Antiplatelet Action of Chloramine Derivatives of Adenosine Phosphates and Their Chemical Activity in Relation to Sulfur-Containing Compounds.

We studied the properties of N6-chloroadenosine phosphates (ATP, ADP, and AMP chloramines) as compounds with potentially increased antiplatelet efficacy determined by their binding to the plasma membrane of platelets. Chloramine derivatives of ATP, ADP, and AMP do not differ in their optical absorption characteristics: their absorption spectra are in the range of 220-340 nm with a maximum at 264 nm. Chloramines of adenosine phosphates are characterized by high reactivity with respect to thiol compounds. In particular, the rate constants of the reaction of N6-chloroadenosine-5'-diphosphate with N-acetylcysteine, reduced glutathione, dithiothreitol, and cysteine reach 59,000, 250,000, 340,000, and 1,250,000 M-1×sec-1, respectively, and only 1.10±0.02 M-1×sec-1 with methionine. It has been found that N6-chloradenosine-5'-triphosphate is a strong inhibitor of platelet functions: it effectively suppresses ADP-induced cell aggregation (IC50 in the whole blood is 5 µM) and inhibits aggregation of preactivated platelets and induces dissociation of their aggregates.

Piezo-modulated active grating for selecting X-ray pulses separated by one nanosecond.

We present a novel method of temporal modulation of X-ray radiation for time resolved experiments. To control the intensity of the X-ray beam, the Bragg reflection of a piezoelectric crystal is modified using comb-shaped electrodes deposited on the crystal surface. Voltage applied to the electrodes induces a periodic deformation of the crystal that acts as a diffraction grating, splitting the original Bragg reflection into several satellites. A pulse of X-rays can be created by rapidly switching the voltage on and off. In our prototype device the duty cycle was limited to ∼1 ns by the driving electronics. The prototype can be used to generate X-ray pulses from a continuous source. It can also be electrically correlated to a synchrotron light source and be activated to transmit only selected synchrotron pulses. Since the device operates in a non-resonant mode, different activation patterns and pulse durations can be achieved.

Inhibition of plasma coagulation and platelet aggregation with structural analogs of taurine chloramine.

We studied the effects of amide and N-alkyl analogs of taurine chloramine on rabbit plasma coagulation and platelet aggregation. Alkyl analog N-isopropyl-N-chlorotaurine produced greater increase in plasma coagulation time after its activation by the contact method or with thrombin than amide analog N-propionyl-N-chlorotaurine. In case of coagulation induced by the tissue factor, the test analogs produced similar effect. Inhibition of platelet aggregation in platelet-rich plasma under the effect of N-isopropyl-N-chlorotaurine depended on the nature of the agonist. Aggregation was suppressed stronger under conditions of collagen stimulation than in response to ADP agonist. Estimated partial charges of the chlorine atom in amide analogs were 5-fold higher than in N-alkyl analogs. This fact determined the difference in the chemoselective interaction with sulfur-containing amino acid residues in targets and the specific features of anticoagulation and antiaggregant effects of two analogs of taurine chloramine.

[Molecular characteristics and prediction of the reactive properties of the N-chlorotaurine analogs].

A number of molecular characteristics for the N-chlorotaurine structural analogs, amino acid chloramines and relative compounds have been computed by the ab initio method B3LYP/6-31G. In particular, the characteristics were the Mulliken atomic charges for the chloramine part and its adjacent atoms. A quantitative measure of the capabilities of the chloramines to react with the methionine sulfide group or sulfhydryl group of reduced glutathione was their reaction rate constants. The constants available in literature and determined in own experiments have been depicted with an exponential equation of multiple correlation. In the case of a reaction with methionine, the high determination coefficient (R2) was obtained with five independent variables. They were the charges of active chlorine, nitrogen, carbon bonded with nitrogen, a bond length between nitrogen and carbon atoms, and also molecular mass. The equation has been used to predict the rate constant values for the reaction between compounds that contain active chlorine and methionine. The prediction has showed that structural analogs of N-chlorotaurine bearing two methyl groups at beta-carbon of taurine are remarkable for the low value of the discussed rate constant.

[Covalent chloramine inhibitors of blood platelet functions: computational indices for their reactivity and antiplatelet activity].

