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Abstract  Gravitational unloading is known to produce changes in the expression of a number of contractile and regulatory proteins in the soleus muscle. This applies particularly to isoforms of myosin heavy chains (MHC) and SERCA sarcoplasmic reticulum calcium pumps. Unloading increases the resting levels of extracellular calcium in soleus muscle fibers. The present study addresses verification of the hypothesis that changes in the expression of MHC and SERCA isoforms in gravitational unloading are linked with the accumulation of calcium ions in the myoplasm of muscle fibers. It is suggested that specific blockade of L-type calcium channels using nifedipine decreases the myoplasmic calcium ion concentration, thus preventing the development of changes in the expression of MHC and SERCA isoforms. A total of 36 male Wistar rats were divided into three groups: a control group, an unloading group using the Morley-Holton soleus muscle functional unloading model, and an unloading + nifedipine group, where animals received daily nifedipine (7 mg/kg/day) with their drinking water on the background of suspension. The results showed that blockade of L-type calcium channels on the background of gravitational unloading significantly decreased the extent of calcium ion accumulation in the myoplasm of soleus muscle fibers, which partly prevented the transformation of muscle fibers (in relation to the fast and slow isoforms of MHC and SERCA) to the rapid type. There was no nuclear translocation of the greater part of transcription factor NFATc1, as seen on unloading. [ABSTRACT FROM AUTHOR]

Abstract  Effect of functional unloading on the course of degenerative process in muscles was studied in dystrophin-deficient mdx mice. Head down-hanging of animals led to a significant decrease in the cross-section area of muscle fibers, increase in the percentage of fibers with centrally located nuclei and of Evans blue-stained fibers. Gravitation unloading of 12-month-old animals with pronounced manifestations of muscular dystrophy did not inhibit this pathological process. [ABSTRACT FROM AUTHOR]

The study investigated the effects of chronic low-frequency electrical stimulation (CLFES) of the stretched knee extensor muscles versus CLFES without a resistance load. A total of 19 male volunteers were randomized into two groups with similar aerobic and force-velocity properties. In group 1, anterior thigh muscles (the knee extensors) of both legs were exposed to CLFES (15 Hz). In group 2, the CLFES of the knee extensors was combined with stretching. The stimulated subjects demonstrated a marked tendency towards increasing endurance and a significant increase (by 10%) in both succinate dehydrogenase activity and the percentage of muscle fibers containing slow myosin heavy chain (MHC) isoforms. At the same time, the proportion of fibers reacting with the antibody against fast MHC isoforms decreased by 12%. In group 1, the maximum voluntary force (MVF) significantly decreased, the volume of m. quadriceps femoris (as measured by magnetic resonance imaging) remained unchanged, and the size of the fast fibers slightly decreased (by 11%). In group 2, no significant decrease in the MVF was observed, while there was a significant increase in muscle volume and the cross-sectional area of muscle fibers. [ABSTRACT FROM AUTHOR]

