Phosphatidylserine Exposure In Red Blood Cells Research Articles

Deciphering and disrupting PIEZO1-TMEM16F interplay in hereditary xerocytosis

AbstractCell-surface exposure of phosphatidylserine (PS) is essential for phagocytic clearance and blood clotting. Although a calcium-activated phospholipid scramblase (CaPLSase) has long been proposed to mediate PS exposure in red blood cells (RBCs), its identity, activation mechanism, and role in RBC biology and disease remain elusive. Here, we demonstrate that TMEM16F, the long-sought-after RBC CaPLSase, is activated by calcium influx through the mechanosensitive channel PIEZO1 in RBCs. PIEZO1-TMEM16F functional coupling is enhanced in RBCs from individuals with hereditary xerocytosis (HX), an RBC disorder caused by PIEZO1 gain-of-function channelopathy. Enhanced PIEZO1-TMEM16F coupling leads to an increased propensity to expose PS, which may serve as a key risk factor for HX clinical manifestations including anemia, splenomegaly, and postsplenectomy thrombosis. Spider toxin GsMTx-4 and antigout medication benzbromarone inhibit PIEZO1, preventing force-induced echinocytosis, hemolysis, and PS exposure in HX RBCs. Our study thus reveals an activation mechanism of TMEM16F CaPLSase and its pathophysiological function in HX, providing insights into potential treatment.

Butylparaben promotes phosphatidylserine exposure and procoagulant activity of human red blood cells via increase of intracellular calcium levels

Parabens are widely used as preservatives, added to products commonly used by humans, and to which individuals are exposed orally or dermally. Once absorbed into the body, parabens move into the bloodstream and travel through the systemic circulation. We investigated the potential impact of parabens on the enhanced generation of thrombin by red blood cells (RBCs), which are the principal cellular components of blood. We tested the effects of methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), butylparaben (BuP), and p-hydroxybenzoic acid on freshly isolated human RBCs. BuP and simultaneous exposure to BuP and PrP significantly increased phosphatidylserine (PS) externalization to the outer membranes of RBCs. PS externalization by BuP was found to be mediated by increasing intracellular Ca2+ levels in RBCs. The morphological changes in BuP-treated RBCs were observed under an electron microscope. The BuP-exposed RBCs showed increased thrombin generation and adhesion to endothelial cells. Additionally, the externalization of PS exposure and thrombin generation in BuP-treated RBCs were more susceptible to high shear stress, which mimics blood turbulence under pathological conditions. Collectively, we observed that BuP induced morphological and functional changes in RBCs, especially under high shear stress, suggesting that BuP may contribute to the thrombotic risk via procoagulant activity in RBCs.

Silica nanoparticles trigger phosphatidylserine exposure in red blood cells and induce thrombosis risk

Silica nanoparticles (SiNPs) have attracted increasing attention for their health effects due to the increased risk of exposure to human bodies via diverse routes. Considering that SiNPs enter the circulatory system and inevitably encounter red blood cells (RBCs), it is necessary to investigate their risk of causing erythrocytotoxicity. In this study, three sizes of SiNPs (SiNP-60, SiNP-120, and SiNP-200) were tested for their effects on mouse RBCs. The results showed that SiNPs could induce hemolysis, morphological changes, and phosphatidylserine (PS) exposure in RBCs in a particulate size-related manner. Further investigations on the underlying mechanism indicated that SiNP-60 exposure increased intracellular reactive oxidative species (ROS) generation and subsequently caused the phosphorylation of p38 and ERK1/2 in RBCs. The addition of antioxidants or inhibitors of mitogen-activated protein kinase (MAPK) signaling significantly attenuated PS exposure in RBCs and ameliorated SiNP-induced erythrocytotoxicity. Moreover, ex vivo assays using platelet-rich plasma (PRP) showed that SiNP-60-induced PS exposure in RBCs could trigger thrombin-dependent platelet activation. The contrary evidence from the assays of PS blockage and thrombin inhibition further confirmed that SiNP-60-induced platelet activation was dependent on PS externalization in RBCs, concomitantly with thrombin formation. These findings revealed the procoagulant and prothrombotic effects of SiNPs through the regulation of PS externalization in RBCs, and may be of great help in bridging the knowledge gap on the potential cardiovascular hazards of particulate silica from both artificial and naturally occurring origins.

