Subsequent Phosphatidylserine Exposure Research Articles

Impact of Recombinant Factor VIII and Platelet Interaction on Platelet Functionality and Hemophilia a Treatment

Background: Platelets play an important role throughout the various stages of hemostasis. After vascular injury, platelets become activated in a pro-aggregatory state, and subsequently a subpopulation undergoes a phenotype shift to a pro-coagulant state. Pro-coagulant platelets bind to factor VIII (FVIII) and ensure efficient localization of FVIII at the site of injury. It is unclear whether modifications in recombinant FVIII (rFVIII) products impact FVIII-platelet binding and subsequent platelet signaling. Aims: To examine binding of different rFVIII products to platelets in vitro, and the impact of rFVIII on platelet phenotype and intracellular signaling. Methods: Platelet activation: Platelets isolated from healthy donors were activated with thrombin and cross-linked collagen-related peptide (CRP-XL). Platelet-FVIII binding: Activated platelets were incubated with simoctocog alfa, efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol. Binding was quantified by flow cytometry either by immunostaining with an anti-FVIII antibody conjugated to Alexa fluor (AF)647 or direct measurement of rFVIII concentrates labelled with AF647. Phenotype shift: During the shift to a pro-coagulant state, phosphatidylserine (PS) becomes exposed on the platelet membranes, with the extent of PS exposure correlating with the level of FVIII binding. To assess PS exposure, activated platelets were incubated with Annexin V-BV421, a molecular probe with high affinity for PS. Inhibition of integrin αIIbβ3 signaling: Activated platelets were incubated with the integrin αIIbβ3 inhibitor antibody 10E5. Results: Binding to pro-coagulant platelets was significantly higher with simoctocog alfa than with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol irrespective of immunostaining method (p<0.05; Figure 1 shows detection with anti-FVIII antibody conjugated with AF647).To validate the variability in rFVIII-platelet interactions observed, we performed a dynamic platelet-binding assay with the two FVIII products exhibiting the strongest binding (simoctocog alfa and efmoroctocog alfa). The results of the dynamic assay confirmed the results of the static experiments (Figure 1). Exposure of activated platelets to simoctocog alfa and efmoroctocog alfa resulted in increased PS exposure, with a greater effect observed with simoctocog alfa, consistent with the results of the binding experiments. Immunostaining experiments revealed co-localization of simoctocog alfa and clusters of integrin αIIbβ3 in pro-aggregatory platelets. Inhibition of integrin αIIbβ3 by 10E5 decreased binding of simoctocog alfa to platelets and subsequent PS exposure in a dose-dependent manner (Figure 2). Analysis of platelet binding and phenotype shift in patients with hemophilia A are ongoing. Conclusion: Simoctocog alfa demonstrated higher binding to activated platelets in vitro compared with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol, resulting in an increased phenotype shift of platelets from the pro-aggregatory to the pro-coagulant state. The increased binding of simoctocog alfa was associated with a phenotypic shift in platelets as evidenced by increased exposure of PS on the platelet membranes. The binding of simoctocog alfa to platelets was disrupted when integrin αIIbβ3 activation was inhibited, suggesting a role of integrin αIIbβ3 signaling following binding of FVIII to platelets. Variations in platelet binding and signaling between different rFVIIIs might impact their efficacy for the prevention of bleeds. Further experiments are ongoing to assess the impact of FVIII on the release of von Willebrand factor and thrombin generation by activated platelets, and to repeat the FVIII-binding experiments using platelets from healthy donors vs hemophilia A patients.

Interactions between neutrophil extracellular traps and activated platelets enhance procoagulant activity in acute stroke patients with ICA occlusion.

