Flow cytometry for RBC damage and complement activation

While phospholipid asymmetry has been well characterized in red blood cells (RBCs), controversy exists as to what role PIP2 plays in cation-induced phosphatidylserine (PS) exposure. We report that PIP2 can redistribute intracellular cations and thereby lead to a loss of phospholipid asymmetry. Flow cytometry was employed to monitor intracellular cation levels by using the fluorophore Fluo-3 and exposure of PS on the outer surface of the RBC bilayer by using fluorescently labeled annexin V. The addition of PIP2 to RBCs led to a concentration-dependent increase in cytosolic cations and PS exposure. IF RBCs were preincubated with 25 microM neomycin sulfate, an inhibitor of phosphoinositide metabolism, PIP2-induced PS exposure decreased dramatically. If the RBC buffer system contained 2.5 mM EGTA, PS exposure also decreased significantly, suggesting a competition between intracellular Fluo-3 and extracellular EGTA. Together, these data indicate that (1) PS exposure was found in RBCs that exhibited an increased cytosolic cation concentration available for the fluorophore. Fluo-3, (2) both the level of intracellular cations and the movement of PS from the inner to the outer monolayer were affected by the level of PIP2 in the bilayer, (3) the cleavage of PIP2 by a phosphoinositide-specific phospholipase lead to the redistribution of intracellular cations and to an increase in the amount of PS exposed on the outer leaflet of the bilayer, and (4) a transient channel could be formed during the interaction of PIP2 with the RBC membrane which would then allow the transbilayer movement of phospholipids and cations.

A flow cytometry approach for quantitative analysis of cellular phosphatidylserine distribution and shedding

Hydroxyurea Treatment of Children with Sickle Cell Disease Inhibits RBC Membrane Degradation by Secretory Phospholipase a2.

Sepsis Induced Red Cell Dysfunction (SiRD): Physiology and Mechanisms

Phosphatidylserine (PS) exposure on the outer membrane surface is a known storage lesion of red blood cells (RBCs). Procoagulant activity (PCA) of PRBC units has been shown to increase during storage. However, the PCA and the relation to PS of banked RBCs remain unknown. The aim of this study is to investigate the PCA and the correlation with PS exposure on banked RBCs. Human normal leucodepleted RBC concentrates were stored in saline-adenine-glucose-mannitol (SAGM) solution at 4 °C for up to 42 days. PS exposure was measured by lactadherin and annexin V. PCA of RBCs was determined by one-step recalcification assay and further determined by prothrombinase and tenase assays. The correlation between PCA and PS exposure of RBCs was measured by inhibition tests with lactadherin or annexin V. Lactadherin detected 1·5% PS-positive stored RBCs vs 0·5% for annexin V after 14 days of storage, which significantly reached 18·4 vs 4·5% after 42 days of storage. Potent PCA was observed on day 14 and continuously increased. PCA of RBCs on day 42 was inhibited 94% by lactadherin vs 45% by annexin V. Incubation at 37 °C in fresh heparinised plasma partially reversed PS exposure and PCA of RBCs stored for 14 days but had no effect on these parameters for cells stored for 42 days. PCA and PS exposure of stored RBCs increased during storage and showed significant positive correlation. PCA of long-term stored RBCs may not be reversed by transfusion.

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.

Cryptantigens: time to uncover the real significance of T-activation.

It is thought that the clearance of transfused red blood cells (RBCs) is related both to the storage time of the transfusion product and to the inflammatory status of the recipient. We investigated these effects in a randomized, "two-hit," healthy volunteer transfusion model, comparing autologous RBCs that were stored for 35 days with those that were stored for 2 days. Healthy male volunteers donated 1 unit of autologous RBCs either 2 days (2D) or 35 days (35D) before the study date. The experiment was started by infusion of 2 ng/kg lipopolysaccharide ("first hit"). Two hours later, the stored RBCs ("second hit") were reinfused, followed by the labeling of RBCs with biotin. Clearance of biotin-labeled RBCs (BioRBCs) was measured during the 5-hour posttransfusion endotoxemia period along with measurements of phosphatidylserine (PS) exposure, lactadherin binding, and expression of CD47 (cluster of differentiation 47; a transmembrane protein encoded by the CD47 gene). In the 2D stored RBCs group, 1.5% ± 3.4% of infused BioRBCs were cleared from the circulation 5 hours posttransfusion versus 4.8% ± 4.0% in the 35D stored RBCs group (p = 0.1). There were no differences in PS exposure, lactadherin binding, or CD47 expression between fresh and stored RBCs or between pretransfusion and posttransfusion measurements. Our study shows a low clearance of RBCs even during endotoxemia. Furthermore, short-term clearance of BioRBCs during endotoxemia was not related to storage duration. Consistent with these observations, PS exposure, lactadherin binding, and CD47 expression did not differ between 2D and 35D stored cells before or after transfusion. We conclude that, in the presence of endotoxemia, clearance of 35D stored autologous RBCs is not increased compared with 2D stored fresh RBCs.

