Debora Bruzzese

Activation of p38 MAPKinase/cPLA2 pathway in homocysteine-treated platelets.

Summary.  Hyperhomocysteinemia is considered a risk factor in arterial and venous thrombosis. The mechanism by which homocysteine (HCy) supports athereothrombosis is still unknown and may be multifactorial. Earlier in vitro studies demonstrated that HCy induced arachidonic acid release and increased thromboxane B2 (TXB2) formation. In this work, we found that HCy stimulated the rapid and sustained phosphorylation of platelet p38 mitogen‐activated protein kinase (p38 MAPK). The effect was time‐ and dose‐dependent. The HCy effect on p38 MAPK phosphorylation was prevented by N‐acetyl‐l‐cysteine and iloprost and was partially inhibited by nordihydroguaiaretic acid. Moreover, the incubation of platelets with HCy led to the phosphorylation of cytosolic phospholipase A2 (cPLA2). In addition HCy promoted cPLA2 activation, assessed as arachidonic acid release. The cPLA2 phosphorylation and activation were both impaired by the inhibition of  p38 MAPK through SB203580. This effect was not complete, reaching at the most the 50% of the total. In FURA 2‐loaded platelets, HCy induced a dose‐dependent intracellular calcium rise suggesting that the calcium elevation promoted by HCy could participate in the cPLA2 activation, leading to arachidonic acid release and TXB2 formation. In conclusion, our data provide insight into the mechanisms of platelet activation induced by HCy, suggesting that the p38 MAPK/cPLA2 pathway could play a relevant role in platelet hyperactivity described in hyperhomocysteinemia.

Platelet activation by collagen is increased in retinal vein occlusion

Retinal vein occlusion (RVO) is the most common retinal vascular disorder second to diabetic retinopathy. The main risk factors in patients with RVO are hypertension, diabetes, hyperlipidemia, increased blood viscosity and glaucoma. The pathogenesis of RVO has not yet been clarified. In these events platelets could play a very important role. In the present study the platelet response to collagen was deeply investigated. Experiments were carried out on a selected group of RVO patients, which were compared to a group of healthy subjects matched for age, sex, clinical and metabolic characteristics. In resting and activated platelets of both groups of subjects p72syk phosphorylation, phospholipase Cgamma2 phosphorylation, protein kinase C activation, intra-cellular calcium levels and nitric oxide formation were measured. Results show that platelets of patients were more responsive to collagen or ADP than healthy subjects and that the response was significantly different (p < 0.0005) at low concentrations of these agonists. In platelets of patients stimulated with collagen increased phosphorylation of p72syk and phospholipase Cgamma2 was found. Also protein kinase C was more activated in patients. In addition intracellular calcium rise induced by collagen was significantly higher in patients than in healthy subjects. RVO patients showed a lower basal level of nitric oxide both in resting and stimulated platelets compared to healthy subjects. Altogether these results suggest that the platelet hyperaggregability described in patients might be an important factor in the development of RVO contributing to the thrombogenic effects.

A role for PLCγ2 in platelet activation by homocysteine

The aim of this study was to examine the homocysteine effect on phospholipase Cγ2 (PLCγ2) activation and to investigate the signaling pathway involved. We found that homocysteine stimulated the tyrosine phosphorylation and activation of platelet PLCγ2. The tyrosine kinases p60src and p72syk appeared to be involved upstream. Reactive oxygen species were increased in homocysteine treated platelets. Likely oxidative stress could prime the non receptor‐mediated tyrosine kinase p60src, inducing phosphorylation and activation of p72syk. The antioxidant N‐acetyl‐L‐cysteine prevented the activation of these kinases. The phosphorylation and activation of PLCγ2 were greatly reduced by the inhibition of p72syk through piceatannol. Moreover indomethacin diminished the homocysteine effect on p60src, p72syk and PLCγ2, suggesting that thromboxane A2 could be involved. In addition the treatment of platelets with homocysteine caused intracellular calcium rise and protein kinase C activation. Finally homocysteine induced platelet aggregation, that was partially reduced by indomethacin and by N‐acetyl‐L‐cysteine of 35% or 50% respectively, while the PLCγ2 specific inhibitor U73122 diminished platelet response to homocysteine of 70%. Altogether the data indicate that PLCγ2 plays an important role in platelet activation by homocysteine and that the stimulation of this pathway requires signals through oxygen free radicals and thromboxane A2. J. Cell. Biochem. 100: 1255–1265, 2007.

