Analysis of intra-specific variations in the venom of individual snakes based on Raman spectroscopy.

The knowledge of variations in the composition of venoms from different snakes is important from both theoretical and practical points of view, in particular, at developing and selecting an antivenom. Many studies on this topic are conducted with pooled venoms, while the existence and significance of variations in the composition of venoms between individual snakes of the same species are emphasized by many authors. It is important to study both inter- and intra-specific, including intra-population, venom variations, because intra-specific variations in the venom composition may affect the effectiveness of antivenoms as strongly as inter-specific. In this work, based on venom Raman spectroscopy with principal component analysis, we assessed the variations in venoms of individual snakes of the Vipera nikolskii species from two populations and compared these intra-specific variations with inter-specific variations (with regard to the other related species). We demonstrated intra-specific (inter- and intra-population) differences in venom compositions which are smaller than inter-specific variations. We also assessed the compositions of V. nikolskii venoms from two populations to explain inter-population differences. The method used is rapid and requires virtually no preparation of samples, used in extremely small quantities, allowing the venoms of individual snakes to be analyzed. In addition, the method is informative and capable of detecting fairly subtle differences in the composition of venoms.

Efficient Expression in Leishmania tarentolae (LEXSY) of the Receptor-Binding Domain of the SARS-CoV-2 S-Protein and the Acetylcholine-Binding Protein from Lymnaea stagnalis.

Leishmania tarentolae (LEXSY) system is an inexpensive and effective expression approach for various research and medical purposes. The stated advantages of this system are the possibility of obtaining the soluble product in the cytoplasm, a high probability of correct protein folding with a full range of post-translational modifications (including uniform glycosylation), and the possibility of expressing multi-subunit proteins. In this paper, a LEXSY expression system has been employed for obtaining the receptor binding domain (RBD) of the spike-protein of the SARS-CoV-2 virus and the homopentameric acetylcholine-binding protein (AChBP) from Lymnaea stagnalis. RBD is actively used to obtain antibodies against the virus and in various scientific studies on the molecular mechanisms of the interaction of the virus with host cell targets. AChBP represents an excellent structural model of the ligand-binding extracellular domain of all subtypes of nicotinic acetylcholine receptors (nAChRs). Both products were obtained in a soluble glycosylated form, and their structural and functional characteristics were compared with those previously described.

Subtype-Selective Peptide and Protein Neurotoxic Inhibitors of Nicotinic Acetylcholine Receptors Enhance Proliferation of Patient-Derived Glioblastoma Cell Lines.

Glioblastoma multiforme (GBM) is the most aggressive type of brain cancer, with a poor prognosis. GBM cells, which develop in the environment of neural tissue, often exploit neurotransmitters and their receptors to promote their own growth and invasion. Nicotinic acetylcholine receptors (nAChRs), which play a crucial role in central nervous system signal transmission, are widely represented in the brain, and GBM cells express several subtypes of nAChRs that are suggested to transmit signals from neurons, promoting tumor invasion and growth. Analysis of published GBM transcriptomes revealed spatial heterogeneity in nAChR subtype expression, and functional nAChRs of α1*, α7, and α9 subtypes are demonstrated in our work on several patient-derived GBM microsphere cultures and on the U87MG GBM cell line using subtype-selective neurotoxins and fluorescent calcium mobilization assay. The U87MG cell line shows reactions to nicotinic agonists similar to those of GBM patient-derived culture. Selective α1*, α7, and α9 nAChR neurotoxins stimulated cell growth in the presence of nicotinic agonists. Several cultivating conditions with varying growth factor content have been proposed and tested. The use of selective neurotoxins confirmed that cell cultures obtained from patients are representative GBM models, but the use of media containing fetal bovine serum can lead to alterations in nAChR expression and functioning.

Comparison of Conformations and Interactions with Nicotinic Acetylcholine Receptors for E. coli-Produced and Synthetic Three-Finger Protein SLURP-1.