Background: Skeletal muscle resident fibro-adipogenic progenitor cells (FAPs) control skeletal muscle regeneration providing a supportive role for muscle stem cells. Altered FAPs characteristics have been shown for a number of pathological conditions, but the influence of temporary functional unloading of healthy skeletal muscle on FAPs remains poorly studied. This work is aimed to investigate how skeletal muscle disuse affects the functionality and metabolism of FAPs., Methods: Hindlimb suspension (HS) rat model employed to investigate muscle response to decreased usage. FAPs were purified from m. soleus functioning muscle (Contr) and after functional unloading for 7 and 14 days (HS7 and HS14). FAPs were expanded in vitro, and tested for: immunophenotype; in vitro expansion rate, and migration activity; ability to differentiate into adipocytes in vitro; metabolic changes. Crosstalk between FAPs and muscle stem cells was estimated by influence of medium conditioned by FAP's on migration and myogenesis of C2C12 myoblasts. To reveal the molecular mechanisms behind unloading-induced alterations in FAP's functionality transcriptome analysis was performed., Results: FAPs isolated from Contr and HS muscles exhibited phenotype of MSC cells. FAPs in vitro expansion rate and migration were altered by functional unloading conditions. All samples of FAPs demonstrated the ability to adipogenic differentiation in vitro, however, HS FAPs formed fat droplets of smaller volume and transcriptome analysis showed fatty acids metabolism and PPAR signaling suppression. Skeletal muscle unloading resulted in metabolic reprogramming of FAPs: decreased spare respiratory capacity, decreased OCR/ECAR ratio detected in both HS7 and HS14 samples point to reduced oxygen consumption, decreased potential for substrate oxidation and a shift to glycolytic metabolism. Furthermore, C2C12 cultures treated with medium conditioned by FAPs showed diverse alterations: while the HS7 FAPs-derived paracrine factors supported the myoblasts fusion, the HS14-derived medium stimulated proliferation of C2C12 myoblasts; these observations were supported by increased expression of cytokines detected by transcriptome analysis., Conclusion: the results obtained in this work show that the skeletal muscle functional unloading affects properties of FAPs in time-dependent manner: in atrophying skeletal muscle FAPs act as the sensors for the regulatory signals that may stimulate the metabolic and transcriptional reprogramming to preserve FAPs properties associated with maintenance of skeletal muscle homeostasis during unloading and in course of rehabilitation., Competing Interests: Declarations. Ethics approval and consent to participate: All procedures with animals in this study were approved by the Biomedicine Ethics Committee of the Institute of Biomedical Problems of the Russian Academy of Sciences/Physiology section of the Russian Bioethics Committee (protocol no. 581 and 582, 28.05.2021). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Support afferentation in recent years was shown to be a key physiological stimulus controlling postural muscle function, structure and phenotype. Lack of support afferentation under various types of muscle disuse leads to a decline of size and percentage of slow-type fatigue-resistant muscle fibers, which can negatively affect muscle performance and life quality. In this study we simulated support afferentation during rat hindlimb unloading and investigated its effect on postural soleus muscle functional properties and signaling. Plantar mechanical stimulation prevented the unloading-induced muscle fatigue increase, maintained the level of mitochondrial DNA copy number and the percent of slow-type muscle fibers and partially prevented the increase of CpG methylation in pgc 1α promoter region and decline in myonuclear content of several transcriptional activators of slow myosin and PGC1 α expression. So, support afferentation under hindlimb suspension leads to maintaining of a slow-twitch oxidative and fatigue-resistant soleus muscle fibers phenotype., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: Coagulation abnormalities are common following major trauma. The aim of this study was to assess the improvement of trauma-induced coagulopathy (TIC) in an in vitro model., Methods: TIC was created on blood taken from healthy individuals by inducing hemodilution, acidosis, hypothermia and fibrinolysis. Next, blood samples were subjected to rotational thromboelastometry to assess the effect of hemostasis modulators on blood coagulation and fibrinolysis., Results: Introducing to blood fibrinogen at 0.75 mg/mL, prothrombin complex concentrate at 0.66 IU/mL or tranexamic acid at 95 µg/mL increased clot strength. Higher effect was observed by combination of fibrinogen with tranexamic acid and prothrombin complex with tranexamic acid, whereas the maximal effect was achieved using all agents together. Fibrinolysis was inhibited by tranexamic acid and stronger by triple combination of the agents. Selective treating the TIC blood with fibrinogen, prothrombin complex or tranexamic acid at two time lower concentrations did not affect clot strength. Combining fibrinogen with prothrombin complex or with tranexamic acid stimulated clot strength but at lower extent compared to higher concentrations. Lysis onset time was prolonged by tranexamic acid. Maximal effect on both clot formation and fibrinolysis was achieved using all three agents together., Conclusions: Blood clotting stimulation and fibrinolysis inhibition in the TIC model was enough combining subthreshold concentrations of fibrinogen, prothrombin complex and tranexamic acid. Further experiments are warranted in both in vitro and in vivo conditions with minimally effective concentrations of both pro-coagulant and anti-fibrinolytic drugs assuming that this combinatorial approach may not only improve coagulopathy but also minimize the risk of thrombotic complications., (© 2020. Springer-Verlag GmbH Germany, part of Springer Nature.)