BackgroundThe role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in stroke patients caused by thromboembolic occlusion of the internal carotid artery (ICA) remains unclear. Our objectives were to evaluate the critical role of NETs in the induction of hypercoagulability in stroke and to identify the functional significance of NETs during atherothrombosis.MethodsThe levels of NETs, activated platelets (PLTs), and PLT-derived microparticles (PMPs) were detected in the plasma of 55 stroke patients and 35 healthy controls. NET formation and thrombi were analysed using immunofluorescence. Exposed phosphatidylserine (PS) was evaluated with flow cytometry and confocal microscopy. PCA was analysed using purified coagulation complex, thrombin, and fibrin formation assays.FindingsThe plasma levels of NETs, activated PLTs, and PMP markers in the carotid lesion site (CLS) were significantly higher than those in the aortic blood. NETs were decorated with PS in thrombi and the CLS plasma of ICA occlusion patients. Notably, the complementary roles of CLS plasma and thrombin-activated PLTs were required for NET formation and subsequent PS exposure. PS-bearing NETs provided functional platforms for PMPs and coagulation factor deposition and thus increased thrombin and fibrin formation. DNase I and lactadherin markedly inhibited these effects. In addition, NETs were cytotoxic to endothelial cells, converting these cells to a procoagulant phenotype. Sivelestat, anti-MMP9 antibody, and activated protein C (APC) blocked this cytotoxicity by 25%, 39%, or 52%, respectively.InterpretationNETs played a pivotal role in the hypercoagulability of stroke patients. Strategies that prevent NET formation may offer a potential therapeutic strategy for thromboembolism interventions.FundingThis study was supported by grants from the National Natural Science Foundation of China (61575058, 81873433 and 81670128) and Graduate Innovation Fund of Harbin Medical University (YJSKYCX2018-58HYD).

Short-Term Effects of Chlorpromazine on Oxidative Stress in Erythrocyte Functionality: Activation of Metabolism and Membrane Perturbation.

The purpose of this paper is to focus on the short-term effects of chlorpromazine on erythrocytes because it is reported that the drug, unstable in plasma but more stable in erythrocytes, interacts with erythrocyte membranes, membrane lipids, and hemoglobin. There is a rich literature about the side and therapeutic effects or complications due to chlorpromazine, but most of these studies explore the influence of long-term treatment. We think that evaluating the short-term effects of the drug may help to clarify the sequence of chlorpromazine molecular targets from which some long-term effects derive. Our results indicate that although the drug is primarily intercalated in the innermost side of the membrane, it does not influence band 3 anionic flux, lipid peroxidation, and protein carbonylation processes. On the other hand, it destabilizes and increases the autooxidation of haemoglobin, induces activation of caspase 3, and, markedly, influences the ATP and reduced glutathione levels, with subsequent exposure of phosphatidylserine at the erythrocyte surface. Overall our observations on the early stage of chlorpromazine influence on erythrocytes may contribute to better understanding of new and interesting characteristics of this compound improving knowledge of erythrocyte metabolism.

Stimulation of Erythrocyte Cell Membrane Scrambling by Mitotane

Background: Mitotane (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane), a cytostatic drug used for the treatment of adrenocortical carcinomas, is effective by triggering tumor cell apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes, which is typically paralleled by cell shrinkage and breakdown of cell membrane phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca²⁺ concentration ([Ca²⁺]_i). The present study tested, whether treatment of human erythrocytes with mitotane is followed by eryptosis. Methods: [Ca²⁺]_i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. Results: Exposure to mitotane (≥ 5 µg/ml ≈ 16 µM) significantly increased [Ca²⁺]_i, increased annexin V binding and triggered hemolysis, but did not significantly modify forward scatter. The effect on annexin V binding was significantly blunted in the absence of extracellular Ca²⁺. Within 30 min Ca²⁺ ionophore ionomycin (1 µM) decreased forward scatter, an effect virtually abolished in the presence of mitotane (15 µg/ml). Conclusions: Mitotane increases [Ca²⁺]_i with subsequent phosphatidylserine translocation. By the same token mitotane inhibits Ca²⁺ induced cell shrinkage.

