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

Abstract

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.

The mammalian plasma membrane is defined by transmembrane asymmetries in lipid unsaturation, leaflet packing, and protein shape

“…To assess biophysical asymmetry, asymmetric PMs in untreated RBCs were compared with those where asymmetry was pharmacologically abrogated. Specifically, treatment with phorbol myristate acetate (PMA) induces efficient PM scrambling, which is detected by the exposure of PS on the exoplasmic leaflet via the PS-binding probe Annexin V (AnxV) (45). This effect was confirmed, as untreated RBCs were almost exclusively AnxV-negative, whereas PMA-treated RBCs were uniformly AnxV-positive ( Fig 3F, left column).…”

Membrane Remodelling and Vesicle Formation During Ageing of Human Red Blood Cells

“…It could also be speculated that what triggers the system for the final clearance is simply the crossing of a certain threshold of cell size and rigidity, when the now smaller and less deformable, but otherwise healthy RBC, will slow down in crossing the sinusoids allowing more time for the probing by macrophages. The phagocytes, by recognizing a combination of parameters such as size, deformability, the appearance of "eat me signals", perhaps a decrease in "don't eat me" signals (that were progressively lost as part of the previous processing), will now select the cell for removal and engulf it, this time as a whole cell, as opposed to only pinching off parts of its membrane [51][52][53]. These observations all concur to stimulate a reconsideration of the still largely unknown molecular and cellular mechanisms that regulate RBC senescence and clearance.…”

Eryptosis (quasi-apoptosis) the human red blood cells. Its role in medicinal therapy

“…Этот процесс запускает механизм избавления от дефектных клеток, не нарушая целостности клеточной мембраны и не производя выброс цитозольного материала во внешнюю среду [15,243]. Эриптоз эритроцитов характеризуется расстройством асимметрии фосфолипидов клеточной мембраны, в ходе которого происходит перемещение фосфолипида клеточной мембраны фосфатидилсерина от внутреннего слоя билипидной мембраны к внешнему слою, процесс регулируется флиппазой [299,300]. Эта трансформация мембраны похожа на апоптоз ядросодержащих клеток, где флиппаза обслуживает архитектуру фосфолипидов мембраны [96,287].…”