Both research conducted under microgravity conditions and ground-based space analog studies have shown that air pump-based plantar mechanical stimulation (PMS) of cutaneous mechanoreceptors of the sole of the foot is able to increase neuromuscular activity in the musculature of the lower limbs. This type of stimulation is able to attenuate unloading-induced skeletal muscle atrophy and impaired muscle function. The aim of the present study was to evaluate the effects of PMS on anabolic signaling pathways in rat soleus muscle following 7-day hindlimb suspension (HS) and to elucidate if the effects of PMS on anabolic processes would be NO-dependent. The soles of the feet were stimulated with a frequency of 1-s inflation/1-s deflation with a total of 20 min followed by 10 min rest. This cycle was repeated for 4 h each day. We observed a decrease in the soleus muscle mass after 7-day HS, which was not prevented by PMS. We also observed a decrease in slow-type fiber cross-sectional area (CSA) by 56%, which significantly exceeded a decrease (-22%) in fast-type fiber CSA. PMS prevented a reduction in slow-twitch fiber CSA, but had no effect on fast-twitch fiber CSA. PMS prevented a 63% decrease in protein synthesis after 7-day HS as well as changes in several key anabolic signaling regulators, such as p70S6k, 4E-BP1, GSK3β, eEF-2, p90RSK. PMS also prevented a decrease in the markers of translational capacity (18S and 28S rRNA, c-myc, 45S pre-rRNA). Some effects of PMS on anabolic signaling were altered due to NO-synthase inhibitor (L-NAME) administration. Thus, PMS is able to partially prevent atrophic processes in rat soleus muscle during 7-day HS, affecting slow-type muscle fibers. This effect is mediated by alterations in anabolic signaling pathways and may depend on NO-synthase activity.

: Trauma-induced coagulopathy (TIC) occurs commonly as a second event following severe injury. We evaluated the effects of fibrinogen, recombinant factor VIIa and factor XIII on blood clotting and fibrinolysis in an in-vitro TIC model. The TIC model included hemodilution, hyperfibrinolysis, acidosis and hypothermia. The extent of clot formation and fibrinolysis was evaluated using rotational thromboelastometry. Clot strength was increased following spiking the TIC blood with either 1.0 mg/ml fibrinogen, 3.0 μg/ml recombinant factor VIIa or 2.0 IU/ml factor XIII. Maximal effect was achieved by all agents in combination approximating the extent of clot formation to those in normal blood. Fibrinolysis was inhibited by factor XIII, while the reduction was stronger using all agents together. When each of the agents used in two times lower concentrations, clot strength was near to threshold. Fibrinogen and factor XIII but not factor VIIa exerted stimulation of clot strength, whereas synergistic effect of fibrinogen and factor XIII was observed. Maximal effect was achieved combining all agents. The antifibrinolytic effect was observed only by co-administration of fibrinogen, factor XIII and factor VIIa. On the basis of our study, we suggest that stimulation of clot formation and inhibition of fibrinolysis may be achieved by combination of FG, rFVIIa an FXIII using each of them at minimal effective concentration. Taken into consideration, multifactorial TIC pathogenesis, this approach may be preferable for improving coagulopathy than separate blood spiking with the essayed factors at high concentrations.

