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.

Use of an oral stable isotope label to confirm variation in red blood cell mean age that influences HbA1c interpretation.

HbA1c is commonly used to monitor glycemic control. However, there is growing evidence that the relationship between HbA1c and mean blood glucose (MBG) is influenced by variation in red blood cell (RBC) lifespan in hematologically normal individuals. Correction of HbA1c for mean RBC age (MRBC ) requires a noninvasive, accurate, and affordable method to measure RBC survival. In this study, we evaluated whether a stable isotope approach would satisfy these requirements. RBC lifespan and MRBC were determined in a group of nine hematologically normal diabetic and nondiabetic subjects using oral (15) N-glycine to label heme in an age cohort of RBC. The MRBC was 58.7 ± 9.1 (2SD) days and RBC lifespan was 106 ± 21 (2SD) days. This degree of variation (±15-20%) is consistent with previous studies using other techniques. In a subset of seven subjects, MRBC determined with the biotin label technique were available from approximately five years prior, and strongly correlated with the stable isotope values (R(2) = 0.79). This study suggests that the MRBC is stable over time but varies substantially among individuals, and supports the importance of its variation in HbA1c interpretation. The characteristics of the stable isotope method support its suitability for studies to directly evaluate the impact of variation in MRBC on the interpretation of HbA1c.

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.

Red cell life span heterogeneity in hematologically normal people is sufficient to alter HbA1c.

Although red blood cell (RBC) life span is a known determinant of percentage hemoglobin A1c (HbA1c), its variation has been considered insufficient to affect clinical decisions in hematologically normal persons. However, an unexplained discordance between HbA1c and other measures of glycemic control can be observed that could be, in part, the result of differences in RBC life span. To explore the hypothesis that variation in RBC life span could alter measured HbA1c sufficiently to explain some of this discordance, we determined RBC life span using a biotin label in 6 people with diabetes and 6 nondiabetic controls. Mean RBC age was calculated from the RBC survival curve for all circulating RBCs and for labeled RBCs at multiple time points as they aged. In addition, HbA1c in magnetically isolated labeled RBCs and in isolated transferrin receptor-positivereticulocytes was used to determine the in vivo synthetic rate of HbA1c. The mean age of circulating RBCs ranged from 39 to 56 days in diabetic subjects and 38 to 60 days in nondiabetic controls. HbA1c synthesis was linear and correlated with mean whole blood HbA1c (R(2) = 0.91). The observed variation in RBC survival was large enough to cause clinically important differences in HbA1c for a given mean blood glucose.

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.

Targeting and cytotoxicity of SapC-DOPS nanovesicles in pancreatic cancer.

Only a small number of promising drugs target pancreatic cancer, which is the fourth leading cause of cancer deaths with a 5-year survival of less than 5%. Our goal is to develop a new biotherapeutic agent in which a lysosomal protein (saposin C, SapC) and a phospholipid (dioleoylphosphatidylserine, DOPS) are assembled into nanovesicles (SapC-DOPS) for treating pancreatic cancer. A distinguishing feature of SapC-DOPS nanovesicles is their high affinity for phosphatidylserine (PS) rich microdomains, which are abnormally exposed on the membrane surface of human pancreatic tumor cells. To evaluate the role of external cell PS, in vitro assays were used to correlate PS exposure and the cytotoxic effect of SapC-DOPS in human tumor and nontumorigenic pancreatic cells. Next, pancreatic tumor xenografts (orthotopic and subcutaneous models) were used for tumor targeting and therapeutic efficacy studies with systemic SapC-DOPS treatment. We observed that the nanovesicles selectively killed human pancreatic cancer cells in vitro by inducing apoptotic death, whereas untransformed cells remained unaffected. This in vitro cytotoxic effect correlated to the surface exposure level of PS on the tumor cells. Using xenografts, animals treated with SapC-DOPS showed clear survival benefits and their tumors shrank or disappeared. Furthermore, using a double-tracking method in live mice, we showed that the nanovesicles were specifically targeted to orthotopically-implanted, bioluminescent pancreatic tumors. These data suggest that the acidic phospholipid PS is a biomarker for pancreatic cancer that can be effectively targeted for therapy utilizing cancer-selective SapC-DOPS nanovesicles. This study provides convincing evidence in support of developing a new therapeutic approach to pancreatic cancer.

The effect of fetal hemoglobin on the survival characteristics of sickle cells.

The determinants of sickle red blood cell (RBC) life span have not been well-defined but may include both intrinsic factors (eg, the tendency to sickle) and extrinsic factors (eg, the capacity of the reticuloendothelial system to remove defective RBCs). Fetal hemoglobin (HbF) is heterogeneously distributed among sickle RBCs; F cells contain 20% to 25% HbF, whereas the remainder have no detectable HbF (non-F cells). Autologous sickle RBCs were labeled with biotin and reinfused to determine overall survival, non-F- and F-cell survival, and time-dependent changes in HbF content (%HbF) for the surviving F cells. A total of 10 patients were enrolled, including 2 who were studied before and after the percentage of F cells was increased by treatment with hydroxyurea. As expected, F cells survived longer in all subjects. Non-F-cell survival correlated inversely with the percentage of F cells, with the time for 30% cell survival ranging from 6 days in patients with more than 88% F cells to 16 days in patients with less than 16% F cells. As the biotin-labeled RBCs aged in the circulation, the HbF content of the surviving F-cell population increased by 0.28%/d +/- 0.21%/d, indicating that within the F-cell population those with higher HbF content survived longer.

Phosphatidylserine externalization in sickle red blood cells: associations with cell age, density, and hemoglobin F.

Phosphatidylserine (PS) is normally confined to the cytoplasmic leaflet of the red blood cell (RBC) membrane, but some sickle RBCs expose PS in the outer leaflet (PS+ cells). This study examined the relationships among PS externalization, fetal hemoglobin content, hydration state, and cell age. Sickle RBCs exhibit a wide range of PS externalization. Those with low-level exposure (type 1 PS+) include many young transferrin-receptor-positive (TfR+) cells. This is not specific for sickle cell disease because many nonsickle TfR+ cells are also PS+. RBCs with higher PS exposure (type 2 PS+) appear to be more specific for sickle cell disease. Their formation is most likely sickling dependent because type 2 PS+ dense sickle cells have a lower percentage of fetal hemoglobin (HbF) than PS- cells in the same density fraction (1.7 vs 2.9; n = 8; P <.01). In vivo experiments using biotin-labeled sickle cells showed a sharp decrease in the percentage of circulating, labeled PS+ cells in the first 24 hours after reinfusion. This decrease was confined to type 1 PS+ cells and was thus consistent with the reversal of PS exposure in very young cells. As the labeled cells aged in the circulation, the percentages of type 1 and type 2 PS+ cells increased. These studies indicate that PS externalization in sickle cells may be low level, as observed in many immature cells, or high level, which is associated with dehydration and appears to be more specific for sickle RBCs.