Dual role of the p38 MAPK/cPLA2MAPK/cPLA_2 pathway in the regulation of platelet apoptosis induced by ABT-737 and strong platelet agonists

p38 Mitogen-activated protein (MAP) kinase is involved in the apoptosis of nucleated cells. Although platelets are anucleated cells, apoptotic proteins have been shown to regulate platelet lifespan. However, the involvement of p38 MAP kinase in platelet apoptosis is not yet clearly defined. Therefore, we investigated the role of p38 MAP kinase in apoptosis induced by a mimetic of BH3-only proteins, ABT-737, and in apoptosis-like events induced by such strong platelet agonists as thrombin in combination with convulxin (Thr/Cvx), both of which result in p38 MAP kinase phosphorylation and activation. A p38 inhibitor (SB202190) inhibited the apoptotic events induced by ABT-737 but did not influence those induced by Thr/Cvx. The inhibitor also reduced the phosphorylation of cytosolic phospholipase $A_2$ (cPLA2), an established p38 substrate, induced by ABT-737 or Thr/Cvx. ABT-737, but not Thr/Cvx, induced the caspase 3-dependent cleavage and inactivation of cPLA2. Thus, p38 MAPK promotes ABT-737-induced apoptosis by inhibiting the cPLA2/arachidonate pathway. We also show that arachidonic acid (AA) itself and in combination with Thr/Cvx or ABT-737 at low concentrations prevented apoptotic events, whereas at high concentrations it enhanced such events. Our data support the hypothesis that the p38 MAPK-triggered arachidonate pathway serves as a defense mechanism against apoptosis under physiological conditions

Spectroscopy 19 (2005) 235-246 235 IOS Press A new method for studying platelets, based upon the low-angle light scattering technique. 1. Theoretical and experimental foundations of the method

Abstract. 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 (t 1/2 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 EC 50 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 EC 50 and maximum velocity of aggregation (Umax) values. The method has potential applications in pharmacology and toxicology research and in clinical practice, as a simple and highly sensitive functionality test for platelets

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+]iunder ATP and ionomycin action induced reversible changes of cell volume: fast shrinking was followed by swelling. Thimerosal caused a reversible change in EATC volume with high amplitude; endoplasmic reticulum played the key role in this response. Having obtained kinetic parameters of changes in cell volume under activation of the cells, quantitative measurements of K+, Na+and anion flows responsible for this process can then be obtained. In spite of some fundamental differences in the behavior of cells of different dimensions there are many similarities, and there is a good theoretical background for dealing with both small and large cells.</jats:p

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 EC50for 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.</jats:p

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 antiaggregantin vitro, and subsequentin vivostudies 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 normalizein vitrothe hypersensitive status of platelets taken from the patients with preeclampsia.</jats:p

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

One of a series of six papers on development and application of a low-angle light scattering technique (2005-2007). A diagnostic tool with a wide-range of biomedical applications