Igor Mindukshev

Low angle light scattering analysis: a novel quantitative method for functional characterization of human and murine platelet receptors

Abstract Background: Determinations of platelet receptor functions are indispensable diagnostic indicators of cardiovascular and hemostatic diseases including hereditary and acquired receptor defects and receptor responses to drugs. However, presently available techniques for assessing platelet function have some disadvantages, such as low sensitivity and the requirement of large sample sizes and unphysiologically high agonist concentrations. Our goal was to develop and initially characterize a new technique designed to quantitatively analyze platelet receptor activation and platelet function on the basis of measuring changes in low angle light scattering. Methods: We developed a novel technique based on low angle light scattering registering changes in light scattering at a range of different angles in platelet suspensions during activation. Results: The method proved to be highly sensitive for simultaneous real time detection of changes in size and shape of platelets during activation. Unlike commonly-used methods, the light scattering method could detect platelet shape change and aggregation in response to nanomolar concentrations of extracellular nucleotides. Furthermore, our results demonstrate that the advantages of the light scattering method make it a choice method for platelet receptor monitoring and for investigation of both murine and human platelets in disease models. Conclusions: Our data demonstrate the suitability and superiority of this new low angle light scattering method for comprehensive analyses of platelet receptors and functions. This highly sensitive, quantitative, and online detection of essential physiological, pathophysiological and pharmacological-response properties of human and mouse platelets is a significant improvement over conventional techniques.

A new method for studying platelets, based upon the low-angle light scattering technique. 1. Theoretical and experimental foundations of the method

A new method is presented for simultaneous monitoring of changes in shape and aggregation of platelets. The signal of light scattering alterations at angles below six degrees was shown to be determined by platelet aggregation dynamics (aggregation, disaggregation, coagulation). Over a range of larger angles (6-15 degrees), cell shape changes also contributed to the signal: (i) spherization, and (ii) pseudopodia formation. The first stage was shown to be fast (t1/2 of few seconds) and correlated with (Ca 2+ ) increase. It was characterised by a narrow signal fluctuation and by a rapid increase (30-40%) in signal intensity. During the second stage, which was much slower, the signal decreased describing the aggregation process. The EC50 value for ADP-induced spherization was 40 nmol l −1 . Aggregation kinetics in saline solution under turbulent flow showed second order kinetics in relation to initial cell concentration. The rate constant depended on stirring conditions and on calcium concentration in the medium. Standardisation of the testing conditions made it possible to characterize the initial functional state of platelets by their sensitivity to agonists, with estimation of EC50 and maximum velocity of aggregation (Umax )v alues. The method has potential applications in pharmacology and toxicology research and in clinical practice, as a simple and highly sensitive functionality test for platelets.

A new method for studying platelets, based upon the low-angle light scattering technique. 2. Application of the method in experimental toxicology and clinical pathology.

A new method for studying platelets based on low-angle light scattering has been applied to studies in experimental toxicology and clinical pathology, using animal and human platelets. Index EC50 for ADP within control groups was estimated to be in the range of 100–180 nmol l–1 (healthy men, rabbits, rats). The refractory state of platelets was obtained by use ADP or ATP concentrations that just caused platelet activation. The time constant and the half-cycle of the refractory state of platelets were calculated. Kinetic parameters of platelet aggregation at interference of hemostasis (rabbit) and the influence of the age factors on the aggregation indices of platelets (rat) were studied. The method of low-angle light scattering was used as a diagnostic tool in experimental low-level intoxication by organophosphates and for interpretation of the pathogenesis of delayed effects. In human patients with ischemic heart disease or with prosthetic heart valves, significant changes in the functional state of platelets were observed. A model for cooperative binding of receptors with ligands on platelets from pregnant women with preeclampsia was developed.

Necrotic and apoptotic volume changes of red blood cells investigated by low-angle light scattering technique

A low-angle light scattering technique, which has been applied previously to studies of blood platelets and Ehrlich ascite tumor cells, revealed differences in the dynamics of necrotic and apoptotic red blood cell death. Under hypotonic loading or in ammonia medium, red blood cells (RBC) swelled to a critical size (diameter approximately 13 µm) prior to hemolysis (necrosis). Under acidic loading, hemolysis occurred with less pronounced swelling of cells (diameter approximately 10 µm). Apoptosis induced by a calcium ionophore resulted in initial formation of echinocytes, followed by development of rounded red blood cells with uneven membrane, capable of agglomeration. In such a way, RBC aggregation can precede the final stages of the RBC apoptosis when small cellular fragments are generated. On the basis of erythrograms of the cells hemolysing in ammonia medium, the echinocytic (preapoptotic) and stomatocytic (prenecrotic) RBC were discerned due to the very high resistance of apoptotic RBC to osmotic (ammonia) loading.

A new method for studying platelets, based upon the low-angle light scattering technique. 3. Aggregation hypersensitivity of platelets (ADP agonist) and search for corrective agents.

A new method for studying platelets based on low-angle light scattering has previously revealed that platelets taken from pregnant women with preeclampsia are hypersensitive to ADP, with aggregation developing at concentrations of 7–15 nmol l−1. The method has been applied to further studies in experimental toxicology and clinical pathology. Toxicological experiments with fluoroacetate (FA), an inhibitor of TCA cycle, showed that the platelet hypersensitivity could also be caused by energy depletion. In modeling experiments, the low-angle light scattering method was applied to assessment of potential corrective agents of the pathological states related to hypersensitivity of platelets. Sodium glutamate (SG) was shown to be a potent antiaggregant in vitro, and subsequent in vivo studies demonstrated that SG can apparently serve as anaplerotic agent and normalize the platelet status of rats intoxicated with FA. Donators of nitric oxide (NO), such as isosorbide-5′-dinitrate, can also normalize in vitro the hypersensitive status of platelets taken from the patients with preeclampsia.