Adhesion of Annexin 7 Deficient Erythrocytes to Endothelial Cells

Annexin 7 deficiency has previously been shown to foster suicidal death of erythrocytes or eryptosis, which is triggered by increase of intracellular Ca2+ concentration ([Ca2+]i) and characterized by cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. Eryptosis following increase of [Ca2+]i by Ca2+ ionophore ionomycin, osmotic shock or energy depletion was more pronounced in erythrocytes from annexinA7-deficient mice (anxA7−/−) than in erythrocytes from wild type mice (anxA7+/+). As phosphatidylserine exposure is considered to mediate adhesion of erythrocytes to the vascular wall, the present study explored adhesion of erythrocytes from anx7−/− and anx7+/+-mice following increase of [Ca2+]i by Ca2+ ionophore ionomycin (1 µM for 30 min), hyperosmotic shock (addition of 550 mM sucrose for 2 hours) or energy depletion (removal of glucose for 12 hours). Phosphatidylserine exposing erythrocytes were identified by annexin V binding, cell volume estimated from forward scatter in FACS analysis and adhesion to human umbilical vein endothelial cells (HUVEC) utilizing a flow chamber. As a result, ionomycin, sucrose addition and glucose removal all triggered phosphatidylserine-exposure, decreased forward scatter and enhanced adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC), effects significantly more pronounced in anx7−/− than in anx7+/+-erythrocytes. Following ischemia, morphological renal injury was significantly higher in anx7−/− than in anx7+/+-mice. The present observations demonstrate that enhanced eryptosis of annexin7 deficient cells is paralleled by increased adhesion of erythrocytes to the vascular wall, an effect, which may impact on microcirculation during ischemia.

Stimulation of Platelet Death by Vancomycin

Background/Aims: Side effects of vancomycin, a widely used antibiotic, include thrombocytopenia. The vancomycin-induced thrombocytopenia has been attributed to immune reactions. At least in theory, thrombocytopenia could result in part from the triggering of apoptosis, which results in cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. The cell membrane scrambling could be initiated by a signaling involving increase of cytosolic Ca²⁺ activity, ceramide formation, mitochondrial depolarization and/or caspase activation. Vancomycin has indeed been shown to trigger neutrophil apoptosis. An effect of vancomycin on platelet apoptosis has, however, never been tested. The present study thus explored the effect of vancomycin on platelet activation and apoptosis. Methods: Human blood platelets were exposed to vancomycin and forward scatter was utilized to estimate cell volume, annexin V-binding to quantify phosphatidylserine (PS) exposure, Fluo-3 AM fluorescence to estimate cytosolic Ca²⁺ activity ([Ca²⁺]_i), antibodies to quantify ceramide formation and immunofluorescence to quantify protein abundance of active caspase-3. Results: A 30 minutes exposure to vancomycin (≥1 µg/ ml) decreased cell volume, triggered annexin V-binding, increased [Ca²⁺]_i, activated caspase 3, stimulated ceramide formation, triggered release of thromboxane B₂, and upregulated surface expression of CD62P (P-selectin) as well as activated integrin α_llbβ₃. Annexin V-binding and upregulation of CD62P (P-selectin) and integrin α_llbβ₃ was significantly blunted by removal of extracellular Ca²⁺. Annexin V-binding was not significantly blunted by pan-caspase inhibitor zVAD-FMK (1 µM). In conclusion, vancomycin results in platelet activation and suicidal platelet death with increase of [Ca²⁺]_i, caspase-3 activation, cell membrane scrambling and cell shrinkage. Activation and cell membrane scrambling required the presence of Ca²⁺, but not activation of caspases. Conclusion: Vancomycin exposure leads to platelet activation and apoptosis.

Increased Cation Conductance in Human Erythrocytes Artificially Aged by Glycation

Excessive glucose concentrations foster glycation and thus premature aging of erythrocytes. The present study explored whether glycation-induced erythrocyte aging is paralleled by features of suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface and cell shrinkage. Both are triggered by increases of cytosolic Ca(2+) concentration ([Ca(2+)](i)), which may result from activation of Ca(2+) permeable cation channels. Glycation was accomplished by exposure to high glucose concentrations (40 and 100 mM), phosphatidylserine exposure estimated from annexin binding, cell shrinkage from decrease of forward scatter, and [Ca(2+)](i) from Fluo3-fluorescence in analysis via fluorescence-activated cell sorter. Cation channel activity was determined by means of whole-cell patch clamp. Glycation of total membrane proteins, immunoprecipitated TRPC3/6/7, and immunoprecipitated L-type Ca(2+) channel proteins was estimated by Western blot testing with polyclonal antibodies used against advanced glycation end products. A 30-48-h exposure of the cells to 40 or 100 mM glucose in Ringer solution (at 37 degrees C) significantly increased glycation of membrane proteins, hemoglobin (HbA(1c)), TRPC3/6/7, and L-type Ca(2+) channel proteins, enhanced amiloride-sensitive, voltage-independent cation conductance, [Ca(2+)](i), and phosphatidylserine exposure, and led to significant cell shrinkage. Ca(2+) removal and addition of Ca(2+) chelator EGTA prevented the glycation-induced phosphatidylserine exposure and cell shrinkage after glycation. Glycation-induced erythrocyte aging leads to eryptosis, an effect requiring Ca(2+) entry from extracellular space.