SLURP-1 is a three-finger human protein targeting nicotinic acetylcholine receptors (nAChRs). The recombinant forms of SLURP-1 produced in E. coli differ in added fusion fragments and in activity. The closest in sequence to the naturally occurring SLURP-1 is the recombinant rSLURP-1, differing by only one additional N-terminal Met residue. sSLURP-1 can be prepared by peptide synthesis and its amino acid sequence is identical to that of the natural protein. In view of recent NMR analysis of the conformational mobility of rSLURP-1 and cryo-electron microscopy structures of complexes of α-bungarotoxin (a three-finger snake venom protein) with Torpedo californica and α7 nAChRs, we compared conformations of sSLURP-1 and rSLURP-1 by Raman spectroscopy and CD-controlled thermal denaturation, analyzed their competition with α-bungarotoxin for binding to the above-mentioned nAChRs, compared the respective receptor complexes with computer modeling and compared their inhibitory potency on the α9α10 nAChR. The CD revealed a higher thermostability of sSLURP-1; some differences between sSLURP-1 and rSLURP-1 were observed in the regions of disulfides and tyrosine residues by Raman spectroscopy, but in binding, computer modeling and electrophysiology, the proteins were similar. Thus, sSLURP-1 and rSLURP-1 with only one additional Met residue appear close in structure and functional characteristics, being appropriate for research on nAChRs.

Dual Modulator of ASIC Channels and GABAA Receptors from Thyme Alters Fear-Related Hippocampal Activity.

Acid-sensing ion channels (ASICs) are proton-gated ion channels that mediate nociception in the peripheral nervous system and contribute to fear and learning in the central nervous system. Sevanol was reported previously as a naturally-occurring ASIC inhibitor from thyme with favorable analgesic and anti-inflammatory activity. Using electrophysiological methods, we found that in the high micromolar range, the compound effectively inhibited homomeric ASIC1a and, in sub- and low-micromolar ranges, positively modulated the currents of α1ß2γ2 GABAA receptors. Next, we tested the compound in anxiety-related behavior models using a targeted delivery into the hippocampus with parallel electroencephalographic measurements. In the open field, 6 µM sevanol reduced both locomotor and θ-rhythmic activity similar to GABA, suggesting a primary action on the GABAergic system. At 300 µM, sevanol markedly suppressed passive avoidance behavior, implying alterations in conditioned fear memory. The observed effects could be linked to distinct mechanisms involving GABAAR and ASIC1a. These results elaborate the preclinical profile of sevanol as a candidate for drug development and support the role of ASIC channels in fear-related functions of the hippocampus.

Analogs of 6-Bromohypaphorine with Increased Agonist Potency for α7 Nicotinic Receptor as Anti-Inflammatory Analgesic Agents.

Hypaphorines, tryptophan derivatives, have anti-inflammatory activity, but their mechanism of action was largely unknown. Marine alkaloid L-6-bromohypaphorine with EC50 of 80 µM acts as an agonist of α7 nicotinic acetylcholine receptor (nAChR) involved in anti-inflammatory regulation. We designed the 6-substituted hypaphorine analogs with increased potency using virtual screening of their binding to the α7 nAChR molecular model. Fourteen designed analogs were synthesized and tested in vitro by calcium fluorescence assay on the α7 nAChR expressed in neuro 2a cells, methoxy ester of D-6-iodohypaphorine (6ID) showing the highest potency (EC50 610 nM), being almost inactive toward α9α10 nAChR. The macrophages cytometry revealed an anti-inflammatory activity, decreasing the expression of TLR4 and increasing CD86, similarly to the action of PNU282987, a selective α7 nAChR agonist. 6ID administration in doses 0.1 and 0.5 mg/kg decreased carrageenan-induced allodynia and hyperalgesia in rodents, in accord with its anti-inflammatory action. Methoxy ester of D-6-nitrohypaphorine demonstrated anti-oedemic and analgesic effects in arthritis rat model at i.p. doses 0.05-0.26 mg/kg. Tested compounds showed excellent tolerability with no acute in vivo toxicity in dosages up to 100 mg/kg i.p. Thus, combining molecular modelling and natural product-inspired drug design improved the desired activity of the chosen nAChR ligand.

