Intracellular Calcium and Phosphatidylserine Exposure in the red Blood Cells

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1 Intracellular Calcium and Phosphatidylserine Exposure in the red Blood Cells Biotechnology Seminar 2 Yaser Alkhaled

2 Table of Content 1. Introduction Membrane of red blood cell Exposure of phosphatidylserine and intracellular ca References

3 1. Introduction Erythrocytes differentiated from stem cells in bone marrow through process named erythropoiesis to form mature RBCs. Erythrocytes initiate their formation in the yolk sac (3 to 4 week old human embryo), then transfer into liver. At the end of the pregnancy stage, erythrocytes are formed in the bone marrow (6). The life span of human red blood cells is about days. They are relative simple since they don t have nuclei (7) and flexible biconcave discus with diameter about 8µM. Therefore, they enable to enter the capillaries easily. Afterwards, their membrane becomes more rigid and then they are eliminated from the circulation by the macrophage. The main function of erythrocytes is the gases exchange in the body, they transfer the oxygen from the lung into the tissues and take the generated carbon dioxide, which result from the metabolic process (5). Renal erythropoietin is an important hormone, which plays an important role in the blood formation via stimulation the stem cells in bone marrow. In case of decreasing of the oxygen level (hypoxia), the kidneys sense and secret hormone erythropoietin (EPO) into the blood circulation, which induce the hematopoietic growth factor to produce the erythrocytes into the circulation to correct the tissues hypoxia (8). 3

4 Fig(1). Role of the erythropoietin in the blood formation (8). 2. Membrane of red blood cell The human membrane of red blood cells consists of lipid, protein and carbohydrate. Phospholipid and cholesterol are the main lipid in the cell membrane. The lipids are asymmetrically distributed through the biological cell membrane, where the phosphatidylcholin (PC) and the sphengomyelin (SM) are located predominantly in the outer layer, whereas phosphtidylethanolamin(pe), phosphtidylinositols(pi), and phoshatidylserine(ps) are found mostly in the inner leaflet of bilayer (4). The membrane proteins composed of an external hydrophobic portion attached with carbohydrate, an internal hydrophilic portion. Under the lipids bilayers found several of proteins spectrin, ankyrin and actin, these proteins forms filamentous network know as cytoskeleton. The cytoskeleton interact with the lipids bilayers 4

5 and the integral proteins to maintenance the membrane integrity, also it play an important role in the red blood cells shape and their flexibility (1). Fig(2). Structure of the proteins cytoskeleton (1). The membrane phospholipids are regulated by three different proteins, flippase, floppase and scramblase. As shown in the figure(3), the aminophospholipide translocase (flippase)is responsible for the transferring phosphatidylethanolamine (PE) and phpsphatidylserine (PS) from the outer to the inner leaflet against their concentration gradient, whereas the protein floppase flop the phospholipids from one side of bilayer to the other, especially sphingomyelin(sm) and phoshatidylcholine(pc) from the inner leaflet to the outer surface. Scramlase is responsible for the translocation phospholipids between two layers of cell membrane (flip-flop) (4). 5

6 Fig(3). Phospholipids bilayer of cell membrane (4). 3. Exposure of phosphatidylserine and intracellular ca 2+ In the normal conditions the phosphatidylserine (PS) located in the inner leaflet membrane red blood cells, the exposure of phosphatidylserine(ps) on the outer surface of erythrocytes considers as a marker for eryptosis, while in the nucleated cells serve as a signal for apoptosis. This signal recognized by macrophage to remove these cells from circulation blood, the eryptosis cell characterized by membrane blebbing, phosphatidylserine(ps) exposure and vesicle formation (2). As shown in the figure (4), the activation of the platelets under certain condition leads to release prostaglandin E2 and lysophosphtidic acid (LPA). These substances consider as mediators, which activate a nonselective voltage dependent cation (NSVDC) channel leading to an influx of extracellular Ca 2+. The increase of intracellular Ca 2+ activates the Gardios channel and scramblase, whereas aminophosphlipid translocase (flippase) inhibited the activation of 6

7 Gardos channel leads to an efflux of intracellular KCL and subsequently lead to shrinking the cell (7). The exposure of phosphatidylserine on the outer leaflet lead to adhesion of the cell into the endothelium in some diseases like malaria, diabetes, thalassemia and sickle cell anemia (2). Fig(4). Signaling cascade for PS exposure in red blood cells (7). Furthermore, the activation of the erythrocytes by various activators leads to elevation in the intracellular calcium content. This elevation differs according to the kind of the activator and rising the intracellular calcium which activates the calcium pump. As shown in the figure(5) the treatment of the RBCs with lysophosphatidic acid(lpa) lead to direct increasing in the intracellular Ca 2+ and reached to 80%, in comparison with the Ca 2+ ionophore A23187 the elevation reached to 99.2%. Whereas, the activation of the cells by phorbol-12 myristate-13 acetate PMA the increasing in the intracellular Ca 2+ is less than the 7

8 lysophosphatidic acid LPA and Ca 2+ ionophore A23187 with percentage about 40% (2). Fig(5). Intracellular Ca 2+ of RBCs activate with 2.5LPA,2 A23187,6 PMA (2). The annexin V used to detect the exposure of phosphatidylserine since its specifically binding to the exposed phosphatidylserine. The erythrocytes are activated by lysophosphatidic acid (LPA), Ca 2+ ionophore A23187 or phorbol-12 myristate-13 acetate(pma) in the presence of 2mM of extracellular Ca2+ which leads to phoshatidylserine exposure in the outer leaflet of RBCs membrane with different values. As shown in the fig (6) the activation of RBCs by LPA, A23187 and PMA in presence and in the absence of extracellular Ca 2+. In case of the presence of Ca 2+ the phoshatidylserine exposure about 35% which were activated by LPA and about 20% with A In addition, the treatment with PMA showed a significant increase of phoshatidylserine exposure (80%) and higher than the stimulation by LPA or A In the absence of extracellular Ca 2+ the results were different, there were no phoshatidylserine exposure during the treatment of RBCs by the activators LPA and A In contrast to that, the activation by PMA there 8

9 was a significant increase in phoshatidylserine exposure in the outer leaflet of RBCs membrane about 50%, but this was lower than in the present of 2+ (2). extracellular a Ca Fig (6) phoshatidylserine exposure in RBCs in the presence and absence extracellular Ca 2+ (2).. 9

10 References 1.J. E. Smith Erythrocyte Membrane: Structure, Function, and Pathophysiology Vet Pathol : Nguyen D,B,., Wagner-Britz, L, Maia S, Steffen P, Wagner C, Kaestner L., 4 and Bernhardt I Regulation of Phosphatidylserine Exposure in Red Blood Cells 3.Kaestner, L., Steffen, P, Nguyen, D.B., Wang, J., Wagner-Britz, L., Jung, A., Wagner. C., Bernhardt, I Lysophosphatidic acid induced red blood cell aggregation in vitro. Bioelectrochemistry 87 (2011), R. F. A. Zwaal*, P. Comfurius and E. M. Bevers Surface exposure of phosphatidylserine in pathological cells 5.S. Peter Klinken Red blood cells 6. K. M. MORITZ,1 GAIK BEE LIM,2 AND E. M. WINTOUR1 Developmental regulation of erythropoietin and erythropoiesis 7. Nguyen, D.B Phosphatidylserine exposure in red blood cells: A suggestion for the active role of red blood cells in blood clot formation. Dissertation, Universität des Saarlandes, Jerry L. Spivak THE ANAEMIA OF CANCER DEATH BY A THOUSAND CUTS 2005 ( ) 10

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