Ankyrin Exposure By Activated PKC Triggers The Erythrophagocytosis Of Macrophages

Although erythrocytes cannot synthesize proteins, erythrocyte aging and removal influence important processes in the body. Every day, approximately 2×1011 human erythrocytes are being destroyed and replenished throughout the 100–120 d lifespan of these cells. Thus, macrophages play an important role in mediating the sequestration of aged or diseased erythrocytes. In addition, the mechanisms for macrophage erythrophagocytosis must be very robust. The presence of phosphatidylserine(PS) at the outer leaflet of erythrocytes is documented as a signal for the clearing of circulating erythrocytes. Macrophages recognize, engulf, and degrade erythrocytes by using specific receptors for PS. In erythrocytes, RGD-containing ankyrin exposed on the cell surface can also trigger αvβ3 integrin-mediated erythrophagocytosis. The cell surface exposure of RGD-containing ankyrin triggers αvβ3 integrin-mediated erythrophagocytosis and activated PKC induces programmed cell death. Thus, in this paper we assessed the relationship between PMA-induced activation of PKC and ankyrin-exposing cells. We report here that activated PKC stimulated the influx of calcium into a subpopulation of erythrocytes and induced protein phosphorylation, ultimately leading to ankyrin exposure. We conclude that PKC activation is associated with ankyrin exposure, and this may provide an underlying molecular mechanism for removal aged or diseased erythrocytes.In the present study, the PKC-specific activator PMA induced ankyrin exposure in a subpopulation of erythrocytes, and the subpopulation of erythrocytes with ankyrin exposure was diminished through PMA incubation in the presence of two inhibitors, which indicate that PMA-induced ankyrin exposure is specific and PKC mediated. However, ankyrin was not exposed in all cells, and the increased size of ankyrin exposure was dependent on PMA dose and time. Repeat experiments using the same erythrocyte samples showed similar subpopulations of erythrocytes with ankyrin exposure. Meanwhile, the erythrocytes from different donors presented significant differences. These results suggest that the degree of PKC modulation varies in different erythrocyte specimens and different specimens have individual PMA-induced properties. In addition, the expression level or the remaining activity of PKC in erythrocytes varies among the subsets of erythrocytes. These observations support the present finding that not all erythrocytes have the same PKC content. PMA-induced ankyrin exposure prompts erythrocyte adhesion to macrophages. Pre-incubation of erythrocytes with purified ankyrin or αvβ3 integrin significantly inhibited erythrocytes-macrophages adhesion in a concentration dependent fashion, and their combined inhibitory effects were greater than the alone one. These results further support that ankyrin exposure plays a potential role in erythrocytes-macrophages adhesion, and also suggest that ankyrin exposure may be a new mechanism for erythrophagocytosis.Without major calcium stores in the erythrocytes, the elevation of intracellular calcium levels requires increased calcium influx. PMA stimulates calcium influx into a subpopulation of erythrocytes and causes programmed cell death. In the present study, these findings indicate that calcium internalization is fundamental to ankyrin exposure. And ankyrin exposure is not directly correlated with elevated calcium levels, and calcium internalization does not necessarily induce ankyrin exposure. Considering that chelerythrine chloride is an inhibitor of the active(phosphorylation) site of PKC, we speculated that membrane protein phosphorylation participates in ankyrin exposure. Ankyrin phosphorylation also may be required for ankyrin exposure induced by PMA.Erythrocytes were separated by density gradient centrifugation, and then aged erythrocytes were used to detect the level of calcium, ankryrin phosphorylation and ankyrin exposure. These experimental findings reflect that ankyrin exposure as a signal for the clearing of circulating erythrocytes, and further support that calcium internalization and ankyrin phosphorylation induced ankyrin exposure.Also, the deformability of erythrocytes was tested. Results show that ankyrin exposure significantly reduced the deformation properties of erythrocytes. Ankyrin phosphorylation by PMA resulted in the decreased affinity of membrane skeleton proteins, but not directly related to the erythrocyte deformability. Further study found that the changes in the binding affinity of the SAB domain via 4.1R phosphorylation play an important role in the development of erythrocyte deformability. Thus, decreased deformability of erythrocyte by PKC promotes the adhesion between macrophages and red blood cell.In summary, we proved that activated PKC by PMA induces ankyrin exposure on the erythrocyte surface in the presence of extracellular calcium. Although calcium internalization is required for ankyrin exposure, this process does not necessarily induce ankyrin exposure. In addition, phosphorylation alone is not sufficient to induce ankyrin exposure in the absence of extracellular calcium. Over all, PMA induces calcium influx into the erythrocytes, leading to the activation of calcium-dependent enzymes, and PMA induces the phosphorylation of membrane proteins, ultimately inducing ankyrin exposure and erythrophagocytosis. Hence, the calcium influx into the erythrocytes and the ankyrin phosphorylation induced by PMA probably participate in ankyrin exposure. This study may provide insights into the molecular mechanisms of removing aged or diseased erythrocytes.