Differential Erythrocyte Phosphatidylserine Exposure Is Associated with Variations in Clinical and Haemostatic Parameters: A Study Involving E-β and β Thalassemia Compared to Normal Population

Introduction: Thalassemia is characterized by an ineffective erythropoiesis (IE). Increased exposure of phosphatidylserine (PS) on red blood cell (RBC) surface following membrane injury by free radical generation is an important pathway of IE triggering apoptotic signal, which is also associated with hypercoagulable state of thalassemia. The current study aimed to assess PS exposure on RBC surface using annexin-V as a surrogate marker comparing thalassemic patients with healthy controls based on different clinical & laboratory parameters. Methods: The assay was based on flowcytometric detection using FITC-conjugated annexin-V antibody for PS exposure and PC-7 conjugated glycophorin A for RBC identification. The study cohort consisted of 60 Thalassemia patients including Beta (10, 17%) and E-Beta (50, 83%), attending Department of Haematology at NRS Medical College & Hospital, Kolkata, India between March 2020 to December 2021 and matched healthy controls (n= 30). Patients were broadly segregated based on age, sex, Thalassaemia type (Beta/E-Beta), transfusion dependent (TDT) / Non-transfusion dependent (NTDT) and splenectomy status. Enrolled patient and control population were assessed for PS exposure using flow cytometer from Navios EX, Beckman Coulter and its correlation with clinical parameters like transfusion dependence, thrombotic episodes, splenectomy status; as well as laboratory parameters including hemoglobin (Hb) level, prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, serum ferritin level. These were assessed using a battery of statistical tools. Independent t test and Mann-Whitney U test were done for two groups and ANOVA for more than two groups. Qualitative variables were correlated using Chi-Square test. Multivariate linear regression was used to analyze the correlation between multiple variables. P-value <0.05 was considered as statistically significant. Result: The median annexin expression was significantly high (P<0.001) in thalassemia patients with Beta (18.15%) and E-Beta (9.2%), compared to control (0.4%) although the difference was not significant between Beta and E-Beta. Similarly, a significant difference (P<0.001) in annexin-V expression was observed in TDT (18%) compared to NTDT (5.4%) as well as in splenectomised (23%; IQR 19.3-28) and non-splenectomised (8.1%; IQR 4.2-18.3) groups (P=0.002). The TDT patients showed a significant risk of iron overload characterized as serum ferritin >1000 ng/ml (37/38, 97.4%) as compared to NTDT (3/22, 13.7%), and also associated with significantly increased annexin expression (18% vs 5.4%, P<0.001). Comparable observations were obtained for pooled thalassemic population with significantly low Hb, prolonged PT and aPTT (Hb: 7.2 ± 1.3 gm/dl, PT: 17.4 ± 4.4 sec, aPTT: 45.6 ± 24.9 sec) compared to healthy control (Hb: 12.6 ± 0.7 gm/dl, PT: 12.6 ± 0.8 sec, aPTT: 26.6 ± 1.9 sec). Similar variations were also significant between TDT & NTDT. The former observation was in concordance to annexin expression as well showing significantly high (P<0.001) annexin-V expression in individuals with low hemoglobin ≤7 gm/dl, prolonged PT and aPTT. Significant annexin expression was not observed with fibrinogen level and no obvious clinical manifestations of hypercoagulability in any patients were detected. Conclusion: Therefore, it can be said that RBC PS exposure was significantly high in Thalassaemia patients and it is associated with degree of anemia, transfusion dependence, iron overload, prolonged PT / aPTT and splenectomy status. There is no significant difference in PS expression between E-Beta and Beta population. But contrary to the available literature annexin expression was more in TDT compared to NTDT patients in our study. This discrepancy might be due to suboptimal transfusions in TDT patients leading to inadequate suppression of ineffective erythropoiesis and incomplete replacement of damaged RBCs by healthy erythrocytes. Studies involving larger population are needed to validate this observation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effects of exhaustive exercise on coagulation state in rats