Escherichia coli α-Hemolysin Triggers Shrinkage of Erythrocytes via KCa3.1 and TMEM16A Channels with Subsequent Phosphatidylserine Exposure

alpha-Hemolysin from Escherichia coli (HlyA) readily lyse erythrocytes from various species. We have recently demonstrated that this pore-forming toxin provokes distinct shrinkage and crenation before it finally leads to swelling and lysis of erythrocytes. The present study documents the underlying mechanism for this severe volume reduction. We show that HlyA-induced shrinkage and crenation of human erythrocytes occur subsequent to a significant rise in [Ca(2+)](i). The Ca(2+)-activated K(+) channel K(Ca)3.1 (or Gardos channel) is essential for the initial shrinkage, because both clotrimazole and TRAM-34 prevent the shrinkage and potentiate hemolysis produced by HlyA. Notably, the recently described Ca(2+)-activated Cl(-) channel TMEM16A contributes substantially to HlyA-induced cell volume reduction. Erythrocytes isolated from TMEM16A(-/-) mice showed significantly attenuated crenation and increased lysis compared with controls. Additionally, we found that HlyA leads to acute exposure of phosphatidylserine in the outer leaflet of the plasma membrane. This exposure was considerably reduced by K(Ca)3.1 antagonists. In conclusion, this study shows that HlyA triggers acute erythrocyte shrinkage, which depends on Ca(2+)-activated efflux of K(+) via K(Ca)3.1 and Cl(-) via TMEM16A, with subsequent phosphatidylserine exposure. This mechanism might potentially allow HlyA-damaged erythrocytes to be removed from the bloodstream by macrophages and thereby reduce the risk of intravascular hemolysis.

Modulation of suicidal erythrocyte cation channels by an AMPA antagonist

In neurons alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are heteromeric cation channels composed of different sub-units, including GluA1-GluA4. When expressed without GluA2, AMPA receptors function as Ca2+-permeable cation channels. In erythrocytes, activation of Ca2+-permeable cation channels triggers suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with subsequent exposure of phosphatidylserine at the cell surface. Activators of the channels and thus eryptosis include removal of extracellular Cl− (replaced by gluconate) and energy depletion (removal of glucose). The present study explored whether GluA1 is expressed in human erythrocytes and whether pharmacological AMPA receptor inhibition modifies Ca2+ entry and suicidal death of human erythrocytes. GluA1 protein abundance was determined by confocal microscopy, phosphatidylserine exposure was estimated from annexin V binding, cell volume from forward scatter in FACS analysis, cytosolic Ca2+ concentration from Fluo3 fluorescence and channel activity by whole-cell patch-clamp recordings. As a result, GluA1 is indeed expressed in the erythrocyte cell membrane. The AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) inhibited the cation channels following Cl− removal and the eryptosis following Cl− removal or energy depletion. The present study reveals a novel action of AMPA receptor antagonists and raises the possibility that GluA1 or a pharmacologically related protein participates in the regulation of Ca2+ entry into and suicidal death of human erythrocytes.

Inhibition of Suicidal Erythrocyte Death by Xanthohumol

Xanthohumol is a proapoptotic hop-derived beer component with anticancer and antimicrobial activities. Similar to nucleated cells, erythrocytes may undergo suicidal cell death or eryptosis, which is triggered by oxidative stress (tert-butylhydroperoxide, TBOOH) or energy depletion (removal of glucose). The triggers increase cytosolic Ca(2+) concentration, leading to activation of Ca(2+)-sensitive K(+) channels with subsequent cell shrinkage and to cell membrane scrambling with subsequent phosphatidylserine exposure at the erythrocyte surface. Eryptotic cells are cleared from the circulating blood, leading to anemia, and may adhere to the vascular wall, thus impeding microcirculation. The present experiments explored whether xanthohumol influences eryptosis using flow cytometry. Exposure of human erythrocytes to 0.3 mM TBOOH or incubation in glucose-free solution significantly increased Fluo3 fluorescence (Ca(2+) concentration) as well as annexin V-binding (cell membrane scrambling) and decreased forward scatter (cell volume), effects significantly blunted by xanthohumol. In conclusion, xanthohumol is a potent inhibitor of suicidal erythrocyte death in vitro.