Recombinant Laccase: I. Enzyme cloning and characterization

We obtained structural and functional characterization of a recombinant Laccase from Rigidoporus lignosus (formerly Rigidoporus microporus), a white‐rot basidiomycete, by means of circular dichroism (CD) spectra, cyclic voltammetry (CV) and biochemical assays. Here we report the optimization of expression and purification procedures of a recombinant Laccase expressed in supercompetent Escherichia coli cells. We amplified the coding sequence of Laccase using PCR from cDNA and cloned into a bacterial expression system. The resulting expression plasmid, pET‐28b, was under a strong T7/Lac promoter induced by IPTG (isopropyl‐β‐d‐thiogalactoipyranoside). We obtained purification by fast protein liquid chromatography (FPLC) method. We recorded the variation of the current of a solution containing purified Laccase with increasing Syringaldazine (SGZ) concentration using a potentiometer as proof of principle, showing its compatibility with the development of a new enzymatic biosensor for medical purposes, as described in Part II. J. Cell. Biochem. 114: 599–605, 2013.

Adenylic Dinucleotides Produced by CD38 Are Negative Endogenous Modulators of Platelet Aggregation

Diadenosine 5′,5‴-P1,P2-diphosphate (Ap2A) is one of the adenylic dinucleotides stored in platelet granules. Along with proaggregant ADP, it is released upon platelet activation and is known to stimulate myocyte proliferation. We have previously demonstrated synthesis of Ap2A and of two isomers thereof, called P18 and P24, from their high pressure liquid chromatography retention time, by the ADP-ribosyl cyclase CD38 in mammalian cells. Here we show that Ap2A and its isomers are present in resting human platelets and are released during thrombin-induced platelet activation. The three adenylic dinucleotides were identified by high pressure liquid chromatography through a comparison with the retention times and the absorption spectra of purified standards. Ap2A, P18, and P24 had no direct effect on platelet aggregation, but they inhibited platelet aggregation induced by physiological agonists (thrombin, ADP, and collagen), with mean IC50 values ranging between 5 and 15 μm. Moreover, the three dinucleotides did not modify the intracellular calcium concentration in resting platelets, whereas they significantly reduced the thrombin-induced intracellular calcium increase. Through binding to the purinergic receptor P2Y11, exogenously applied Ap2A, P18, and P24 increased the intracellular cAMP concentration and stimulated platelet production of nitric oxide, the most important endogenous antiaggregant. The presence of Ap2A, P18, and P24 in resting platelets and their release during thrombin-induced platelet activation at concentrations equal to or higher than the respective IC50 value on platelet aggregation suggest a role of these dinucleotides as endogenous negative modulators of aggregation.

Langmuir-Blodgett based lipase nanofilms of unique structure-function relationship.

Proteins represent versatile building blocks for realization of nanostructured materials of unique structure-function relationship to be applied in nanobiotechnology. Following a recent work [Bruzzese, D., Pastorino, L., Pechkova, E., Sivozhelezov, Nicolini, C., Increase of catalytic activity of lipase towards olive oil by Langmuir-Film Immobilization of Lipase, Enzyme and Microbial Technology, submitted for publication.], the Langmuir-Blodgett technique was utilized to develop nanostructured crystal materials based on enzymes interfacially activated with olive oil as substrate. Particularly, thin films of lipase from both Mucor miehei and Candida rugosa were fabricated and characterized by UV-vis spectroscopy, Atomic force microscopy and biochemical assays. As the first step the M. miehei protein films were studied at the air-water interface and then transferred onto a solid support for further characterization of the enzymatic activity also versus surface pressure, proving that Langmuir-Blodgett film provides a better catalytic effect in lipase than a mere oil-water boundary. Moreover, improvement of lipase catalytic performance was achieved for the M. miehei versus the C. rugosa, despite its almost random distribution of hydrophobic patches and the low purity of its preparation.