Red Blood Cell Scramblase Activation Is Induced by Phorbol Ester and Prevented by Inhibition of Protein Kinase C

Phosphatidylserine (PS) is normally confined to the inner leaflet of the red blood cell (RBC) membrane, but is externalized in many sickle RBC. This external PS may contribute to decreased sickle RBC survival, increased thrombogenesis, and increased sickle RBC-endothelial interaction. Aminophospholipid Translocase (APLT) and Phospholipid Scramblase (PLSCR) are thought to regulate the distribution of PS between the inner and outer membrane leaflets. APLT maintains phospholipid (PL) asymmetry by returning externalized PS to the inner membrane leaflet. PLSCR disrupts PL asymmetry by causing nonspecific PL equilibration across the membrane. The mechanism(s) of PLSCR activation have not been defined, but previous studies have demonstrated PS externalization as a result of treatment with the potent PKC activator, PMA (phorbol-12-myristate-13-myristate). We examined the effect of PMA on PLSCR activation (measured by NBD-PC internalization) in normal RBC in the presence or absence of the PKC inhibitor, chelerythrine chloride. In response to PMA, PLSCR is significantly and rapidly activated as measured by the fraction of NBD-PC internalized after six minutes compared to a DMSO control (0.267±0.046 vs. 0.056±0.011, n=5, p<0.001). There was also a significant increase in PS externalization (Annexin V binding) in response to PMA stimulation after thirty minutes compared to a DMSO control (14.76±4.80% vs. 1.50±1.55%, n=5, p=0.021). PMA-dependent NBD-PC internalization and PS externalization were significantly inhibited in the presence of chelerythrine chloride (0.180±0.031 vs. 0.267±0.046, n=5, p=0.016 for NBD-PC internalization at six minutes and 3.51±0.92% vs. 14.76±4.80%, n=5, p=0.011 for PS externalization at thirty minutes). These data provide evidence that PKC is involved in the regulation of scramblase activity and thus the distribution of PL across the RBC membrane. Future treatment strategies for sickle cell disease could include PKC inhibitors that would prevent PS externalization and potentially result in decreased sickle RBC-endothelial interactions, decreased thrombogenesis, and prolonged sickle RBC survival.