The quantum mechanics computation of the reactivities of chloramine derivatives of amino acids and taurine has been accomplished. A pair of computational indices that reflect a predisposition of alpha amino acid chloramines to chemical decay have been revealed. One of the indices was the dihedral angle for the chain of four atoms: carbons at beta- and alpha-positions, carbon of the carboxyl group, and carbonyl oxygen. The second index was the sum of partial charges for three or two carbon atoms in the chain. The amino acid chloramines with high values of the indices showed enhanced stability. Partial charges for active chlorine in known chloramines having different structures have been computed. The charges correlate with the rate constants of the reaction between chloramines and the thiol group of reduced glutathione. New derivatives of taurine chloramines have been constructed via the introduction of different substituents into the chloramine part. Among them, the amidoderivatives had the greatest charges of active chlorine (0.19-0.23). It was found in the study of the reactions of N-acetyl-N-chlorotaurine and N-propyonyl-N-chlorotaurine with amino acids and peptides possessing the thiol, thioester, or disulphide groups that the amidoderivatives manifested the thiol chemoselectivity. N-Acetyl-N-chlorotaurine and N-propionyl-N-chlorotaurine suppress the aggregation activity of blood platelets under their activation by the agonists ADP and collagen. It is not excluded that the amidoderivatives studied prevent platelet aggregation by a modification of the critical thiol group in the purine receptor P2Y12.

Antiaggregant effect of taurine chloramines in the presence of serum albumin.

The effects of taurine chloramine derivatives on initial aggregation of isolated platelets suspended in buffered saline were studied. Inhibition of ADP-induced aggregation in pure cell suspension depended on the structure of chloramine antiaggregants. The most effective of them was N,N-dichlorotaurine; its concentration needed for 50% inhibition of aggregation was about 0.1 mM. Weaker antiaggregants N-chloro-N-methyltaurine and N-chlorotaurine in a final concentration of 0.5 mM reduced platelet aggregation by only 10%. The studied chloramines considerably differed by their characteristics (velocity of the reaction with sulfur-containing groups of atoms). N,N-dichlorotaurine exhibited the weakest reactivity with methionine thioester group. In turn, the velocity constant with reduced glutathione was by 2-3 orders of magnitude higher than that of other chloramines. Antiaggregant effect of taurine chloramine derivatives was 2-fold higher in the presence of serum albumin, presumably due to special interactions of taurine chloramines in complex with albumin with platelets.

[Amino acid chloramines and chlorimines as antiplatelet agents: reactive properties and mechanism of action].

Oxidative modifications of thiols, disulfide, and thioester atomic groups in proteins, peptides, and amino acids induced by chloramines or chloramine derivatives of amino acids and other reactive oxidants are considered. In the case of disulfide and thiol groups, production of sulfur-reactive groups may take place, such as disulphide S-oxides and sulphenic groups. Various chloramines and chloramines differently modify sulfur-containing groups. For example, N,N-dichlorotaurine rapidly modifies the thiolgroup in reduced glutathione and N-chloroglycine readily oxidizes the thioester group in methionine. Amino acid chloramines inhibit platelet aggregation by modifying S-containing centres. Autodecay of amino acid chloramines does not affect aggregation as follows from the absence of positive correlation between chloramines decay rate and antiplatelet activity. N,N-dichlorotaurine and its chlorimine derivatives are characterized by high stability and have good prospects as potential antiaggregants.

[Initial selectivity of the antiplatelet covalent action of biogenic chloramines on platelet-rich plasma].