Amyloid Induced Suicidal Erythrocyte Death

Amyloid peptides are known to induce apoptosis in a wide variety of cells. Erythrocytes may similarly undergo suicidal death or eryptosis, which is characterized by scrambling of the cell membrane with subsequent exposure of phosphatidylserine (PS) at the cell surface. Eryptosis is triggered by increase of cytosolic Ca²⁺ activity and by activation of acid sphingomyelinase with subsequent formation of ceramide. Triggers of eryptosis include energy depletion and isosmotic cell shrinkage (replacement of extracellular Cl^- by impermeable gluconate for 24 h). The present study explored whether amyloid peptide Aβ _1-42 could trigger eryptosis and to possibly identify underlying mechanisms. Erythrocytes from healthy volunteers were exposed to amyloid and PS-exposure (annexin V binding), cell volume (forward scatter), cytosolic Ca²⁺ activity (Fluo3 fluorescence) and ceramide formation (anti-ceramide antibody) were determined by FACS analysis. Exposure of erythrocytes to the amyloid peptide Aβ _1-42 (? 0.5 µM) for 24 h significantly triggered annexin V binding, an effect mimicked to a lesser extent by the amyloid peptide Aβ _1-40 (1 µM). Aβ _1-42 (? 1.0 µM) further significantly decreased forward scatter of erythrocytes. The effect of Aβ _1-42 (? 0.5 µM) on erythrocyte annexin V binding was paralleled by formation of ceramide but not by significant increase of cytosolic Ca²⁺ activity. The presence of Aβ _1-42 further significantly enhanced the eryptosis following Cl^- depletion but not of glucose depletion for 24 hours. The present observations disclose a novel action of Aβ _1-42, which may well contribute to the pathophysiological effects of amyloid peptides, such as vascular complications in Alzheimer''s disease.

Mechanisms and Significance of Eryptosis

Suicidal death of erythrocytes (eryptosis) is characterized by cell shrinkage, membrane blebbing, activation of proteases, and phosphatidylserine exposure at the outer membrane leaflet. Exposed phosphatidylserine is recognized by macrophages that engulf and degrade the affected cells. Eryptosis is triggered by erythrocyte injury after several stressors, including oxidative stress. Besides caspase activation after oxidative stress, two signaling pathways converge to trigger eryptosis: (a) formation of prostaglandin E(2) leads to activation of Ca(2+)-permeable cation channels, and (b) the phospholipase A(2)-mediated release of platelet-activating factor activates a sphingomyelinase, leading to formation of ceramide. Increased cytosolic Ca(2+) activity and enhanced ceramide levels lead to membrane scrambling with subsequent phosphatidylserine exposure. Moreover, Ca(2+) activates Ca(2+)-sensitive K(2+) channels, leading to cellular KCl loss and cell shrinkage. In addition, Ca(2+) stimulates the protease calpain, resulting in degradation of the cytoskeleton. Eryptosis is inhibited by erythropoietin, which thus extends the life span of circulating erythrocytes. Eryptosis may be a mechanism of defective erythrocytes to escape hemolysis. Conversely, excessive eryptosis favors the development of anemia. Conditions with excessive eryptosis include iron deficiency, lead or mercury intoxication, sickle cell anemia, thalassemia, glucose 6- phosphate dehydrogenase deficiency, malaria, and infection with hemolysin-forming pathogens.