Critical role of Th17 cells in development of autoimmune hemolytic anemia

Enterohemorrhagic Escherichia coli (EHEC) causes thrombotic microangiopathy, yet the red blood cell (RBC)-centered mechanism has remained unclear. We identify the RTX-family hemolysin EhxA as the driver of RBC-mediated thrombogenesis. Deletion of ehxA abolishes Ca2+ influx, phosphatidylserine (PS) exposure, progression from discocyte to echinocyte to spherocyte, thrombin generation, RBC-endothelium adhesion, and RBC aggregation. Genetic complementation restores these readouts to wild type, and purified EhxA in bacteria-free assays recapitulates them while localizing to intact RBC membranes. By contrast, Δstx2 mutants do not elicit these RBC phenotypes, distinguishing this pathway from Shiga toxin-dependent effects. Multiple regression quantifies the link between PS exposure, morphology, and procoagulant outputs. In rats, infection with wild type increased RBC remodeling and venous thrombosis, whereas infection with ΔehxA did not. Together, the data define an EhxA-Ca2+-PS pathway that drives RBC structural remodeling and procoagulant activation during EHEC infection and nominate RTX toxins as targets for preventing toxin-induced coagulopathies.

The three-dimensional structure of the PMCA pump has not been solved, but its basic mechanistic properties are known to repeat those of the other Ca(2+) pumps. However, the pump also has unique properties. They concern essentially its numerous regulatory mechanisms, the most important of which is the autoinhibition by its C-terminal tail. Other regulatory mechanisms involve protein kinases and the phospholipids of the membrane in which the pump is embedded. Permanent activation of the pump, e.g. by calmodulin, is physiologically as harmful to cells as its absence. The concept is now emerging that the global control of cell Ca(2+) may not be the main function of the pump; in some cell types, it could even be irrelevant. The main pump role would be the regulation of Ca(2+) in cell microdomains in which the pump co-segregates with partners that modulate the Ca(2+) message and transduce it to important cell functions.

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

Recognition of the exposure of phosphatidylserine (PS) on the outer surface of plasma membrane has been implicated in the phagocytosis of aged/apoptotic cells. Because oxidized low-density lipoprotein (OxLDL) has been reported to block the phagocytosis, here we examined whether lectin-like OxLDL receptor 1 (LOX-1), the OxLDL receptor in endothelial cells, mediates phagocytosis of aged/apoptotic cells by endothelial cells. Cultured bovine aortic endothelial cells (BAE) and Chinese hamster ovary (CHO) cells expressing bovine LOX-1 (BLOX-1-CHO), but not wild-type CHO-K1 cells, bound aged red blood cells (RBC) and apoptotic cells, which were further phagocytosed. The binding of aged RBC and the phagocytosis of apoptotic cells were inhibited by OxLDL, acetyl LDL, and other LOX-1 ligands in both BAE and BLOX-1-CHO. mAb against LOX-1 blocked the binding of aged RBC to BAE, suggesting a role for LOX-1 in the recognition of aged cells. The recombinant soluble LOX-1 inhibited the interactions of aged/apoptotic cells with both BLOX-1-CHO and BAE and distinguished aged RBC from native RBC and apoptotic cells from native cells. PS liposome inhibited these LOX-1-mediated interactions with aged/apoptotic cells, suggesting LOX-1 recognizes PS of the apoptotic cells. PS exposed on the surface of apoptotic cells is known to be procoagulant. Accordingly, increased OxLDL may be one of the reasons for enhanced coagulation in atherosclerosis, inhibiting the removal of apoptotic cells.

A Nonselective Cation Permeabilization Pathway (Pcat) of Human Red Blood Cells (RBCs) Activated by Ca2+ and by Isotonic Dehydration.

Background/Aims: Sickle cell disease (SCD) also known as sickle cell anemia is one of the most worldwide-disseminated hereditary hemoglobinopathies. It is caused by a single amino acid substitution at the sixth residue of β globin gene (Glu6Val), which results in an abnormal hemoglobin called hemoglobin S (HbS). The acceleration of HbS polymerization induces rigid and dysfunctional erythrocytes that play a central role in acute and chronic clinical manifestations of SCD(Conran et al.,2009). Vaso-occlusion and hemolytic anemia are the hallmarks of SCD. We aim to explore the cellular environment of red blood cells to explain the physiopathology of SCD. In fact, the life span of circulating erythrocytes in healthy individuals vary from 100 to 120 days. In SCD, red blood cells undergo a form of cell death, namely, eryptosis before they reach their full life span. Eryptosis is triggered by a wide variety of factors as hyperosmolarity, oxidative stress and energy depletion. It is characterized by the presence of membrane blebbing, cell shrinkage, and phosphatidylserine (PS) exposure (Lang et al.,2012). In this study, we will explore the mechanism of triggering of eryptosis in Sickle cell disease. Methods: Following clinical diagnosis, 50 homozygous SCD patients and 30 healthy donors were identified for hematological and cellular assays. Flow cytometry was performed in order to determine the viability parameters of erythrocytes. The morphology of red blood cells and the externalization of phosphatidylserine was detected by labeling red blood cells with Annexine V. Moreover, we had identified intracellular calcium concentration and ceramide level by labeling erythrocytes with Fluo3-am and anti-ceramide antibodies. Finally, we had quantified reactive oxygen species (ROS) by labeling red blood cells with CM-H2DCFDA. Results and Discussion: Eryptosis in sickle cell patients is accelerated. In fact, PS (+) red blood cells are more present in patients than in healthy subjects. Therefore, eryptosis is triggered by oxidative stress, which stimulates the increase of calcium activity and subsequent externalization of PS and red blood cells shrinkage in sickle cell patients. However, the pathway of ceramide can also be considered a potential stimulating factor of eryptosis in SCD. Eryptosis ensures healthy erythrocyte quantity in circulation, whereas excessive eryptosis is the cause of acute anemia and may contribute to vaso-occlusive crisis in SCD patients.