Differentiation of snake venom using Raman spectroscopic analysis.

Snake venoms are complex mixtures of different substances, proteins being their predominant components. To study the composition of venoms, methods based on chromatographic separation and mass spectrometric analysis are currently used, requiring the application of a number of sophisticated instruments. To assess the composition of snake venoms, we propose an alternative method based on Raman spectroscopy, which is an express method to study the structural features of different substances, including proteins. The method does not require preliminary preparation of the samples, which are used in small quantities; this makes Raman spectroscopy extremely attractive for venom research. In this work, we have carried out Raman spectroscopic studies on a number of dry venoms from various venomous snakes. Based on the obtained Raman spectra, with the help of mathematical methods of dimensionality reduction and clustering, differentiation of venoms reflecting their composition and the assignment of the venom producing snake to the corresponding family or even genus were performed. The proposed method can be used to analyze both the composition of and variations in venoms of different snake species, including rare and endangered ones.

Toxins' classification through Raman spectroscopy with principal component analysis.

The method based on the combination of Raman spectroscopy and principal component analysis (PCA) was applied to the set of peptide and protein toxins from animal venoms and to synthetic analogues of peptides. The study demonstrated the possibility of toxin classification according to their primary and secondary structures based on Raman spectroscopy. The method described here allows discrimination of snake venom three-finger toxins from predatory marine mollusks α-conotoxins. Moreover, PCA of the Raman spectra of toxins revealed differences within the group of three-finger toxins and also within the group of conotoxins, related to their spatial structure. In particular, on the basis of the developed technique it is possible to distinguish the disulfide isomers of the same peptide toxin. The results obtained have been confirmed by bioinformatic methods. So, we have proposed a method for the rapid analysis of newly discovered venom-derived protein or peptide toxins by establishing their similarity with other already studied toxins by referring to a particular class. Taking into account a low specimen consumption by Raman spectroscopy, the proposed method could represent a first step in the study of toxins from rare and/or endangered venomous animals. The ability to distinguish configuration of disulfide bonds allows to synthesize the correct isomer of the toxin.

Marine Origin Ligands of Nicotinic Receptors: Low Molecular Compounds, Peptides and Proteins for Fundamental Research and Practical Applications.

The purpose of our review is to briefly show what different compounds of marine origin, from low molecular weight ones to peptides and proteins, offer for understanding the structure and mechanism of action of nicotinic acetylcholine receptors (nAChRs) and for finding novel drugs to combat the diseases where nAChRs may be involved. The importance of the mentioned classes of ligands has changed with time; a protein from the marine snake venom was the first excellent tool to characterize the muscle-type nAChRs from the electric ray, while at present, muscle and α7 receptors are labeled with the radioactive or fluorescent derivatives prepared from α-bungarotoxin isolated from the many-banded krait. The most sophisticated instruments to distinguish muscle from neuronal nAChRs, and especially distinct subtypes within the latter, are α-conotoxins. Such information is crucial for fundamental studies on the nAChR revealing the properties of their orthosteric and allosteric binding sites and mechanisms of the channel opening and closure. Similar data are provided by low-molecular weight compounds of marine origin, but here the main purpose is drug design. In our review we tried to show what has been obtained in the last decade when the listed classes of compounds were used in the nAChR research, applying computer modeling, synthetic analogues and receptor mutants, X-ray and electron-microscopy analyses of complexes with the nAChRs, and their models which are acetylcholine-binding proteins and heterologously-expressed ligand-binding domains.

Peptides from the Sea Anemone Metridium senile with Modified Inhibitor Cystine Knot (ICK) Fold Inhibit Nicotinic Acetylcholine Receptors.