Stimulation of erythrocyte phosphatidylserine exposure by chlorpromazine

Side effects of treatment with chlorpromazine include anaemia which could result from decreased formation or accelerated clearance of circulating erythrocytes. Recently, a novel mechanism leading to erythrocyte clearance has been disclosed. Osmotic shock, oxidative stress and glucose deprivation lead to activation of cation channels, Ca 2+ entry, activation of a Ca 2+-sensitive erythrocyte scramblase and subsequent exposure of phosphatidylserine at the erythrocyte surface. As macrophages are equipped with phosphatidylserine receptors, they bind, engulf and degrade phosphatidylserine exposing cells. The present experiments have been performed to explore whether chlorpromazine triggers phosphatidylserine exposure of erythrocytes. The phosphatidylserine exposure was estimated from annexin binding as determined in fluorescence activated cell sort (FACS) analysis. A 24 h exposure to glucose-free medium decreased cytosolic ATP levels, decreased cellular levels of reduced glutathione (GSH) and increased annexin binding. The effect on annexin binding and ATP but not on GSH was significantly enhanced in the presence of chlorpromazine (10 μM). Higher concentrations of chlorpromazine (40 μM) increased cytosolic Ca 2+ activity. Osmotic shock and Cl − removal similarly increased annexin binding, effects again being enhanced in the presence of chlorpromazine. In conclusion, the present observations point to a novel side effect of chlorpromazine, i.e. increased sensitivity of erythrocytes to glucose deprivation. The effect could well contribute to the known anaemia observed in the treatment with this antipsychotic drug.

Protein kinase C mediates erythrocyte “programmed cell death” following glucose depletion

Glucose depletion of erythrocytes leads to activation of Ca2+-permeable cation channels, Ca2+ entry, activation of a Ca2+-sensitive erythrocyte scramblase, and subsequent exposure of phosphatidylserine at the erythrocyte surface. Ca2+ entry into erythrocytes was previously shown to be stimulated by phorbol esters and to be inhibited by staurosporine and chelerythrine and is thus thought to be regulated by protein phosphorylation/dephosphorylation, presumably via protein kinase C (PKC) and the corresponding phosphoserine/threonine phosphatases. The present experiments explored whether PKC could contribute to effects of energy depletion on erythrocyte phosphatidylserine exposure and cell volume. Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorter analysis. Removal of extracellular glucose led to depletion of cellular ATP, stimulated PKC activity, led to translocation of PKCalpha, enhanced serine phosphorylation of membrane proteins, decreased cell volume, and increased annexin binding, the latter effect being blunted but not abolished in the presence of 1 microM staurosporine or 50 nM calphostin C. The PKC stimulator phorbol-12-myristate-13-acetate (3 microM) and the phosphatase inhibitor okadaic acid (1-10 microM) mimicked the effect of glucose depletion and similarly led to translocation of PKCalpha and enhanced serine phosphorylation, increased annexin binding, and decreased forward scatter, the latter effects being abrogated by PKC inhibitor staurosporine (1 microM). Fluo-3 fluorescence measurements revealed that okadaic acid also enhanced erythrocyte Ca2+ activity. The present observations suggest that protein phosphorylation and dephosphorylation via PKC and the corresponding protein phosphatases contribute to phosphatidylserine exposure and cell shrinkage after energy depletion.

Inhibition of erythrocyte “apoptosis” by catecholamines

Osmotic shock, oxidative stress and Cl- removal activate a non-selective Ca2+-permeable cation conductance in human erythrocytes. The entry of Ca2+ leads to activation of a scramblase with subsequent exposure of phosphatidylserine at the cell surface. Phosphatidylserine mediates binding to phosphatidylserine receptors on macrophages which engulf and degrade phosphatidylserine exposing cells. Moreover, phosphatidylserine exposure may lead to adherence of erythrocytes to the vascular wall. In the present study, we explored whether activation of the non-selective cation conductance and subsequent phosphatidylserine exposure might be influenced by catecholamines. Phosphatidylserine exposure has been determined by FITC-annexin V binding while cell volume was estimated from forward scatter in FACS analysis. Removal of Cl- enhanced annexin binding and decreased forward scatter, an effect significantly blunted by the beta agonist isoproterenol (IC50 approx. 1 microM). Fluo-3 fluorescence measurements revealed an increase of cytosolic Ca2+ activity following Cl- removal, an effect again significantly blunted by isoproterenol exposure (10 microM). Whole-cell patch-clamp experiments performed in Cl- free bath solution indeed disclosed a time-dependent inactivation of a non-selective cation conductance following isoproterenol exposure (10 microM). Phenylephrine (IC50<10 microM), dobutamine (IC50 approx. 1 microM) and dopamine (IC50 approx. 3 microM) similarly inhibited the effect of Cl- removal on annexin binding and forward scatter. In conclusion, several catecholamines inhibit the Cl- removal-activated Ca2+ entry into erythrocytes, thus preventing increase of cytosolic Ca2+ activity, subsequent cell shrinkage and activation of erythrocyte scramblase. The catecholamines thus counteract erythrocyte phosphatidylserine exposure and subsequent clearance of erythrocytes from circulating blood.

