Activation of protein kinase C by phorbol ester increases red blood cell scramblase activity and external phosphatidylserine.

Externalization of phosphatidylserine (PS) is thought to contribute to sickle cell disease (SCD) pathophysiology. The red blood cell (RBC) aminophospholipid translocase (APLT) mediates the transport of PS from the outer to the inner RBC membrane leaflet to maintain an asymmetric distribution of PL, while phospholipid scramblase (PLSCR) equilibrates PL across the RBC membrane, promoting PS externalization. We previously identified an association between PS externalization level and PLSCR activity in sickle RBC under basal conditions. Other studies showed that activation of protein kinase C (PKC) by PMA (phorbol-12-myristate-13-acetate) causes increased external PS on RBC. Therefore, we hypothesized that PMA-activated PKC stimulates PLSCR activity in RBC and thereby contributes to increased PS externalization. In the current studies, we show that PMA treatment causes immediate and variable PLSCR activation and subsequent PS externalization in control and sickle RBC. While TfR+ sickle reticulocytes display some endogenous PLSCR activity, we observed a robust activation of PLSCR in sickle reticulocytes treated with PMA. The PKC inhibitor, chelerythrine (Chel), significantly inhibited PMA-dependent PLSCR activation and PS externalization. Chel also inhibited endogenous PLSCR activity in sickle reticulocytes. These data provide evidence that PKC mediates PS externalization in RBC through activation of PLSCR.

Changes in the properties of normal human red blood cells during in vivo aging.

The changes in red blood cells (RBC) as they age and the mechanisms for their eventual removal have been of interest for many years. Proposed age-related changes include dehydration with increased density and decreased size, increased membrane IgG, loss of membrane phospholipid asymmetry, and decreased activity of KCl cotransport. The biotin RBC label allows unambiguous identification of older cells and exploration of their properties as they age. Autologous normal human RBC were labeled ex vivo and, after reinfusion, compared with unlabeled RBC throughout their lifespan. RBC density increased with age, with most of the change in the first weeks. Near the end of their lifespan, RBC had increased surface IgG. However, there was no evidence for elevated external phosphatidylserine (PS) even though older RBC had significantly lower activity of aminophospholipid translocase (APLT). KCl cotransport activity persisted well past the reticulocyte stage, but eventually decreased as the RBC became older. These studies place limitations on the use of density fractionation for the study of older human RBC, and do not support loss of phospholipid asymmetry as a mechanism for human RBC senescence. However, increased levels of IgG were associated with older RBC, and may contribute to their removal from the circulation.

Aminophospholipid translocase and phospholipid scramblase activities in sickle erythrocyte subpopulations.

Phosphatidylserine (PS) externalization may contribute to Sickle Cell Disease (SCD) characteristics including thrombogenesis, endothelial adhesion and shortened red blood cell (RBC) lifespan. Aminophospholipid translocase (APLT) returns externalized PS to the inner membrane, and phospholipid scramblase (PLSCR) equilibrates phospholipids (PL) across the membrane. APLT inhibition and PLSCR activation appear to be important for PS externalization. We examined relationships between APLT, PLSCR and external PS in mature sickle RBC and reticulocytes. Normally-hydrated sickle RBC without external PS had active APLT and inactive PLSCR. PS-exposing sickle RBC had inhibited APLT and active PLSCR. Sickle reticulocytes had active APLT and active PLSCR independent of external PS. Sickle RBC dehydrated in vivo had the highest proportion of PS-exposing RBC and markedly inhibited APLT. Normal and sickle RBC dehydrated in vitro had moderately decreased APLT. Rehydration resulted in significant recovery of APLT in RBC previously dehydrated in vitro, but not in sickle RBC dehydrated in vivo. These findings indicate that (i) PS externalization in mature sickle RBC depends on the balance between APLT and PLSCR activities, (ii) PS externalization in sickle reticulocytes depends primarily on PLSCR activation and (iii) APLT inhibition in sickle RBC dehydrated in vivo is due to dehydration itself and other factors.