Nicotinic acetylcholine receptors (nAChRs) play an important role in the functioning of the central and peripheral nervous systems, and other organs of living creatures. There are several subtypes of nAChRs, and almost all of them are considered as pharmacological targets in different pathological states. The crude venom of the sea anemone Metridium senile showed the ability to interact with nAChRs. Four novel peptides (Ms11a-1-Ms11a-4) with nAChR binding activity were isolated. These peptides stabilized by three disulfide bridges have no noticeable homology with any known peptides. Ms11a-1-Ms11a-4 showed different binding activity towards the muscle-type nAChR from the Torpedo californica ray. The study of functional activity and selectivity for the most potent peptide (Ms11a-3) revealed the highest antagonism towards the heterologous rat α9α10 nAChR compared to the muscle and α7 receptors. Structural NMR analysis of two toxins (Ms11a-2 and Ms11a-3) showed that they belong to a new variant of the inhibitor cystine knot (ICK) fold but have a prolonged loop between the fifth and sixth cysteine residues. Peptides Ms11a-1-Ms11a-4 could represent new pharmacological tools since they have structures different from other known nAChRs inhibitors.

Snake Toxins Labeled by Green Fluorescent Protein or Its Synthetic Chromophore are New Probes for Nicotinic acetylcholine Receptors.

Fluorescence can be exploited to monitor intermolecular interactions in real time and at a resolution up to a single molecule. It is a method of choice to study ligand-receptor interactions. However, at least one of the interacting molecules should possess good fluorescence characteristics, which can be achieved by the introduction of a fluorescent label. Gene constructs with green fluorescent protein (GFP) are widely used to follow the expression of the respective fusion proteins and monitor their function. Recently, a small synthetic analogue of GFP chromophore (p-HOBDI-BF2) was successfully used for tagging DNA molecules, so we decided to test its applicability as a potential fluorescent label for proteins and peptides. This was done on α-cobratoxin (α-CbTx), a three-finger protein used as a molecular marker of muscle-type, neuronal α7 and α9/α10 nicotinic acetylcholine receptors (nAChRs), as well as on azemiopsin, a linear peptide neurotoxin selectively inhibiting muscle-type nAChRs. An activated N-hydroxysuccinimide ester of p-HOBDI-BF2 was prepared and utilized for toxin labeling. For comparison we used a recombinant α-CbTx fused with a full-length GFP prepared by expression of a chimeric gene. The structure of modified toxins was confirmed by mass spectrometry and their activity was characterized by competition with iodinated α-bungarotoxin in radioligand assay with respective receptor preparations, as well as by thermophoresis. With the tested protein and peptide neurotoxins, introduction of the synthetic GFP chromophore induced considerably lower decrease in their affinity for the receptors as compared with full-length GFP attachment. The obtained fluorescent derivatives were used for nAChR visualization in tissue slices and cell cultures.

Snake venom phospholipase A2s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2.

The COVID-19 pandemic caused by SARS-CoV-2 requires new treatments both to alleviate the symptoms and to prevent the spread of this disease. Previous studies demonstrated good antiviral and virucidal activity of phospholipase A2s (PLA2s) from snake venoms against viruses from different families but there was no data for coronaviruses. Here we show that PLA2s from snake venoms protect Vero E6 cells against SARS-CoV-2 cytopathic effects. PLA2s showed low cytotoxicity to Vero E6 cells with some activity at micromolar concentrations, but strong antiviral activity at nanomolar concentrations. Dimeric PLA2 from the viper Vipera nikolskii and its subunits manifested especially potent virucidal effects, which were related to their phospholipolytic activity, and inhibited cell-cell fusion mediated by the SARS-CoV-2 spike glycoprotein. Moreover, PLA2s interfered with binding both of an antibody against ACE2 and of the receptor-binding domain of the glycoprotein S to 293T/ACE2 cells. This is the first demonstration of a detrimental effect of PLA2s on ß-coronaviruses. Thus, snake PLA2s are promising for the development of antiviral drugs that target the viral envelope, and could also prove to be useful tools to study the interaction of viruses with host cells.