Stimulation of erythrocyte ceramide formation by platelet-activating factor

Osmotic erythrocyte shrinkage leads to activation of cation channels with subsequent Ca2+ entry and stimulates a sphingomyelinase with subsequent formation of ceramide. Ca2+ and ceramide then activate a scramblase leading to breakdown of phosphatidylserine asymmetry of the cell membrane. The mediators accounting for activation of erythrocyte sphingomyelinase and phosphatidylserine exposure remained elusive. The study demonstrates that platelet-activating factor (PAF) is released from erythrocytes upon hyperosmotic cell shrinkage. The experiments further disclose the presence of PAF receptors in erythrocytes and show that PAF stimulates the breakdown of sphingomyelin and the release of ceramide from erythrocytes at isotonic conditions. PAF further triggers cell shrinkage (decrease of forward scatter) and phosphatidylserine exposure (annexin binding) of erythrocytes. The stimulation of annexin-binding is blunted by a genetic knockout of PAF receptors, by the PAF receptor antagonist ABT491 or by inhibition of sphingomyelinase with urea. In conclusion, PAF activates an erythrocyte sphingomyelinase and the then formed ceramide leads to the activation of scramblase with subsequent phosphatidylserine exposure.

Independent mechanisms for macrophage binding and macrophage phagocytosis of damaged erythrocytes. Evidence of receptor cooperativity.

The binding and phagocytosis of oxidatively damaged red blood cells (OxRBCs) by mouse peritoneal macrophages can be inhibited by oxidatively modified LDL (OxLDL), implying some commonality at their receptor-binding domains. Studies from many different laboratories support the view that OxRBC binding is due to the disruption of plasma membrane phospholipid asymmetry and the subsequent exposure of phosphatidylserine (PS) on the outer membrane leaflet. Presumably, oxidation of LDL creates a surface structure on it in some way homologous to the PS-rich domain on OxRBCs. Apoptotic cells in some instances are also recognized because of PS exposure on the outer leaflet of the membrane, and apoptotic cells are a common feature of atherosclerotic lesions. In the present studies, the mechanisms of binding and internalization of cells recognized by virtue of their membrane PS were studied using OxRBCs or vanadate-treated erythrocytes (VaRBCs) as models. Disruption of phospholipid asymmetry with vanadate produced cells that were bound by macrophages in the same divalent cation-dependent manner as OxRBCs. However, whereas OxRBCs were rapidly phagocytosed, VaRBCs were not. Stimulation of mouse macrophages with phorbol myristate acetate resulted in a concentration-dependent induction of phagocytosis of bound VaRBCs, an effect that could be prevented by the protein kinase C inhibitor staurosporine. Because phagocytosis of OxRBCs occurred unassisted, we speculated that there must be additional membrane changes induced by oxidation (over and above the disruption of phospholipid asymmetry) that contribute to phagocytosis of OxRBCs, possibly resulting in the ligation of a distinct receptor that does not necessarily contribute to adherence. This proposal is supported by the finding that ligation of macrophage Fc gamma receptors by the anti-Fc gamma RII/RIII antibody 2.4G2 triggers the phagocytosis of bound VaRBCs. Phagocytosis is also triggered by subthreshold opsonization of VaRBC, i.e., by antibody concentrations that do not by themselves cause binding and phagocytosis of native RBCs. Finally, treatment with low concentrations of glutaraldehyde, which causes membrane protein cross-linking, promotes the phagocytosis of VaRBCs, but, at the low concentration used, has little or no effect on binding and phagocytosis of native RBCs. We suggest that the internalization of damaged cells, bound because of PS exposure, requires the cooperation of a PS-binding receptor with at least one additional receptor to trigger an intracellular signaling pathway to initiate phagocytosis.