Objective: To investigate the effects of one-time exhaustive exercise on coagulation state in rats and its mechanism. Methods: Forty-eight SD rats were randomly divided into control group and exhaustive exercise group, 24 rats in each group. Rats in exhaustive exercise group were trained with treadmill training for 25～50 min at a time on non-slope treadmill and the initial speed of 5 m/min was uniformly accelerated to 25 m/min until the rats exhausted. Thrombelastography (TEG) was used to monitor the coagulation function of rats after training. The ligation model of inferior vena cava (IVC) was established to evaluate thrombosis. The phosphatidylserine (PS) exposure and Ca2+ concentration were detected by flow cytometry. The production of FXa and thrombin was detected by microplate reader. The clotting time was measured by using coagulometer. Results: Compared with the control group, the blood of rats in the exhaustive exercise group exhibited hypercoagulable state. The probability of thrombus formation, weight, length and ratio in the exhaustive exercise group were significantly higher than those in the control group (P＜0.01). The levels of PS exposure and intracellular Ca2+ concentration of red blood cells (RBCs) and platelets in the exhaustive exercise group were increased significantly (P＜0.01). The blood clotting time of RBCs and platelets was shortened (P＜0.01), and the production of FXa and thrombin was increased significantly (P＜0.01) in the exhausted exercise group, and both were inhibited by lactadherin (Lact, P＜0.01). Conclusion: The blood of exhaustive exercise rats is in a hypercoagulable state and the risk of thrombosis is increased. The increased PS exposure of RBCs and platelets caused by exhaustive exercise may be an important mechanism of thrombosis.

TMEM16F mediated phosphatidylserine exposure and microparticle release on erythrocyte contribute to hypercoagulable state in hyperuricemia

The link between hyperuricemia (HUA) and the risk of venous thromboembolism (VTE) has been well established. However, the mechanisms of thrombus generation and the effect of HUA on procoagulant activity (PCA) of erythrocytes remain unclear no matter in uremia or hyperuricemia. Here, phosphatidylserine (PS) exposure, microparticles (MPs) release, cytosolic Ca2+, TMEM16F expression, reactive oxygen species (ROS) and lipid peroxidation of erythrocyte were detected by flow cytometer. PCA was assessed by coagulation time, purified coagulation complex and fibrin production assays. The fibrin formation was observed by scanning electron microscopy (SEM). We found that PS exposure, MPs generation, TMEM16F expression and consequent PCA of erythrocyte in HUA patients significantly increased compared to those in healthy volunteers. Furthermore, high UA induced PS exposure, and MPs release of erythrocyte in concentration and time-dependent manners in vitro, which enhanced the PCA of erythrocyte and was inhibited by lactadherin, a PS inhibitor. Additionally, using SEM, we also observed compact fibrin clots with highly-branched networks and thin fibers supported by red blood cells (RBCs) and RBC-derived MPs (RMPs). Importantly, we demonstrated UA enhanced the production of ROS and lipid peroxidation and reduced the generation of glutathione (GSH) of erythrocyte, which enhanced TMEM16F activity and followed PS externalization and RMPs formation. Collectively, these results suggest that Ca2+-dependent TMEM16F activation may be responsible for UA-induced PS exposure and MPs release of RBC, which thereby contribute to the prothrombotic risk in HUA.

Insights Into the Mechanisms of Brain Endothelial Erythrophagocytosis.

