EC50 of PAF. However, total PAF is in a normal range and this probably can explain one of the reasons this TD patient has no CAD.

In Vitro Effects of Anti-Glaucomatous Eye Drops on Platelet-Activating Factor and its Metabolism

Abstract Purpose: The purpose of this study is to determine the effect of various commonly used antiglaucoma eye drops on inflammatory mediators such as the platelet activating factor (PAF). Methods: Various intraocular pressure (IOP) lowering drops were tested to examine their inhibitory effect on PAF. Multiple eye drops were tested in washed rabbit platelets (WRPs) in order to determine the interaction between these eye drops and the inhibition of PAF in the PAF-induced platelet aggregation model. In addition, we examined the eyedrops’ effect on PAF-metabolism, through in vitro analysis on PAF basic metabolic enzymes (PAF-CPT, lyso PAF-AT, and PAF-AH). Results: Latanoprost (Xalatan) was found to be the most potent in inhibiting PAF, suggesting that it is the most effective in decreasing IOP amongst the eye drops tested. Conversely, dorzolamide hydrochloride-timolol (Cosopt) exhibited the least anti-PAF action. Conclusions: This is the first study examine the relationship between PAF activity and glaucoma medication. Potency in PAF inhibition may be related to drop efficacy.

ÃÂn Vivo Effects of Tenofovir-DF/Emtricitabine and Abacavir/Lamivudine withAtazanavir-R on Platelet Activating Factor Metabolism in HIV Naive Patients

Antiretroviral therapy (ΑRT) has successfully decreased AIDS morbidity and mortality and increased the lifespan of HIV patients to several decades. However, numerous factors contribute with unknown mechanisms to chronic immune activation and inflammation leading to severe “non-AIDS morbidities’’. Platelet Activating Factor (PAF) is a potent lipid inflammatory mediator with important role in the ‘’non-AIDS morbidities’’. The purpose of this study was to investigate whether tenofovir-DF/emtricitabine and abacavir/lamivudine with atazanavir boosted ritonavir (ART_A and ART_B, respectively) affect in vitro PAF activity and in vivo PAF levels and metabolism. In this intent, the two ART regimens were examined in vitro against platelet aggregation induced by PAF. In addition, PAF levels and PAF metabolic enzymes were determined in HIV-1 infected volunteers before and after the initiation of antiretroviral therapy for a 12-month period. The in vitro results showed that ritonavir was the most potent inhibitor against PAF induced platelet aggregation while abacavir presented the less potent action. The in vivo results showed that tenofovir-DF/emtricitabine with atazanavir-r seems not to affect PAF levels and metabolism while abacavir/lamivudine with atazanavir-r increased bound and total PAF blood levels, PAF biosynthesis in platelets and also decreased Lp-PLA2 activity. In addition, ART_B revealed higher lyso-PAF-AT specific activity at 3rd, 6th and 9th month (p3=0.04, p6=0.04 and p9=0.03) compared to ART_A. In conclusion, there is a direct relation between in vitro and in vivo effect of antiretrovirals on PAF and abacavircontaining regimen activates PAF biosynthesis leading to elevated PAF levels.

Anti-platelet effects of anti-glaucomatous eye drops: an in vitro study on human platelets

Purpose Altered platelet aggregability has been implicated in the pathogenesis of glaucoma. This study aims to investigate the anti-platelet potential of intraocular pressure lowering drops, with the possibility of establishing it as an additional mechanism of anti-glaucomatous action. Materials and methods The anti-aggregating effects of a series of anti-glaucomatous eye drops were determined on human platelets in the platelet aggregation model, using four known aggregating factors (platelet activating factor [PAF], adenosine diphosphate [ADP], thrombin receptor-activating peptide [TRAP], and arachidonic acid [AA]). Results Almost all of the tested samples inhibited platelet aggregation induced by PAF, ADP, TRAP, and AA, except for Alphagan, which did not demonstrate inhibition of ADP- and TRAP-induced aggregation at a wide range of concentrations. Trusopt, Betoptic, and Azarga eye drops were the most potent inhibitors of all four aggregating factors, while Alphagan was the least potent (P<0.05). Conclusion This study shows that anti-glaucomatous eye drops possess anti-platelet effects, and this was shown for the first time by experimenting on human platelets.