Nitric oxide (NO), produced by NO-synthases via L-arginine oxidation, is an essential trigger for signaling processes involved in structural and metabolic changes in muscle fibers. Recently, it was shown that L-arginine administration prevented the decrease in levels of the muscle cytoskeletal proteins, desmin and dystrophin, in rat soleus muscle after 14 days of hindlimb unloading. Therefore, in this study, we investigated the effect of L-arginine administration on the degree of atrophy changes in the rat soleus muscles under unloading conditions, and on the content, gene expression, and phosphorylation level of titin, the giant protein of striated muscles, able to form a third type of myofilaments-elastic filaments. A 7-day gravitational unloading [hindlimb suspension (HS) group] resulted in a decrease in the soleus weight:body weight ratio (by 31.8%, p < 0.05), indicating muscle atrophy development. The content of intact titin (T1) decreased (by 22.4%, p < 0.05) and the content of proteolytic fragments of titin (T2) increased (by 66.7%, p < 0.05) in the soleus muscle of HS rats, compared to control rats. The titin gene expression and phosphorylation level of titin between these two groups were not significantly different. L-Arginine administration under 7-day gravitational unloading decreased the degree of atrophy changes and also prevented the decrease in levels of T1 in the soleus muscle as compared to HS group. Furthermore, L-arginine administration under unloading resulted in increased titin mRNA level (by 76%, p < 0.05) and decreased phosphorylation level of T2 (by 28%, p < 0.05), compared to those in the HS group. These results suggest that administration of L-arginine, the NO precursor, under unloading decreased the degree of atrophy changes, increased gene expression of titin and prevented the decrease in levels of T1 in the rat soleus muscle. The results can be used to search for approaches to reduce the development of negative changes caused by gravitational unloading in the muscle., (Copyright © 2019 Ulanova, Gritsyna, Salmov, Lomonosova, Belova, Nemirovskaya, Shenkman and Vikhlyantsev.)

The prevailing myosin isoform [myosin heavy chain (MyHC)] in a skeletal muscle determines contractile properties of the muscle. Under actual or simulated microgravity conditions such as human bed rest or rat hindlimb unloading, decrease in expression of MyHC of the slow type [MyHC I(β)] has been observed. It was demonstrated that increasing sensory input by performing plantar mechanical stimulation (PMS) on the soles of the feet results in an increase in neuromuscular activation of the lower limb muscles and may prevent slow-to-fast fiber type shift. The calcineurin-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway is the main cascade regulating MyHC I(β) expression. The present study was aimed to analyze the states of the calcineurin-NFATc1 signaling cascade under conditions of PMS during rat hindlimb unloading. Male Wistar rats were randomly assigned to vivarium control groups and 1-day unloading (1HS), 3-day unloading (3HS), 1HS+PMS, and 3HS+PMS groups. We found that both 1 day and 3 days of unloading caused decrease in MyHC I(β) mRNA expression and decrease in glycogen synthase kinase-3β phosphorylation (Ser 9) that brought about the kinase activation, and these effects of unloading were prevented by PMS. Three days of unloading also caused increase in expression of calsarcin-2 (myozenin-I), which was found to be the endogenous calcineurin inhibitor. The level of calsarcin-2 expression in the 3HS+PMS group did not differ from the control group. Therefore, we conclude that PMS upregulates the calcineurin-NFATc1 signaling pathway and prevents unloading-induced MyHC I(β) decrease. NEW & NOTEWORTHY It is widely accepted that changes in the myosin phenotype during functional unloading (disuse) are determined by a decreased expression of the myosin heavy chain (MyHC) I(β) gene, and this decrease leads to changes of contractile and fatigue characteristics of soleus muscle. The calcineurin-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) pathway is one of the most important signaling cascades regulating slow MyHC isoform expression. The present study is the first to show that plantar mechanical stimulation upregulates calcineurin-NFATc1 signaling in soleus muscles of hindlimb-unloaded rats.

Mechanisms that convert a mechanical signal into a biochemical response in an atrophied skeletal muscle remain poorly understood. The aims of the study were to evaluate a temporal response of anabolic signaling and protein synthesis (PS) to eccentric contractions (EC) in rat soleus during hindlimb unloading (HU); and to assess a possible role of stretch-activated ion channels (SAC) in the propagation of a mechanical signal to mTORC1 following HU. Following HU, an isolated soleus was subjected to EC. Upon completion of EC, muscles were collected for western blot analyses to determine the content/phosphorylation of the key anabolic markers. We found that a degree of EC-induced p70S6K phosphorylation and the rate of PS in the soleus of 3- and 7-day unloaded rats was significantly less than that in control. A decrease in EC-induced phosphorylation of p70S6K, RPS6 and PS in the 7-day unloaded soleus treated with SAC inhibitor did not differ from that of the 7-day unloaded soleus without SAC blockade. The results of the study suggest that (i) HU results in a blunted anabolic response to a bout of EC, (ii) attenuation of mTORC1-signaling and PS in response to EC in unloaded soleus may be associated with inactivation of SAC.

