Pathologies such as malaria, hemorrhagic stroke, sickle cell disease, and thalassemia are characterized by the release of hemoglobin degradation products from damaged RBCs. Hematin (liganded with OH⁻) and hemin (liganded with Cl⁻)—are the oxidized forms of heme with toxic properties due to their hydrophobicity and the presence of redox-active Fe³. In the present study, using the original LaSca-TM laser particle analyzer, flow cytometry, and confocal microscopy, we showed that both hematin and hemin induce dose-dependent RBC spherization and hemolysis with ghost formation. Hematin and hemin at nanomolar concentrations increased [Ca²⁺]_i in RBC; however, spherization and hemolysis occurred in the presence and absence of calcium, indicating that both processes are independent of [Ca²⁺]_i. Both compounds triggered acute phosphatidylserine exposure on the membrane surface, reversible after 60 min of incubation. A comparison of hematin and hemin effects on RBCs revealed that hematin is a more reactive toxic metabolite than hemin towards human RBCs. The toxic effects of heme derivatives were reduced and even reversed in the presence of albumin, indicating the presence in RBCs of the own recovery system against the toxic effects of heme derivatives.

Hemolysis during severe diseases (malaria, hemorrhagic stroke, sickle cell disease, etc.) and blood transfusion induces the release of free hemoglobin, which degrades to highly reactive and toxic compounds—hemin and hematin. Oxidized heme derivatives induce platelet activation, aggregation, and degranulation, leading to prothrombotic and inflammatory events. In the present study, we showed that hematin is a more potent agonist of platelet activation than hemin, and using several methods, including the original laser diffraction method, flow cytometry, and confocal microscopy, we demonstrated that hematin at low doses induces platelet activation and aggregation without reducing cell viability and affecting calcium efflux. On the contrary, hematin at high concentrations triggered phosphatidylserine exposure, severe loss of platelet viability, and calcium dysregulation, which was not inhibited by cGMP/PKG and cAMP/PKA pathways. Additionally, we showed that albumin could initiate disaggregation processes in hematin-activated platelets.

Thymoquinone (TQ), the main bioactive ingredient of Nigella sativa, exhibits numerous pharmacological activities and is used for the prevention of many diseases including hypertension and cancer. However, information concerning the effects of TQ on platelets is limited. In this study, we used the upgraded laser microparticle analyzer LaSca-TMF for simultaneous analysis of platelet shape change, aggregation, and changes in [Ca²⁺]_i. We showed that TQ acutely inhibited platelet aggregation induced by ADP, Trap-6, and CRP; however, the rise of [Ca²⁺]_i was inhibited only in CRP-stimulated platelets, but not in ADP- or Trap-6-stimulated ones. DTT, a thiol-reducing agent, prevented TQ-induced effects in platelets, indicating that protein disulfide isomerases could be involved in the regulation of TQ effects on platelets. Our results, for the first time, demonstrated acute inhibitory effects of TQ on platelet activation induced by GPCRs and ITAM-containing receptors, which were independent of PKA and caspase-3 activation. To the best of our knowledge, this is the first example in which complete inhibition of ADP- and Trap-6-, but not CRP-induced, aggregation is accompanied by high [Ca²⁺]_i levels. Additional experimental approaches are required to explain some effects of TQ on calcium homeostasis and TQ could be a valuable molecule for the analysis of calcium homeostasis in platelets and other cells.

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).