The endothelial cells which form the inner cellular lining of the vasculature can act as non-professional phagocytes to ingest and remove emboli and aged/injured red blood cells (RBCs) from circulation. We previously demonstrated an erythrophagocytic phenotype of the brain endothelium for oxidatively stressed RBCs with subsequent migration of iron-rich RBCs and RBC degradation products across the brain endothelium in vivo and in vitro, in the absence of brain endothelium disruption. However, the mechanisms contributing to brain endothelial erythrophagocytosis are not well defined, and herein we elucidate the cellular mechanisms underlying brain endothelial erythrophagocytosis. Murine brain microvascular endothelial cells (bEnd.3 cells) were incubated with tert-butyl hydroperoxide (tBHP, oxidative stressor to induce RBC aging in vitro)- or PBS (control)-treated mouse RBCs. tBHP increased the reactive oxygen species (ROS) formation and phosphatidylserine exposure in RBCs, which were associated with robust brain endothelial erythrophagocytosis. TNFα treatment potentiated the brain endothelial erythrophagocytosis of tBHP-RBCs in vitro. Brain endothelial erythrophagocytosis was significantly reduced by RBC phosphatidylserine cloaking with annexin-V and with RBC-ROS and phosphatidylserine reduction with vitamin C. Brain endothelial erythrophagocytosis did not alter the bEnd.3 viability, and tBHP-RBCs were localized with early and late endosomes. Brain endothelial erythrophagocytosis increased the bEnd.3 total iron pool, abluminal iron levels without causing brain endothelial monolayer disruption, and ferroportin levels. In vivo, intravenous tBHP-RBC injection in aged (17–18 months old) male C57BL/6 mice significantly increased the Prussian blue-positive iron-rich lesion load compared with PBS-RBC-injected mice. In conclusion, RBC phosphatidylserine exposure and ROS are key mediators of brain endothelial erythrophagocytosis, a process which is associated with increased abluminal iron in vitro. tBHP-RBCs result in Prussian blue-positive iron-rich lesions in vivo. Brain endothelial erythrophagocytosis may provide a new route for RBC/RBC degradation product entry into the brain to produce iron-rich cerebral microhemorrhage-like lesions.

Of membranes and malaria: phospholipid asymmetry in Plasmodium falciparum-infected red blood cells.

Malaria is a vector-borne parasitic disease with a vast impact on human history, and according to the World Health Organisation, Plasmodium parasites still infect over 200 million people per year. Plasmodium falciparum, the deadliest parasite species, has a remarkable ability to undermine the host immune system and cause life-threatening disease during blood infection. The parasite's host cells, red blood cells (RBCs), generally maintain an asymmetric distribution of phospholipids in the two leaflets of the plasma membrane bilayer. Alterations to this asymmetry, particularly the exposure of phosphatidylserine (PS) in the outer leaflet, can be recognised by phagocytes. Because of the importance of innate immune defence numerous studies have investigated PS exposure in RBCs infected with P. falciparum, but have reached different conclusions. Here we review recent advancements in our understanding of the molecular mechanisms which regulate asymmetry in RBCs, and whether infection with the P. falciparum parasite results in changes to PS exposure. On the balance of evidence, it is likely that membrane asymmetry is disrupted in parasitised RBCs, though some methodological issues need addressing. We discuss the potential causes and consequences of altered asymmetry in parasitised RBCs, particularly for in vivo interactions with the immune system, and the role of host-parasite co-evolution. We also examine the potential asymmetric state of parasite membranes and summarise current knowledge on the parasite proteins, which could regulate asymmetry in these membranes. Finally, we highlight unresolved questions at this time and the need for interdisciplinary approaches to uncover the machinery which enables P. falciparum parasites to hide in mature erythrocytes.

Impact of Trail Running Races on Blood Viscosity and Its Determinants: Effects of Distance.

Blood rheology is a key determinant of tissue perfusion at rest and during exercise. The present study investigated the effects of race distance on hematological, blood rheological, and red blood cell (RBC) senescence parameters. Eleven runners participated in the Martigny–Combes à Chamonix 40 km race (MCC, elevation gain: 2300 m) and 12 others in the Ultra-Trail du Mont Blanc (UTMB, 171 km, elevation gain: 10,000 m). Blood samples were collected before and after the races. After the UTMB, the percentage of RBC phosphatidylserine (PS) exposure was not affected while RBC CD235a levels decreased and RBC-derived microparticles increased. In contrast, after the MCC, RBC PS exposure increased, while RBC CD235a and RBC-derived microparticles levels were not affected. The free hemoglobin and hemolysis rate did not change during the races. RBC aggregation and blood viscosity at moderate shear rates increased after the MCC. RBC deformability, blood viscosity at a high shear rate, and hematocrit decreased after the UTMB but not after the MCC. Our results indicate that blood rheology behavior is different between a 40 km and a 171 km mountain race. The low blood viscosity after the ultra-marathon might facilitate blood flow to the muscles and optimize aerobic performance.