Thrombocytopenia (TCP) and Glanzmann thrombasthenia (GT) are typical platelet disorders characterized by mild to severe bleeding. This study aims to create in vitro models of TCP and GT and to correct the impaired clot formation by fibrinogen and coagulation factor XIII. The TCP model (mean platelet count, 16 × 10 9 L -1 ) was produced by differential centrifugation of normal blood followed by mixing plasma with packed cells. The GT model was created by treating normal blood with 50 μg/mL eptifibatide, an inhibitor of platelet integrin αIIbβ3. Clot formation was evaluated in whole blood by rotation thromboelastometry. In both models, the extent of clot strength was two-three times lower compared to normal blood. Fibrinogen and, to a lesser extent, factor XIII stimulated the propagation phase of clot formation both in TCP and GT models. Clot strength in TCP was increased by both agents, while in GT by fibrinogen only. Similar results were obtained in blood from patients with primary immune thrombocytopenia and Glanzmann thrombasthenia. In conclusion, the created models may be useful in the development of new ways to correct the impaired coagulation potential in TCP and GT., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Spaceflight induces bone alterations with site-specific rates of bone loss according to the weight-bearing function of the bone. For the first time, this study aimed to characterize bone microarchitecture and density alterations of three ankle bones (calcaneus, navicular, and talus) of mice after spaceflight and to evaluate the impact of 8 days of Earth reambulation. Ten C57BL/6N male 4-month-old mice flew on the Bion-M1 biosatellite for 1 month; half were euthanized within 24-h of return and half after 8-days recovery on Earth. Bone microarchitecture and quality was assessed by microtomography (μCT). Whole calcaneus bone volume fraction decreased in Flight group (-6.4%, p < 0.05), and worsened in the Recovery group (-11.08%, p < 0.01), when compared to Control group. Navicular and talus trabecular bone volume fraction showed trends toward decrease in Flight and differences reached statistical significance in Recovery group (-8.16%; -8.87%, respectively; p < 0.05) when compared to Control group. At calcaneus, cortical thickness decreased in Recovery vs. Control groups (-11.69%; p < 0.01). Bone surface area, reflecting periosteal bone erosion, significantly increased in all bone sites analyzed. Qualitative analyses of 3-D bone reconstruction revealed local sites of cortical thinning and bone erosion, predominantly at articulations, muscle insertions, and ground contact bone sites. Overall, spaceflight-induced bone loss in ankle bones was site and compartment specific whilst the tissue mineral density of the remaining bone was preserved. Eight days after landing, bone status worsened as compared to immediate return.

Mechanisms of platelet activation are triggered by thrombin, adenosine diphosphate (ADP), epinephrine, thromboxane A 2 , and other soluble agonists which induce signaling via heterotrimeric Gαq, Gαi, and Gα12/13 proteins. We have undertaken a study addressing the contribution of these G proteins to platelet activation and clot formation in the presence of eptifibatide, thus excluding outside-in signaling provided by integrin αIIbβ3-fibrinogen engagement. Selective and combined activation of the G proteins was achieved by using combinations of platelet agonists and inhibitors. Platelet activation in platelet-rich plasma was evaluated by P-selectin expression using flow cytometry. Contribution of platelets to whole blood clotting was assessed by rotation thromboelastometry (ROTEM). Selective signaling of Gαq or Gαi but not Gα12/13 promoted P-selectin expression. Further enhancement of P-selectin expression was achieved by ADP-induced combined signaling of Gαq and Gαi, and to more extent by U46619 at high concentration (1.5 μM) induced combined signaling of Gαq and Gα12/13 while maximal P-selectin expression was achieved by thrombin receptor-activating peptide (TRAP)-induced combined signaling of Gαq, Gαi, and Gα12/13. In ROTEM, selective activation of Gαq, Gαi, or Gα12/13 failed to affect blood clotting. Combined signaling of Gαq and Gαi or Gαq and Gα12/13 or all three G proteins shortened the clotting time and stimulated clot strength. Pretreatment of platelets with acetylsalicylic acid did not change the effect of ADP but inhibited the effect of TRAP. Signaling of Gαq and Gα12/13 triggered by U46619 also stimulated clot formation. Combined signaling of either Gαq and Gαi or Gαq and Gα12/13 is sufficient to stimulate maximal platelet activation and enhanced clot formation in platelets treated with inhibitor of integrin αIIbβ3. It could be suggested that outside-in signaling is not necessarily required to fulfill these platelet functions.