To describe in full the peculiarities of the antiplatelet action of covalent inhibitors on platelet-rich plasma, we have proposed to take into account the initial selectivity that determines the elevated efficacy of inactivation of platelet molecular target (receptor). The quantitative index of initial selectivity is the ratio of rate constant of inactivation of the platelet molecular target to the rate constant of the chemical reaction of an inhibitor with reactive atomic groups in plasma proteins. For the important case of the domination of the inhibitor expenditure in the reaction with plasma proteins, a formula was derived which depicts the dependence of the share of inactivated targets on the concentration of the inhibitor introduced and reactive atomic groups contained in plasma. In the case of chloramine derivatives of amino acids, evidence was obtained indicating that the degree of inhibition of platelet aggregation measured by the turbidimetric method is equal to the square of the share of inactivated receptors. The index of initial selectivity can be evaluated by measuring the degree of inhibition of platelet aggregation and the operating concentration of the inhibitor. According to experimental evidence, the effects of a number of chloramine derivatives of amino acids (biochloramines) on aggregation of platelets stimulated by ADP show selectivity at the molecular target level, so that the index of initial selectivity is greater than 1. The mechanism of the selective action of the biochloramines having significant molecular masses (150-200 Da) probably consists in the inactivation of the molecular target via chemical modification of several reactive atomic groups in its different sites. One may suppose that the biochloramines with lower molecular masses (150-100 Da) exhibit a high anti-aggregatory capacity owing to another mechanism of initial selectivity, which involves the modification of highly sensitive sulfur-containing atomic groups.

[Luminol-enhanced chemiluminescence of rabbit polymorphonuclear leukocytes: the nature of oxidants that directly induce luminol oxidation].

The present work deals with the reaction pathways, including the formation of hydroxyl radicals and chloroamines, which lead to luminol chemiluminescence caused by hypochlorite generation in a suspension of stimulated rabbit polymorphnonuclear leukocyte. Luminol-enhanced (0.02 mM) chemiluminescence of leukocytes stimulated by phorbol 12-myristate 13-acetate does not change in the presence of dimethyl sulfoxide at moderate concentrations (0.02-2.6 mM) at which it must show the specific ability to scavenge hydroxyl radicals. It suggests that no generation of hydroxyl radical with the participation of hypochlorite and superoxide anion takes place after the stimulation of polymorphnonuclear leukocytes. A high dimethyl sulfoxide concentrations (260 mM) a significant fall in chemiluminescence intensity, due to direct interaction of the scavenger with hypochlorite, is observed. Chemiluminescence intensity rose if luminol was added to a leukocyte suspension preliminary stimulated for 10 min. The effect results from the accumulation of hydrogen peroxide but not chloroamines. Exogenic amino acids and taurin at high concentrations (3-15 mM) weaken the chemiluminescence. The data obtained suggest that chemiluminescence in the system studied results predominantly from the direct initial reaction of hypochlorite with luminol. The chemiluminescence intensity is enhanced by hydrogen peroxide via the oxidation of luminol oxidation products.

[Mechanism of action of biogenic chloramines and hypochlorite on initial aggregation of blood platelets].

The antiaggregant action of two reactive oxidants N,N-dichlorotaurine (chloramine of biogenic type) and sodium hypochlorite on the initial ADP-induced aggregation of rabbit blood platelets has been studied. Platelet aggregation in the reconstructed platelet-rich plasma (PRP) was measured by the nephelometric method, and the aggregation index was an increase in the intensity of small-angle light scattering. The introduction of chloramine at comparatively small concentrations (no greater than 1 mM active chlorine) directly into the reconstructed platelet-rich plasma induces the suppression of the initial aggregation (formation of small aggregates) several times stronger than in the case of its preliminary incubation with plasma alone. This suggests that N,N-dichlorotaurine exerts its antiaggeregant action on the platelet-rich plasma by direct interaction with cells. The effects of the inhibition of platelet aggregation in two variants of introduction of high concentrations of N,N-dichlorotaurine do not significantly differ. In this case a great amount of residual chloramine remains in the plasma, which just induces the suppression of platelet aggregation during subsequent reconstruction of the platelet-rich plasma. Similar data have been obtained in the study of the antiaggregant action of hypochlorite. N,N-Dichlorotaurine and hypochlorite at final concentrations of 0.2-0.3 and 0.15 mM, respectively, inhibit strongly the initial aggregation of isolated platelets (approximately 2 x 10(8) cells in 1 ml) preliminarily activated for 1.5 min by the addition of 100-500 nM ADP. However, the antiaggregants show a more profound suppression of aggregation of unstimulated platelets. The antiaggregant effects of N,N-dichlorotaurine and hypochlorite are probably due to the oxidative modification of sulfur-containing groups in platelet plasmatic membrane.

[Chemiluminescence in a stimulated polymorphonuclear leukocytes--luminol system: suppression by thiols].