CHAPTER 7 – Russian VX

Publisher Summary This chapter describes the chemistry, analysis, toxicity, monitoring, and regulatory hygiene, and therapy of Russian VX nerve agent. One of the most abundant and most toxic chemical warfare agents in the chemical arsenals of the USA and Russia is VX and Russian VX respectively, whose development in the middle of the 20th century signified the peak of warfare chemistry. The arbitrary name VX relates to a group of O, S-diesters of methylphosphonic acid ROPO(CH3)S(CH2)2N(R1)2. V-gases are low-volatile liquids with high boiling points and, therefore, they are much more persistent than higher volatility organophosphorus (OP) agents of the G-series, such as sarin, soman, or tabun. V-series compounds are more toxic than OP nerve agents of the G-series. Poisoning occurs irrespective of route of exposure; specifically inhalation, ingestion of vaporous and liquid agents through intact or injured skin or eye mucosa, and on contact with contaminated surfaces. The most effective antidote complex for treating acute intoxications with RVX consists of an antagonist of M-cholinoceptors, a reversible inhibitor of cholinesterase, and a reactivator of cholinesterase. In addition, anticonvulsants can be used in cases where convulsions occur.

Polarographic and spectroscopic studies of the effects of fluoroacetate/fluorocitrate on cells and mitochondria

Experiments were performed with rat liver mitochondria, Ehrlich ascite tumor cells (EATC) and cardiomyocytes, exposed to fluoroacetate (FA) or fluorocitrate (FC) in vitro. The effects of FA developed at much higher concentrations in comparison with FC and was dependent upon respiratory substrates: with pyruvate, FA induced a slow oxidation of pyridine nucleotides (NAD(P)H) and inhibition of respiration. NAD(P)H oxidation was prevented by incubation of mitochondria with cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore. Studies of the NAD(P)H level and calcium response generated in EATC under activation with ATP via the metabotropic P2Y receptor, revealed a loss of NAD(P)H from mitochondria resulting in a shift in the balance of mitochondrial and cytosolic NAD(P)H on exposure to FA. An increase of cytosolic (Ca 2+ ) was observed in the cell lines exposed to FA and is explained by activation of plasma membrane calcium channels; this mechanism could have an impact on amplitude and rate of Ca 2+ waves in cardiomyocytes, and cause the hyper- sensitivity of platelets reported on earlier. Highlighting the reciprocal relationship between intracellular NAD(P)H and calcium balance, we discuss metabolic pathway modulation in the context of development of an effective therapy for FA poisoning.

Application of a low-angle light scattering technique to cell volume and cell signaling studies on Ehrlich ascite tumor cells

A method for studying cells based on low-angle light scattering was applied to cell volume and cell signaling studies on Ehrlich ascite tumor cells (EATC). Changes in the volume of EATC were measured in hypotonic medium, as well as after activation with exogenous ATP, ionomycin and thimerosal. Increase of [Ca2

Chapter 24 – Flow Cytometry and Light Scattering Technique in Evaluation of Nutraceuticals

Abstract Toxic and mechanistic properties of nutraceuticals are not as extensively studied as those of pharmaceuticals. Flow cytometry is among the most popular techniques to investigate the modes and mechanisms of cytotoxic action of chemical compounds, although flow cytometry methods are not suitable for online registration of fast changes of cell volumes. The latter is especially important for some types of cells, such as human erythrocytes with no nuclei and mitochondria. This chapter describes a new device (LaSca, Biomedsystems Ltd., Russia) for the registration of kinetic changes in cell volume, morphology, and aggregation. In addition, flow cytometry methods of cell viability assessment are overviewed, with attention given to mechanisms of cell death and survival, and some cellular studies of nutraceuticals using light scattering and flow cytometry techniques are summarized.

Protein kinase A activation by the anti-cancer drugs ABT-737 and thymoquinone is caspase-3-dependent and correlates with platelet inhibition and apoptosis

Chemotherapy-induced thrombocytopenia is a common bleeding risk in cancer patients and limits chemotherapy dose and frequency. Recent data from mouse and human platelets revealed that activation of protein kinase A/G (PKA/PKG) not only inhibited thrombin/convulxin-induced platelet activation but also prevented the platelet pro-coagulant state. Here we investigated whether or not PKA/PKG activation could attenuate caspase-dependent apoptosis induced by the anti-cancer drugs ABT-737 (the precursor of navitoclax) and thymoquinone (TQ), thereby potentially limiting chemotherapy-induced thrombocytopenia. This is particularly relevant as activation of cyclic nucleotide signalling in combination chemotherapy is an emerging strategy in cancer treatment. However, PKA/PKG-activation, as monitored by phosphorylation of Vasodilator-stimulated phosphoprotein (VASP), did not block caspase-3-dependent platelet apoptosis induced by the compounds. In contrast, both substances induced PKA activation themselves and PKA activation correlated with platelet inhibition and apoptosis. Surprisingly, ABT-737- and TQ-induced VASP-phosphorylation was independent of cAMP levels and neither cyclases nor phosphatases were affected by the drugs. In contrast, however, ABT-737- and TQ-induced PKA activation was blocked by caspase-3 inhibitors. In conclusion, we show that ABT-737 and TQ activate PKA in a caspase-3-dependent manner, which correlates with platelet inhibition and apoptosis and therefore potentially contributes to the bleeding risk in chemotherapy patients.