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The purpose of the study was to assess the rate of protein synthesis (PS) and elucidate signalling pathways regulating PS in mouse soleus (Sol) and tibialis anterior (TA) muscles following chronic hypergravity (30-day centrifugation at 2G). The content of the key signalling proteins of the various anabolic signalling pathways was determined by Western-blotting. The rate of PS was assessed using in-vivo SUnSET technique. An exposure to 2G centrifugation did not induce any significant changes in the rate of PS as well as phosphorylation status of the key anabolic markers (AKT, p70s6k, 4E-BP1, GSK-3beta, eEF2) in Sol. On the contrary, a significant 55% increase in PS (p < 0.05) was found in TA. The cause of such a rise in PS could be associated with an increase in AKT (+72%, p < 0.05), GSK-3beta (+60%, p < 0.05) and p70s6k (+40%, p < 0.05) phosphorylation, as well as a decrease in eEF2 phosphorylation (-46%, p < 0.05) as compared to control values. Thus, the results of our study indicate that 30-day 2G centrifugation induces a distinct anabolic response in mouse Sol and TA muscles. The activation of the PS rate in TA could be linked to an up-regulation of both mTORC1-dependent and mTORC1-independent signalling pathways.

The weightless environment during spaceflight induces site-specific bone loss. The 30-day Bion-M1 mission offered a unique opportunity to characterize the skeletal changes after spaceflight and an 8-day recovery period in mature male C57/BL6 mice. In the femur metaphysis, spaceflight decreased the trabecular bone volume (-64% vs. Habitat Control), dramatically increased the bone resorption (+140% vs. Habitat Control) and induced marrow adiposity invasion. At the diaphysis, cortical thinning associated with periosteal resorption was observed. In the Flight animal group, the osteocyte lacunae displayed a reduced volume and a more spherical shape (synchrotron radiation analyses), and empty lacunae were highly increased (+344% vs. Habitat Control). Tissue-level mechanical cortical properties (i.e., hardness and modulus) were locally decreased by spaceflight, whereas the mineral characteristics and collagen maturity were unaffected. In the vertebrae, spaceflight decreased the overall bone volume and altered the modulus in the periphery of the trabecular struts. Despite normalized osteoclastic activity and an increased osteoblast number, bone recovery was not observed 8 days after landing. In conclusion, spaceflight induces osteocyte death, which may trigger bone resorption and result in bone mass and microstructural deterioration. Moreover, osteocyte cell death, lacunae mineralization and fatty marrow, which are hallmarks of ageing, may impede tissue maintenance and repair.