Novel Three-Finger Neurotoxins from Naja melanoleuca Cobra Venom Interact with GABAA and Nicotinic Acetylcholine Receptors.

Cobra venoms contain three-finger toxins (TFT) including α-neurotoxins efficiently binding nicotinic acetylcholine receptors (nAChRs). As shown recently, several TFTs block GABAA receptors (GABAARs) with different efficacy, an important role of the TFTs central loop in binding to these receptors being demonstrated. We supposed that the positive charge (Arg36) in this loop of α-cobratoxin may explain its high affinity to GABAAR and here studied α-neurotoxins from African cobra N. melanoleuca venom for their ability to interact with GABAARs and nAChRs. Three α-neurotoxins, close homologues of the known N. melanoleuca long neurotoxins 1 and 2, were isolated and sequenced. Their analysis on Torpedocalifornica and α7 nAChRs, as well as on acetylcholine binding proteins and on several subtypes of GABAARs, showed that all toxins interacted with the GABAAR much weaker than with the nAChR: one neurotoxin was almost as active as α-cobratoxin, while others manifested lower activity. The earlier hypothesis about the essential role of Arg36 as the determinant of high affinity to GABAAR was not confirmed, but the results obtained suggest that the toxin loop III may contribute to the efficient interaction of some long-chain neurotoxins with GABAAR. One of isolated toxins manifested different affinity to two binding sites on Torpedo nAChR.

α-Conotoxins and α-Cobratoxin Promote, while Lipoxygenase and Cyclooxygenase Inhibitors Suppress the Proliferation of Glioma C6 Cells.

Among the brain tumors, glioma is the most common. In general, different biochemical mechanisms, involving nicotinic acetylcholine receptors (nAChRs) and the arachidonic acid cascade are involved in oncogenesis. Although the engagement of the latter in survival and proliferation of rat C6 glioma has been shown, there are practically no data about the presence and the role of nAChRs in C6 cells. In this work we studied the effects of nAChR antagonists, marine snail α-conotoxins and snake α-cobratoxin, on the survival and proliferation of C6 glioma cells. The effects of the lipoxygenase and cyclooxygenase inhibitors either alone or together with α-conotoxins and α-cobratoxin were studied in parallel. It was found that α-conotoxins and α-cobratoxin promoted the proliferation of C6 glioma cells, while nicotine had practically no effect at concentrations below 1 µL/mL. Nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor, and baicalein, a 12-lipoxygenase inhibitor, exerted antiproliferative and cytotoxic effects on C6 cells. nAChR inhibitors weaken this effect after 24 h cultivation but produced no effects at longer times. Quantitative real-time polymerase chain reaction showed that mRNA for α4, α7, ß2 and ß4 subunits of nAChR were expressed in C6 glioma cells. This is the first indication for involvement of nAChRs in mechanisms of glioma cell proliferation.

Point Mutations of Nicotinic Receptor α1 Subunit Reveal New Molecular Features of G153S Slow-Channel Myasthenia.

Slow-channel congenital myasthenic syndromes (SCCMSs) are rare genetic diseases caused by mutations in muscle nicotinic acetylcholine receptor (nAChR) subunits. Most of the known SCCMS-associated mutations localize at the transmembrane region near the ion pore. Only two SCCMS point mutations are at the extracellular domains near the acetylcholine binding site, α1(G153S) being one of them. In this work, a combination of molecular dynamics, targeted mutagenesis, fluorescent Ca2+ imaging and patch-clamp electrophysiology has been applied to G153S mutant muscle nAChR to investigate the role of hydrogen bonds formed by Ser 153 with C-loop residues near the acetylcholine-binding site. Introduction of L199T mutation to the C-loop in the vicinity of Ser 153 changed hydrogen bonds distribution, decreased acetylcholine potency (EC50 2607 vs. 146 nM) of the double mutant and decay kinetics of acetylcholine-evoked cytoplasmic Ca2+ rise (τ 14.2 ± 0.3 vs. 34.0 ± 0.4 s). These results shed light on molecular mechanisms of nAChR activation-desensitization and on the involvement of such mechanisms in channelopathy genesis.