Abstract TP478: Mechanisms of Brain Endothelial Erythrophagocytosis: Insights Into the Pathogenesis of Cerebral Microbleeds

Background: Cerebral microbleeds, MRI signatures of cerebral microhemorrhage, are thought to arise from brain microvessel disruption. Our prior work suggests an alternate origin for some microbleeds, emphasizing brain endothelial erythrophagocytosis (BEE) producing microhemorrhage-like lesions (“pseudo-microbleeds”) in absence of vascular disruption (Frontiers Cell Neurosci 9-18; PMID: 30237761). We demonstrated an erythrophagocytic phenotype of brain microvascular endothelium for oxidatively-stressed red blood cells (RBC), and migration of iron-rich hemoglobin across the brain endothelium. However, cellular mechanisms underlying BEE and hemoglobin passage across brain endothelium are not defined. Herein we investigated a) role of oxidative stress and RBC phosphatidylserine (PS) exposure in BEE and b) intracellular endosomal trafficking of RBC within brain endothelial cells. Methods: Murine brain endothelial cells (bEnd.3 cells) were incubated with 2x10 6 mouse RBC treated with 3mM tert-butylhydroperoxide (t-BHP, an oxidative stressor) or sterile PBS (control) for 48h at 37°C, in the presence of 7.5 μg annexin V to cloak PS, or vitamin C (15μM-1500μM) to reduce reactive oxygen species (ROS). BEE was evaluated by hematoxylin &amp; eosin stain and diaminofluorene assay for hemoglobin. RBC ROS levels, PS exposure, and cell viability were measured. bEnd.3 cells were immunostained to visualize RBC in early and late endosomes, the latter being the presumed site of RBC degradation post-erythrophagocytosis. Results: tBHP induced both ROS production and PS exposure in RBC. There was a 9-fold increase (p&lt;0.001) in BEE of t-BHP-RBC compared with control. Vitamin C reduced RBC ROS levels (70%, p&lt;0.001) and PS exposure (27%, p&lt;0.001), while annexin V blocked RBC PS exposure (65%, p&lt;0.001). BEE was significantly attenuated by annexin V (63%, p&lt;0.001) and vitamin C (39%, p&lt;0.001). No change in bEnd.3 viability was observed and t-BHP-RBC localized to both early and late endosomes. Conclusions: These data demonstrate the importance of RBC PS exposure in BEE, and describe the intracellular trafficking of RBC in brain endothelial cells. These findings provide insights into the development of microhemorrhage-like lesions and cerebral microbleeds.

Pro-Coagulant and Pro-Thrombotic Effects of Paclitaxel Mediated by Red Blood Cells.

Paclitaxel is one of the most widely used anti-cancer drugs, but numerous case reports of thrombotic events in the cancer patients using paclitaxel raise concern over its pro-thrombotic risk. We investigated whether paclitaxel can elicit pro-thrombotic properties in red blood cells (RBCs) through phosphatidylserine (PS) exposure and microvesicle (MV) release. In freshly isolated human RBCs, paclitaxel induced thrombin generation through PS exposure and MV release, whereas either coagulation factors or platelets were unaffected. Paclitaxel-induced PS exposure in RBC was mediated by scramblase activation which was induced by calcium-independent protein kinase C (PKC)ζ activation. Paclitaxel also increased RBC-endothelial cell adhesion and RBC aggregate formation which can also contribute to thrombosis. Indeed, intravenous administration of paclitaxel to rats induced PS exposure and PKCζ activation in RBCs in vivo which ultimately promoted venous thrombus formation. These results demonstrated that paclitaxel may elicit pro-thrombotic properties in RBCs through PS exposure and MV release, which can ultimately promote thrombus formation.

Platelet-RBC interaction mediated by FasL/FasR induces procoagulant activity important for thrombosis.