Background: Trauma-induced coagulopathy (TIC) is commonly seen among patients with severe injury. The dynamic process of TIC is characterized by variability of the features of the disease., Methods: A model of TIC was created. Hemodilution was produced by mixing the blood with 40% Tris/saline solution, fibrinolysis by treating the blood with 160 ng/mL tPA, acidosis by adding 1.2 mg/mL lactic acid achieving pH 7.0 to 7.1, and hypothermia by running the assay at 31°C. Intact blood tested at 37°C served as control. Clot formation was evaluated using rotation thromboelastometry. Platelet adhesion and aggregation were assayed at a shear rate of 1800 s(-) using Impact-R device., Results: Clotting time was not affected by any of the TIC constituents used. Clotting initiation was reduced by hemodilution and further reduced by additive hypothermia. The propagation phase of blood clotting was reduced by hemodilution, further reduced by additive hypothermia, and maximally reduced if additionally combined with fibrinolysis. No effect of fibrinolysis on clot propagation was observed at 37°C. Maximum clot firmness was reduced by hemodilution, further reduced by additive fibrinolysis, and maximally reduced if additionally combined with hypothermia. No effect of hypothermia on clot strength was observed in the absence of fibrinolysis. Platelet adhesion (percentage of surface coverage) and aggregation (aggregate size) under flow condition were reduced by hemodilution and further reduced by additive acidosis. Introduction of tPA to diluted blood had no effect on platelet function., Conclusion: The study revealed a differential effect of TIC constituents-hemodilution, hypothermia, fibrinolysis, and acidosis-on clot formation and platelet function. The effect of one factor may influence that of another factor. These data may be helpful to better understand the pathogenesis of TIC and to elaborate an individually tailored treatment strategy., Level of Evidence: A new model of TIC is created. Contribution of various constituents to pathogenesis of TIC and their interactions are evaluated.

Effective platelet function requires formation of a physical link between fibrin(ogen), integrin αIIbβ3, and cytoplasmic actin filaments. We investigated the role of the Gαq, Gαi, and Gα12/13 families of heterotrimeric GTP-binding proteins (G proteins) in the assembly of a ligand-αIIbβ3-actin cytoskeleton complex. Selective and combined activation of the G proteins was achieved by using combinations of various platelet agonists and inhibitors. Formation and stability of fibrinogen-αIIbβ3 interaction were evaluated by the extent of platelet aggregation and the rate of eptifibatide-induced platelet disaggregation; association of αIIbβ3 with the cytoskeleton was analyzed by western blot. Formation of the fibrin-αIIbβ3-actin cytoskeleton complex was evaluated by rotational thromboelastometry assay in which clot formation was induced by the mixture of reptilase and factor XIIIa. We demonstrated that involvement of heterotrimeric G proteins in the formation of the ligand-αIIbβ3-cytoskeleton complex depends on whether fibrinogen or fibrin serves as the integrin ligand. Formation of the fibrinogen-αIIbβ3-cytoskeleton complex requires combined activation of at least two G protein pathways while the maximal αIIbβ3-cytoskeleton association and the strongest αIIbβ3-fibrinogen binding supporting irreversible platelet aggregation require combined activation of all three-Gαq, Gαi, and Gα12/13-G protein families. In contrast, formation of the fibrin-αIIbβ3-cytoskeleton complex mediating clot retraction is critically dependent on the activation of the Gαi family, especially on the activation of Gαz.

Background: Between stages of Mohs micrographic surgery, the wound is dressed and the patient waits for the histopathological results.
, Objective: To investigate the efficacy of a hemostatic-anesthetic solution-impregnated gauze in decreasing bleeding between Mohs stages.
, Materials and Methods: Twenty patients were treated with a hemostatic-anesthetic solution composed of tranexamic acid, adrenaline, and lidocaine (TAL), and 20 others were treated with a saline solution for control. At the second Mohs stage, size measurements of the blood stain on a Telfa pad and the defect were recorded. The Rotation Thromboelastometry Method (ROTEM) was used to investigate a possible effect of lidocaine and adrenaline on the clot stability induced by tranexamic acid.
, Results: The ratio of blood stain size to Mohs defect size in the hemostatic anesthetic solution group was 1:1.47, whereas the ratio in the control saline group was 1:3.37 (P&lt;.001). Results of the ROTEM test showed that lidocaine and adrenaline did not interfere with the effect of tranexamic acid on clot formation and stability.
, Conclusion: The application of gauze impregnated with tranexamic acid, adrenaline, and lidocaine on a surgical wound may be effective in reducing bleeding between Mohs stages. , , J Drugs Dermatol. 2016;15(7):851-855.