Phospholipase D Activity Is Essential for PKC-Mediated Phosphatidylserine Exposure.
Authors: DeJong Kitty , Rahbar Shahrzad A. , Kuypers Frans A. 
Published: April 2020

Abstract
The adverse effects of red blood cells (RBC) that expose phosphatidylserine (PS) in the sickle cell circulation are well recognized. However, the mechanism that underlies the formation of these cells is not well understood. We have shown previously that activation of protein kinase C (PKC) using phorbol myristate acetate (PMA) stimulates RBC to expose PS on a subset of cells. This process was dependent on Ca2+-influx induced by this stimulation. PKC inhibitors were shown to counteract this process and also reduce PS exposure induced by high levels of intracellular Ca2+. Furthermore, PIP2 administered to the RBC similarly induces PS exposure. Because both PKC and PIP2 can stimulate phospholipase D (PLD), we investigated the role of PLD in signal transduction initiated phospholipid scrambling. PLD cleaves phospholipids to produce phosphatidic acid (PA), which can be further metabolized to lysophosphatidic acid, an important lipid mediator, or to diacylglycerol (DAG), itself a PKC activator. In presence of primary alcohols such as ethanol or 1-butanol, PLD generates phosphatidylethanol or phosphatidylbutanol, respectively. Using annexin V, we measured the fraction of PS-exposing cells after treatment of RBC with PMA in presence of these primary alcohols. Treatment with 1% ethanol as well as 0.2% 1-butanol (at 0.4% hematocrit) completely abolished the generation of PS exposure in PMA-treated cells, indicating that the formation of PA by PLD was essential for scrambling. Anandamides are structural analogues of arachidonoyl ethanolamide, an endocannabinoid. Since these structures provide feedback inhibition of the PLD-isoform that produces these compounds in the brain and were found effective on plant PLD isoforms, we evaluated their effect on PLD-mediated PS scrambling in our assay. Short-chain lauroyl ethanolamide as well as arachidonoyl ethanolamide itself induced concentration-dependent inhibition of PMA-induced PS exposure. This suggests that these compounds indeed inhibit the isoforms of PLD present in the RBC, and confirms the importance of PLD activity for PKC-mediated PS exposure. Furthermore we evaluated the effect of propranolol, which inhibits phosphatidate phosphatase, the enzyme that converts PA to DAG. PMA-induced PS exposure was more than 80% inhibited by propranolol, indicating that the conversion of DAG from PLD-generated PA is essential for PKC-mediated PS exposure. PLD inhibitors did not inhibit Ca2+-induced scrambling initiated with Ca-ionophore, indicating that loading RBC with high levels of intracellular Ca2+ can override the requirement for PLD activation, possibly because DAG is formed through direct activation of phospholipase C. These results show that PLD activation is essential for PMA-induced Ca-influx and PS exposure, and that the subsequent formation of DAG from PA is a critical step in this process. Altered Ca2+ homeostasis, increased PA generation and increased PKC activation are observed in sickle RBC. Our data indicate that activation of signal transduction pathways plays an important role in generating PS exposure in sickle cells.