Data are accumulating on the hydrolytic activity of serum albumin towards esters and organophosphates. Previously, with the help of the technology of proton nuclear magnetic resonance (¹H NMR) spectroscopy, we observed the yield of acetate in the solution of bovine serum albumin and p-nitrophenyl acetate (NPA). Thus, we showed that albumin possesses true esterase activity towards NPA. Then, using the methods of molecular docking and molecular dynamics, we established site Sudlow I as the catalytic center of true esterase activity of albumin. In the present work, to expand our understanding of the molecular mechanisms of albumin pseudoesterase and true esterase activity, we investigated—in experiments in vitro and in silico—the interaction of anticoagulant warfarin (WRF, specific ligand of site Sudlow I) and benzodiazepine diazepam (DIA, specific ligand of site Sudlow II) with albumins of different species, and determined how the binding of WRF and DIA affects the hydrolysis of NPA by albumin. It was found that the characteristics of the binding modes of WRF in site Sudlow I and DIA in site Sudlow II of human (HSA), bovine (BSA), and rat (RSA) albumins have species differences, which are more pronounced for site Sudlow I compared to site Sudlow II, and less pronounced between HSA and RSA compared to BSA. WRF competitively inhibits true esterase activity of site Sudlow I towards NPA and does not affect the functioning of site Sudlow II. Diazepam can slow down true esterase activity of site Sudlow I in noncompetitive manner. It was concluded that site Sudlow I is more receptive to allosteric modulation compared to site Sudlow II.

Upon activation, platelets undergo rapid phenotypic transitions to maintain hemostasis, yet the kinetics governing these transitions remain poorly quantified. We present an integrated experimental and mathematical model describing platelet transitions between resting, activated, aggregating, inhibited, and exhausted phenotypes, determined by experiment rate constants for these reactions. Theoretical simulations of platelet transitions accurately describe the independently determined experimental read-out. Platelet aggregation under the conditions used directly correlates with the activation of αIIbβ3 integrins, demonstrating that the parameters of platelet aggregation achieved by the laser diffraction technique can be used for the evaluation of the rapid activation and deactivation kinetics of αIIbβ3 integrins. We demonstrate that platelet desensitization occurs at multiple activation stages, with distinct kinetic profiles for shape change and integrin deactivation. We also show that even 5 s of receptor-mediated PKA activation (iloprost) is sufficient for a complete inhibition of ADP-induced platelet aggregation. However, when iloprost was added after platelet stimulation by ADP, platelet activation was not fully inhibited, and after 180 s, aggregation became irreversible. The presented data help to understand the mechanisms of platelet transition between different phenotypes. The model effectively characterizes key physiological phenotypes and can serve as a modular framework for integration into more comprehensive models.

The health of animal populations can reflect ongoing changes in their environment. Various physiological parameters can be used to assess individual and population conditions. These parameters may be interrelated or independent, allowing for organisms to respond flexibly to environmental changes. We investigated the dynamics of the leukocyte profile (total number and relative proportions of various types of leukocytes) in an urban population of the Great tit (Parus major) during two stages of the annual cycle: autumn migration and wintering. We also analyzed the relations between the leukocyte profile and the energy state, hematocrit, and the levels of blood metabolites (glucose and β-hydroxybutyrate). We found the total number of leukocytes and lymphocytes to be higher during autumn migration compared to wintering, which indicates a more active state of the immune system in Great tits during migration. An increased number of lymphocytes (specific immunity) can reflect high probability of encountering already familiar infections during the relatively short-distance migrations typical for this species. The ratio of heterophils to lymphocytes (H : L ratio, a measure of long-term physiological stress) in this population was higher in winter than during autumn migration. We assume that this may be caused by low temperatures and increased competition for food in wintering birds. The total number of leukocytes correlated positively with energy reserves and negatively with the blood glucose level, possibly indicating a coordinated response of these physiological parameters to environmental cues. Our results also show that birds that have adequate energy reserves and low blood glucose concentrations (i.e., are in good condition) maintain a more active immune system. Reduced energy reserves (e.g., due to unfavorable feeding conditions, higher energy costs of both locomotor activity and thermogenesis in cold weather) caused an increase in blood glucose concentrations and a decrease in the number of leukocytes. The H : L ratio was not correlated with other physiological parameters, showing that the level of long-term physiological stress was related to neither the individual energetic condition nor the level of metabolism in Great tits under the observed environmental conditions. Taken together, our results show a low level of long-term physiological stress in the studied population of Great tits.

Platelet activation, shape change and aggregation are active processes that can be significantly dependent on the ambient temperature. However, there are conflicting data in the literature regarding the effect of temperature on platelets. In our work, we used a laser diffraction method to investigate the influence of temperature on the reaction of human platelets activated byvarious agonists: ADP, U46619 (thromboxane mimetic), and thrombin (TRAP-6), that act through G-protein coupled receptors, and collagen, that activates the immunoglobulin receptor GPVI. For agonists that activate G-protein coupled receptors, we showed that an increase in temperature causes an acceleration of the initial platelet activation (shape change) and has nosignificant effect on agonist sensitivity (EC₅₀). At the same time, hypothermia at low doses of such agonists potentiates platelet aggregation, which differs significantly from the effect of collagen. With increasing temperature, collagen accelerates platelet aggregation over the entire range of temperatures investigated. In this paper, we showed that the effect of temperature on platelet activation processes depends on both the dose of agonists and the type of activated receptors. In our study, we included healthy volunteers after obtaining a written informed consent. Blood samples were taken in accordance with the guidelines of the I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences and the Declaration of Helsinki. Studies with human erythrocytes were approved by the Ethics Committee of the I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences (Protocol No.3–03 dated 2 March 2021 and Protocol No.1–04 dated 7 April 2022).

Oxidative stress (OS) has a significant impact on the lifespan and physical fitness of living organisms. It is commonly associated with ageing and can lead to changes in the functionality of red blood cells (RBCs). The precise mechanisms underlying these changes are not fully understood. Unlike mammals, avian RBCs have a nucleus and functional mitochondria that regulate the cellular response to oxidative stress. In this study, we examined the effects of OS on red blood cells from adult female quail (Coturnix japonica, n=12). We used flow cytometry to analyze the formation of OS-induced microparticles and RBC transformation. We also evaluated band 3 clustering and phosphatidylserine externalization at the cell surface using eosin-5-maleimide and Annexin-V fluorescent probes, respectively. In addition, we analyzed band 3 clustering using confocal microscopy. We used a laser diffraction-based method to analyze cell deformability, and we characterized hemoglobin species spectrophotometrically. We found that OS caused band 3 clustering, microparticle formation, and phosphatidylserine release onto the cell membrane. The microparticles formed under the influence of oxidants differed from those formed under the influence of A23187 (calcium ionophore). The rate of microparticle formation and the onset of osmotic rigidity depended on the oxidant concentration. Erythrocyte-derived microparticles contained hemoglobin oxidized to hemichrome (HbChr). Overall, these findings demonstrate that avian erythrocytes undergo different processes during oxidative stress, depending on the level of oxidation. These differences are due to variations in cellular transformations and the formation of different types of microparticles. This research was supported by the Russian Fund for Basic Researches (grant no. 23-15-00142)