The effect of some scavengers of thiol nature, which eliminate all reactive oxygen species and oxidants with reactive chlorine, on the luminol-enhanced chemiluminescence of polymorphonuclear leukocytes was studied. The use of two scavengers of this type (penetrating and not penetrating into the cell) made it possible to separate the luminescence of cell structures from the luminescence generated by oxidants in the surrounding medium. It was found that about a half of luminol luminescence is due to its oxidation in the medium surrounding the cell, and it is completely inhibited by the nonpenetrating reduced glutathione. The cell itself is a source of a considerable portion of luminescence, and this luminescence is quenched by penetrating sulfhydryl compounds such as dithiothreitol and N-acethyl cysteine. Reduced glutathione, which penetrates into cells and whose action is due only to the sulfhydryl group, is recommended as a candidate for the selective neutralization of extracellular oxidants.

PUVA-induced erythema and changes in mechanoelectrical properties of skin. Inhibition by tocopherols.

Influence of antioxidants on two phototoxic effects of 8-methoxypsoralen (8-MOP) was studied: erythema and changes in mechanoelectrical properties of skin. alpha-Tocopherol and its analogs with shortened lateral hydrocarbon chains at C2-atoms of chromane groups (chromanols) were used as antioxidants. alpha-Tocopherol and its analogs inhibited both phototoxic effects of 8-MOP. Inhibition was observed only if antioxidants were present in skin during irradiation. When applied after irradiation these antioxidants produce no inhibitory effect. The antioxidant antierythemal action depends greatly on their concentration. The protective effects is maximal at antioxidant concentrations 2.5 . 10(-10) - 5 . 10(-9) mol . cm-2 of skin, at concentrations higher than 5 . 10(-9) mol . cm-2 the protective action is decreased. The protective effect of antioxidants depends on the irradiation dose.

Electromigration in Al thin films induced by surface acoustic waves: application to imaging.

The propagation of a high amplitude surface acoustic wave in an Al thin film induces a large-scale electromigration phenomenon resulting in a permanent etching of the acoustic field in the film. The etched patterns depend on the time of propagation and on the acoustic characteristics. Preliminary observations of a few grooved structures in Al films have been performed by different techniques. A first explanation of this phenomenon based on dynamical Grinfeld instabilities is proposed. By providing permanent pictures of acoustic fields emitted by transducers, this effect could be used to perform imaging of surface acoustic wave propagation.

[Relation between erythrocyte photodamage and the intensity of ultraviolet radiation]. / Zavisimost' fotopovrezhdeniia éritrotsitov ot intensivnosti ul'trafioletovogo oblucheniia.

Photoperoxidation of lipids (PPOL) in erythrocytes membranes and photohemolysis are essentially weakened with an increase of UV - irradiation intensity at constant irradiation dose. The initial part of the relationship between PPOL product concentration or rate of hemolysis and irradiation intensity is of a linear pattern. This is explained by the fact that the breakage of the chains of lipid free radical photooxidation on endogenous alpha-tocopherol is more effective than disproportionation of peroxide free radicals.

[Photobiological processes in biomembranes under the effect of ultraviolet radiation on animal cells, tissues, and organs]. / Fotobiologicheskie protsessy v biomembranakh pri deistvii ul'trafioletovogo izlucheniia na kletki, tkani i organy zhivotnykh.

The current knowledge about the mechanisms of the lipid photoperoxidation (LPPO) and the role of the process in the effects of the UV-irradiation of cells and tissues is reviewed. The LPPO in biomembranes occurs as a result of the lipid hydroperoxide phototransformation to free radicals and the antioxidant photolysis. The LPPO significantly depends on the structural state of biomembranes. The UV-radiation stimulates the two types of dark lipid peroxidation (LPO): the free radical non-enzymatic peroxidation, and the cyclooxygenase peroxidation. The UV-radiation produces increase of the membrane ion permeability, decrease (or enhancement) of the aggregatory interaction of cells, and changes of the membrane enzymatic activity. The LPPO and the dark non-enzymatic LPO play an important role in the changes of the membrane functions under the UV-irradiation. The dark non-enzymatic LPO is important for the development of UV erythema. The cyclooxygenase LPO may be responsible for the curative properties of the UV-irradiated blood.