Interaction of α9α10 Nicotinic Receptors With Peptides and Proteins From Animal Venoms.

Unlike most neuronal nicotinic acetylcholine receptor (nAChR) subunits, α7, α9, and α10 subunits are able to form functional homo- or heteromeric receptors without any ß subunits. While the α7 subtype is widely distributed in the mammalian brain and several peripheral tissues, α9 and α9α10 nAChRs are mainly found in the cochlea and immune cells. α-Conotoxins that specifically block the α9α10 receptor showed anti-nociceptive and anti-hyperalgesic effects in animal models. Hence, this subtype is considered a drug target for analgesics. In contrast to the α9α10-selective α-conotoxins, the three-finger toxin α-bungarotoxin inhibits muscle-type and α7 nAChRs in addition to α9α10 nAChRs. However, the selectivity of α-neurotoxins at the α9α10 subtype was less intensively investigated. Here, we compared the potencies of α-conotoxins and α-neurotoxins at the human α9α10 nAChR by two-electrode voltage clamp analysis upon expression in Xenopus oocytes. In addition, we analyzed effects of several α9α10-selective α-conotoxins on mouse granulocytes from bone marrow to identify possible physiological functions of the α9α10 nAChR subtype in these cells. The α-conotoxin-induced IL-10 release was measured upon LPS-stimulation. We found that α-conotoxins RgIA, PeIA, and Vc1.1 enhance the IL-10 expression in granulocytes which might explain the known anti-inflammatory and associated analgesic activities of α9α10-selective α-conotoxins. Furthermore, we show that two long-chain α-neurotoxins from the cobra Naja melanoleuca venom that were earlier shown to bind to muscle-type and α7 nAChRs, also inhibit the α9α10 subtype at nanomolar concentrations with one of them showing a significantly slower dissociation from this receptor than α-bungarotoxin.

Does Cholinergic Stimulation Affect the P2X7 Receptor-Mediated Dye Uptake in Mast Cells and Macrophages?

Background: Extracellular ATP is a powerful trigger of neuroinflammation by activating immune cells via P2X7 receptors. Acetylcholine and nicotinic agonists inhibit ATP-triggered proinflammatory cytokines via the so-called "cholinergic anti-inflammatory pathway" (CAP). However, it remains unclear as to what stage of ATP-induced signaling cholinergic agents provide this anti-inflammatory effect. Using the specific property of P2X7 receptor to open a pathway permeable to large molecules, associated with activation of inflammasome, we studied the action of cholinergic agents on this key event in CAP activation. Methods: Freshly isolated mouse peritoneal mast cells and primary human macrophages were used. To assess P2X7 channel opening, the permeability to the fluorescent dye YO-PRO1 or ethidium bromide (EtBr) was measured by flow cytometry. Expression of nicotinic receptors was probed in macrophages with the fluorescently labeled α-bungarotoxin or with patch-clamp recordings. Results: ATP opened P2X7 ion channels in mast cells and macrophages permeable to YO-PRO1 or EtBr, respectively. This stimulatory effect in mast cells was inhibited by the specific P2X7 antagonist A839977 confirming that YO-PRO1 uptake was mediated via ATP-gated P2X7 ion channels. Cholinergic agents also slightly induced dye uptake to mast cells but not in macrophages, which expressed functional α7 nicotinic receptors. However, both in mast cells and in macrophages, acetylcholine and nicotine failed to inhibit the stimulatory effect of ATP on dye uptake. Conclusion: These data suggest that in immune cells, cholinergic agents do not act on P2X7 receptor-coupled large pore formation but can mediate the anti-inflammatory effect underlying CAP downstream of ATP-driven signaling.