Red blood cells (RBCs) influence rheology, and release ADP, ATP, and nitric oxide, suggesting a role for RBCs in hemostasis and thrombosis. Here, we provide evidence for a significant contribution of RBCs to thrombus formation. Anemic mice showed enhanced occlusion times upon injury of the carotid artery. A small population of RBCs was located to platelet thrombi and enhanced platelet activation by a direct cell contact via the FasL/FasR (CD95) pathway known to induce apoptosis. Activation of platelets in the presence of RBCs led to platelet FasL exposure that activated FasR on RBCs responsible for externalization of phosphatidylserine (PS) on the RBC membrane. Inhibition or genetic deletion of either FasL or FasR resulted in reduced PS exposure of RBCs and platelets, decreased thrombin generation, and reduced thrombus formation in vitro and protection against arterial thrombosis in vivo. Direct cell contacts between platelets and RBCs via FasL/FasR were shown after ligation of the inferior vena cava (IVC) and in surgical specimens of patients after thrombectomy. In a flow restriction model of the IVC, reduced thrombus formation was observed in FasL-/- mice. Taken together, our data reveal a significant contribution of RBCs to thrombosis by the FasL/FasR pathway.

Donor variation effect on red blood cell storage lesion: a multivariable, yet consistent, story.

Previous studies have shown that baseline hematologic characteristics concerning or influencing red blood cell (RBC) properties might affect storage lesion development in individual donors. This study was conducted to evaluate whether variation in hemolysis, microparticle accumulation, phosphatidylserine (PS) exposure, and other storage lesion-associated variables might be a function of the prestorage hematologic and biologic profiles of the donor. Ten eligible, regular blood donors were paired and studied before donation (fresh blood) and during storage of RBCs in standard blood banking conditions. Plasma and cellular characteristics and modifications were evaluated by standard laboratory and biochemical or biologic analyses as well as by statistical and network analysis tools. Nitrate/nitrite and other bioactive factors exhibited high interdonor variability, which further increased during storage in a donor-specific manner. Storage lesion evaluators, including RBC fragility and PS exposure, fluctuated throughout the storage period in proportion to their values in fresh blood. Donors' levels of phosphatidylserine exposure and hemoglobin F correlated with stored cells' mean cell (RBC) Hb concentration, oxidative stress markers, and cellular fragility. Storage lesion indicators change in an orderly fashion, namely, by following donor-related prestorage attributes. These correlations are illustrated for the first time in "prestorage versus storage" biologic networks, which might help determine the best candidates for in vivo biomarkers of storage quality and provide deeper insight into the apparently complex donor variation effect on the RBC storage lesion.

Novel Insights in the Regulation of Phosphatidylserine Exposure in Human Red Blood Cells

Background/Aims: In previous publications we were able to demonstrate the exposure of phosphatidylserine (PS) in the outer membrane leaflet after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA), phorbol-12 myristate-13acetate (PMA), or 4-bromo-A23187 (A23187). It has been concluded that three different mechanisms are responsible for the PS exposure in human RBCs: (i) Ca2+-stimulated scramblase activation (and flippase inhibition) by A23187, LPA, and PMA; (ii) PKCα activation by LPA and PMA; and (iii) enhanced lipid flip flop caused by LPA. Further studies aimed to elucidate interconnections between the increased Ca2+ content, scramblase- and PKCα-activation. In addition, the role of the Ca2+-activated K+ channel (Gardos channel) activity in the process of PS exposure needs to be investigated. Methods: The intracellular Ca2+ content and the PS exposure of RBCs have been investigated after treatment with LPA (2.5 µM), PMA (6 µM), or A23187 (2 µM). Fluo-4 and annexin V-FITC has been used to detect intracellular Ca2+ content and PS exposure, respectively. Both parameters (Ca2+ content, PS exposure) were studied using flow cytometry. Inhibitors of the scramblase, the PKCα, and the Gardos channel have been applied. Results: The percentage of RBCs showing PS exposure after activation with LPA, PMA, or A23187 is significantly reduced after inhibition of the scramblase using the specific inhibitor R5421 as well as after the inhibition of the PKCα using chelerythrine chloride or calphostin C. The inhibitory effect is more pronounced when the scramblase and the PKCα are inhibited simultaneously. Additionally, the inhibition of the Gardos channel using charybdotoxin resulted in a significant reduction of the percentage of RBCs showing PS exposure under all conditions measured. Similar results were obtained when the Gardos channel activity was suppressed by increased extracellular K+ content. Conclusion: PS exposure is mediated by the Ca2+-dependent scramblase but also by PKCα activated by LPA and PMA in a Ca2+-dependent and a Ca2+-independent manner. Furthermore, we hypothesize that a hyperpolarisation of RBCs caused by the opening of the Gardos channel is essential for the scramblase activity as well as for a fraction of the LPA-induced Ca2+ entry.

