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 osmotic fragility of erythrocytes is a parameter that reflects the ability of cells to endure variations in the osmotic environment. The impairment of this ability is often associated with a variety of pathologies, encompassing hemolytic anemias, malignant tumors, and cardiovascular dysfunctions. Osmotic fragility exhibits variability across different animal taxa and is closely related with ecosystems. We developed the method for assessing osmotic fragility using a laser particle size analyzer, which facilitates real-time kinetic monitoring of cell concentration changes under controlled temperature conditions. The species examined included Homo sapiens, Rattus norvegicus domestica, Coturnix japonica domestica, Rana ridibunda, Carassius carassius, and Lampetra fluviatilis. The methodology was presented in two variants: (1) manual water additions and (2) automated medium dilution. The key parameters characterizing osmotic fragility included H ₅₀ (the osmolality that lysed half of lysis-susceptible cells), H ₉₀ (90% cell lysis), and W (population heterogeneity by the degree of lysis resistance). In terms of H ₅₀ and W, the results obtained via this method did not differ significantly from those obtained by spectrophotometry and flow cytometry. No significant differences were also observed between the outcomes of automated and manual variants of the method. Erythrocytes of aquatic and semiaquatic animals exhibited significantly higher resistance to hypotonic lysis. Among all species examined, amphibian (Rana ridibunda) and lamprey (Lampetra fluviatilis) erythrocytes demonstrated the lowest osmotic fragility. The most pronounced variability in the degree of lysis resistance was detected among amphibians, with an almost twofold difference compared to other taxa examined. While mammalian (human and rat) erythrocytes exhibited similar fragility levels, they were more variable in their resistance profiles. Avian erythrocytes demonstrated a half-lysis occurrence at higher osmolality levels compared to mammalian erythrocytes, although in the domestic quail (Coturnix japonica domestica), erythrocytes lysed over a considerably wider osmotic range and contained a subset of cells resistant to hypotonic lysis. These findings indicate that erythrocytes of lower vertebrates share a lower osmotic fragility compared to those of higher vertebrates, a phenomenon most likely attributable to embryonic characteristics, ecto-/endothermy, and habitat considerations.

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).