[Changes in erythrocyte and thrombocyte aggregation after ultraviolet irradiation]. / Izmenenie agregatsii éritrotsitov i trombotsitov pod deistviem ul'trafioletovogo izlucheniia.

Lipid peroxidation which occurs in blood serum under ultraviolet irradiation was studied. The products of these reaction suppress ADP-induced aggregation of native platelets. The rouleaux-forming capacity increased after UV-irradiation of plasma and serum albumin. Under UV-irradiation the aggregates of albumin molecules are supposed to form the aggregates of albumin molecules which bind the erythrocytes in rouleaux.

[Kinetics of the interaction of ferrous oxide with oxidized lipids and possibility of chiluminescent determination of hydroperoxides]. / Kinetika vzaimodeistviia zakisnogo zheleza s okislennymi lipidami i vozomozhnost' khemiliuminestsentnogo opredeleniia gidroperekisei

The theory shows that for lipids containing hydroperoxides of unsaturated fatty acids there exists a verge concentration of Fe2+: at concentrations of Fe2+ lower than the verge one the peroxidation process develops autocatalytically and at higher concentrations it looks pulseshaped. In the second case the amplitude of pulse concentration of peroxide radicals and the square root from the maximum value of chemiluminescence intensity are proportional to the concentration of hydroperoxides present in the system at the time of ferrous ions injection. The former two values do not depend on the concentration of inoxidized fatty acids. In the experiments of chemiluminescence of partially oxidized linoleic acid and lecithin in methanol-benzene solutions (1 : 2) a perfect correlation between the experimental data and theoretical conclusions was revealed.

[Chemiluminescence in oxidation of luminol by chloramine derivatives of biogenic compounds]. / Khemiliuminestsentsiia pri okislenii liuminola khloraminovymi proizvodnymi biogennykh soedinenii.

Chloramine derivatives of amino acids induce chemiluminescence of a luminol solution. The chemiluminescence is more prolonged than the emission of luminol produced by hypochlorite. Persistent chemiluminescence also appears under the action of hypochlorite on a mixture of luminol and amino acids. It is assumed that the chemiluminescence of luminol in suspensions of stimulated phagocytes may be associated with its oxidation by chloramines.

[Effect of extremely low frequency electromagnetic radiation and ultra-violet radiation on aggregation of thymocytes and erythrocytes]. / Deistvie élektromagnitnogo izlucheniia kraine vysokikh chastot i UF-izlucheniia na agregatsionnoe vzaimodeistvie timotsitov s éritrotsitami.

Electromagnetic radiation of superhigh frequencies (46.12 and 46.19 GHz, 0.3-1 mV/cm2) at an incident dose of about 12 kJ/m2 enhances the ability of isolated rabbit thymocytes for aggregation interaction with homologous erythrocytes. In the case of 46.19 GHz frequency, the stimulatory effect disappears as radiation dose in increased. A radiation of 46.12 GHz stimulates thymocytes also at high radiation doses. Superhigh-frequency radiation enhances the sensitivity of thymocytes to the damaging effect of UV radiation.

[Chemiluminescence of the polymorphonuclear leukocytes-luminol system in the presence of biogenic chloramines]. / Khemiliuminestsentsiia sistemy polimorfnoiadernye leikotsity-liuminol v prisutstvii biogennykh khloraminov.

It was demonstrated that N-chlorphenylalanine and other chloramines strengthen sharply chemiluminescence in the polymorphonuclear leukocytes (PML)-luminol system without special activation of cells. The intensity of chemiluminescence is higher than the intensity of luminol solution emission induced by N-chlorphenylalanine. But it was nearly equal to chemiluminescence intensity of a mixture of luminol, N-chlorphenylalanine and 20-30 nM H2O2. The increase in chemiluminescence in the PML-luminol system in the presence of N-chlorphenylalanine is not related to PML activation but is the result of direct oxidation of luminol by N-chlorphenylalanine. Chloramine derivatives of amino acids and taurine at final concentrations of 0.01-0.1 mM do not suppress luminol chemiluminescence in suspension of PML stimulated by phorbol-12-myristate-13-acetate. At the same time, hypochlorite inhibits sharply luminol emission induced by stimulated cells.