Measurements of Intracellular Ca2+ Content and Phosphatidylserine Exposure in Human Red Blood Cells: Methodological Issues

Background/Aims: The increase of the intracellular Ca²⁺ content as well as the exposure of phosphatidylserine (PS) on the outer cell membrane surface after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA) has been investigated by a variety of research groups. Carrying out experiments, which we described in several previous publications, we observed some discrepancies when comparing data obtained by different investigators within our research group and also between batches of LPA. In addition, we found differences comparing the results of double and single labelling experiments (for Ca²⁺ and PS). Furthermore, the results of PS exposure depended on the fluorescent dye used (annexin V-FITC versus annexin V alexa fluor® 647). Therefore, it seems necessary to investigate these methodological approaches in more detail to be able to quantify results and to compare data obtained by different research groups. Methods: The intracellular Ca²⁺ content and the PS exposure of RBCs separated from whole blood have been investigated after treatment with LPA (2.5 µM) obtained from three different companies (Sigma-Aldrich, Cayman Chemical Company, and Santa Cruz Biotechnology Inc.). Fluo-4 and x-rhod-1 have been used to detect intracellular Ca²⁺ content, annexin V alexa fluor® 647 and annexin V-FITC have been used for PS exposure measurements. Both parameters (Ca²⁺ content, PS exposure) were studied using flow cytometry and fluorescence microscopy. Results: The percentage of RBCs showing increased intracellular Ca²⁺ content as well as PS exposure changes significantly between different LPA manufacturers as well as on the condition of mixing of LPA with the RBC suspension. Furthermore, the percentage of RBCs showing PS exposure is reduced in double labelling compared to single labelling experiments and depends also on the fluorescent dye used. Finally, data on Ca²⁺ content are slightly affected whereas PS exposure data are not affected significantly by the measuring method (flow cytometry, fluorescence microscopy). Conclusion: The LPA batch used and the mixing procedure of LPA and the RBC suspension has to be taken into consideration when comparing results of intracellular Ca²⁺ content and PS exposure of RBCs after LPA activation. In addition, one should consider that the results of single and double labelling experiments might be different depending on the fluorescent dyes used.

Phosphatidylserine Exposure in Human Red Blood Cells Depending on Cell Age

Background/Aims: The exposure of phosphatidylserine (PS) on the outer membrane leaflet of red blood cells (RBCs) serves as a signal for suicidal erythrocyte death or eryptosis, which may be of importance for cell clearance from blood circulation. PS externalisation is realised by the scramblase activated by an increase of intracellular Ca²⁺ content. It has been described in literature that RBCs show an increased intracellular Ca²⁺ content as well as PS exposure when becoming aged up to 120 days (which is their life span). However, these investigations were carried out after incubation of the RBCs for 48 h. The aim of this study was to investigate this effect after short-time incubation using a variety of stimulating substances for Ca²⁺ uptake and PS exposure. Methods: We separated RBCs by age in five different fractions by centrifugation using Percoll density gradient. The intracellular Ca²⁺ content and the PS exposure of RBCs with different age has been investigated after treatment with lysophosphatidic acid (LPA) as well as after activation of protein kinase C (PKC) using phorbol-12 myristate-13 acetate (PMA). For positive control RBCs were treated with 4-bromo-A23187. Measurement techniques included flow cytometry and live cell imaging (fluorescence microscopy). Results: The percentage of RBCs showing increased Ca²⁺ content as well as the PS exposure did not change significantly in dependence on cell age after short-time incubation in control experiments (without stimulating substances) or using LPA or PMA. However, we confirm findings reported that Ca²⁺ content and the PS exposure of RBCs increased after 48 h incubation. Conclusion: No significant differences of intracellular Ca²⁺ content and PS exposure can be seen for RBCs of different age in resting state or after stimulation of Ca²⁺ uptake at short-time incubation.

