Key Points: Temporal profiles of >4000 phosphopeptides after stimulating human platelets (a) with ADP and (b) consecutively with ADP and Iloprost. Reciprocal phosphorylation profiles of ADP and Iloprost point to central players of platelet homeostasis.

Extracellular vesicles (EVs) released by different cell types play an important role in many physiological and pathophysiological processes. In physiological conditions, red blood cell (RBC)-derived EVs compose 4–8% of all circulating EVs, and oxidative stress (OS) as a consequence of different pathophysiological conditions significantly increases the amount of circulated RBC-derived EVs. However, the mechanisms of EV formation are not yet fully defined. To analyze OS-induced EV formation and RBC transformations, we used flow cytometry to evaluate cell esterase activity, caspase-3 activity, and band 3 clustering. Band 3 clustering was additionally analyzed by confocal microscopy. Two original laser diffraction-based approaches were used for the analysis of cell deformability and band 3 activity. Hemoglobin species were characterized spectrophotometrically. We showed that cell viability in tert-Butyl hydroperoxide-induced OS directly correlated with oxidant concentration to cell count ratio, and that RBC-derived EVs contained hemoglobin oxidized to hemichrome (HbChr). OS induced caspase-3 activation and band 3 clustering in cells and EVs. Importantly, we showed that OS-induced EV formation is independent of calcium. The presented data indicated that during OS, RBCs eliminated HbChr by vesiculation in order to sacrifice the cell itself, thereby prolonging lifespan and delaying the untimely clearance of in all other respects healthy RBCs.

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 A₂ (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.

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.

Red blood cells (RBCs) are the most numerous cells in the body and perform gas exchange between all tissues. During the infusion of cancer chemotherapeutic (CT) agents, blood cells are the first ones to encounter aggressive cytostatics. Erythrocyte dysfunction caused by direct cytotoxic damage might be a part of the problem of chemotherapy-induced anemia—one of the most frequent side effects. The aim of the current study is to evaluate the functional status of RBCs exposed to mono and combinations of widely used commercial pharmaceutical CT drugs with different action mechanisms: paclitaxel, carboplatin, cyclophosphamide, and doxorubicin, in vitro. Using laser diffraction, flow cytometry, and confocal microscopy, we show that paclitaxel, having a directed effect on cytoskeleton proteins, by itself and in combination with carboplatin, caused the most marked abnormalities—loss of control of volume regulation, resistance to osmotic load, and stomatocytosis. Direct simulations of RBCs’ microcirculation in microfluidic channels showed both the appearance of a subpopulation of cells with impaired velocity (slow damaged cells) and an increased number of cases of occlusions. In contrast to paclitaxel, such drugs as carboplatin, cyclophosphamide, and doxorubicin, whose main target in cancer cells is DNA, showed significantly less cytotoxicity to erythrocytes in short-term exposure. However, the combination of drugs had an additive effect. While the obtained results should be confirmed in in vivo models, one can envisioned that such data could be used for minimizing anemia side effects during cancer chemotherapy.