High Selectivity of an α-Conotoxin LvIA Analogue for α3ß2 Nicotinic Acetylcholine Receptors Is Mediated by ß2 Functionally Important Residues.

The α3ß2 and α3ß4 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the central and peripheral nervous systems, playing critical roles in various physiological processes and in such pathologies as addiction to nicotine and other drugs of abuse. α-Conotoxin LvIA, which we previously isolated from Conus lividus, modestly discriminates α3ß2 and α3ß4 rat nAChRs exhibiting a ∼17-fold tighter binding to the former. Here, alanine scanning resulted in two more selective analogues [N9A]LvIA and [D11A]LvIA, the former having a >2000-fold higher selectivity for α3ß2. The determined crystal structures of [N9A]LvIA and [D11A]LvIA bound to the acetylcholine-binding protein (AChBP) were followed by homologous modeling of the complexes with the α3ß2 and α3ß4 nAChRs and by receptor mutagenesis, which revealed Phe106, Ser108, Ser113, and Ser168 residues in the ß2 subunit as essential for LvIA binding. These results may be useful for the design of novel compounds of therapeutic potential targeting α3ß2 nAChRs.

Ultrafast, Ultrasensitive Detection and Imaging of Single Cardiac Troponin-T Molecules.

The fluorescence-based methods of single-molecule optical detection have opened up unprecedented possibilities for imaging, monitoring, and sensing at a single-molecule level. However, single-molecule detection methods are very slow, making them practically inapplicable. In this paper, we show how to overcome this key limitation using the expanded laser spot, laser excitation in a nonfluorescent spectral window of biomolecules, and more binding fluorescent molecules on a biomolecule that increases the detection volume and the number of collected photons. We demonstrate advantages of the developed approach unreachable by any other technique using detection of single cardiac troponin-T molecules: (i) 1000-fold faster than by known approaches, (ii) real-time imaging of single troponin-T molecules dissolved in human blood serum, (iii) measurement of troponin-T concentration with a clinically important sensitivity of about 1 pg/mL. The developed approach can be used for ultrafast, ultrasensitive detection, monitoring, and real-time imaging of other biomolecules as well as of larger objects including pathogenic viruses and bacteria.

Assessment of Fibrinogen Macromolecules Interaction with Red Blood Cells Membrane by Means of Laser Aggregometry, Flow Cytometry, and Optical Tweezers Combined with Microfluidics.

An elevated concentration of fibrinogen in blood is a significant risk factor during many pathological diseases, as it leads to an increase in red blood cells (RBC) aggregation, resulting in hemorheological disorders. Despite the biomedical importance, the mechanisms of fibrinogen-induced RBC aggregation are still debatable. One of the discussed models is the non-specific adsorption of fibrinogen macromolecules onto the RBC membrane, leading to the cells bridging in aggregates. However, recent works point to the specific character of the interaction between fibrinogen and the RBC membrane. Fibrinogen is the major physiological ligand of glycoproteins receptors IIbIIIa (GPIIbIIIa or αIIßß3 or CD41/CD61). Inhibitors of GPIIbIIIa are widely used in clinics for the treatment of various cardiovascular diseases as antiplatelets agents preventing the platelets' aggregation. However, the effects of GPIIbIIIa inhibition on RBC aggregation are not sufficiently well studied. The objective of the present work was the complex multimodal in vitro study of the interaction between fibrinogen and the RBC membrane, revealing the role of GPIIbIIIa in the specificity of binding of fibrinogen by the RBC membrane and its involvement in the cells' aggregation process. We demonstrate that GPIIbIIIa inhibition leads to a significant decrease in the adsorption of fibrinogen macromolecules onto the membrane, resulting in the reduction of RBC aggregation. We show that the mechanisms underlying these effects are governed by a decrease in the bridging components of RBC aggregation forces.