Indolic uremic solutes enhance procoagulant activity of red blood cells through phosphatidylserine exposure and microparticle release.

Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients). Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca2+ ([Ca2+]) with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca2+]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process.

Homocysteine induces procoagulant activity of red blood cells via phosphatidylserine exposure and microparticles generation.

Increased homocysteine (Hcy) levels in plasma correlate with the risk of thromboic events. Red blood cells (RBCs), the most abundant blood cells in circulation, also play an active role in the process of thrombus formation. However, the effect of Hcy on procoagulant activity (PCA) of RBCs is unclear. In the present study, RBCs from healthy adults were treated with Hcy (8, 20, 80, 200, 800 μmol/L) for 24 h. Phosphatidylserine (PS) exposure of RBCs and red blood cell-derived microparticles (RMPs) release were detected using Alexa Fluor 488-lactadherin. PCA was assessed by coagulation time and purified clotting complexes testes. We found that Hcy treatment dose dependently enhanced PS exposure and consequent PCA of RBCs. Hcy also elevated the formation of procoagulant RMPs, with statistical significance at 800 μmol/L of Hcy. Moreover, 128 nmol/L lactadherin inhibited about 90% PCA of RBCs and RMPs. Our data suggest that PS exposure and RMPs shedding are key sources for Hcy-induced PCA of RBCs. Lactadherin could be used to modulate the anticoagulant and procoagulant balance in this process.

Inhibitors of second messenger pathways and Ca2+-induced exposure of phosphatidylserine in red blood cells of patients with sickle cell disease

The present work investigates the contribution of various second messenger systems to Ca2+-induced phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell disease (SCD) patients. The Ca2+ dependence of PS exposure was confirmed using the Ca2+ ionophore bromo-A23187 to clamp intracellular Ca2+ over 4 orders of magnitude in high or low potassium-containing (HK or LK) saline. The percentage of RBCs showing PS exposure was significantly increased in LK over HK saline. This effect was reduced by the Gardos channel inhibitors, clotrimazole and charybdotoxin. Nevertheless, although Ca2+ loading in the presence of an outwardly directed electrochemical gradient for K+ stimulated PS exposure, substantial exposure still occurred in HK saline. Under the conditions used inhibitors of other second messenger systems (ABT491, quinacrine, acetylsalicylic acid, 3,4-dichloroisocoumarin, GW4869 and zVAD-fmk) did not inhibit the relationship between [Ca2+] and PS exposure. Inhibitors of phospholipase A2, cyclooxygenase, platelet-activating factor, sphingomyelinase and caspases, therefore, were without effect on Ca2+-induced PS exposure in RBCs, incubated in either HK or LK saline.

Procoagulant activity of erythrocytes and platelets through phosphatidylserine exposure and microparticles release in patients with nephrotic syndrome

Recent studies showed that an imbalance of prothrombotic and antithrombotic factors and impaired thrombolytic activity contribute to the thrombophilia of the nephrotic syndrome (NS). However, it is not clear whether blood cell injury and/or activation is involved in hypercoagulability in NS patients. Our objectives were to study the increase in microparticle (MP) release and phosphatidylserine (PS) exposure on the outer membrane of MP-origin cells in NS patients, and to evaluate their procoagulant activity (PCA). The subjects were patients with membranous nephropathy (MN), minimal change nephrotic syndrome (MCNS) and healthy controls. Analyses of MPs and PS exposure were performed using a flow cytometer. PCA was determined by clotting time and purified coagulation complex assays. We found that lactadherin+ MPs, which derived from red blood cells (RBC), platelet and endothelial cell, increased in NS patients. Moreover, PS exposure on RBCs and platelets in each NS group, especially in MN, are higher than that in controls. MP shedding and PS exposure of RBCs/platelets were highly procoagulant in NS patients. However, blockade of PS with lactadherin inhibited over 90% of PCA while an anti-tissue factor antibody had no significant inhibition effect. Our results demonstrate that the thrombophilic susceptibility of NS may be partly ascribed to MP release and PS exposure of RBCs, platelets and endothelial cells. Lactadherin is a sensitive probe for PS that has high anticoagulant activity.