ALBERT

Description: Sickle red blood cells (RBCs) become depleted of potassium, leading to dehydration and abnormally elevated cellular density. The increased sickling that results is important for both hemolysis and vasocclusion. In this study, sickle cells were subjected to high-speed centrifugation, and the bottom 15% were isolated. This procedure removed light cells and to a variable degree enriched cells that were denser than normal to produce a high-density–enriched (HDE) population of sickle cells. Autologous HDE cells from 3 subjects were labeled with biotin and re-infused. The following determinations were performed: (1) the survival and density changes of HDE cells; (2) the amount of fetal hemoglobin (HbF) in labeled cells after magnetic isolation; (3) the percentage of labeled F cells; (4) the percentage of labeled cells displaying external phosphatidylserine (PS). For patients with 3.5%, 4.5%, and 24% HbF in the HDE RBCs, the circulation half-time was 40, 80, and 180 hours, respectively. The percentage of HbF (measured in all 3 subjects) and of F cells (measured in 2 subjects) in labeled RBCs increased with time after re-infusion, indicating that HDE F cells have longer in vivo survival than HDE non-F cells. The percentage of PS+, biotin-labeled HDE cells showed no consistent increase or decrease with time after re-infusion. These data provide evidence that HDE sickle cells, especially those that do not contain HbF, have a very short in vivo survival, and that the percentage of PS+ cells in a re-infused HDE population does not change in a consistent manner as these cells age in the circulation.

Description: External phosphatidylserine (PS) is present on some sickle RBC and may contribute to thrombogenesis, endothelial adhesion, and shortened RBC lifespan. Phospholipid scramblase (PLSCR) disrupts phospholipid (PL) asymmetry by causing nonspecific PL equilibration across the membrane. Aminophospholipid translocase (APLT) maintains PL asymmetry by returning externalized PS to the inner membrane leaflet. It has been proposed that both APLT inhibition and PLSCR activation are required for PS externalization. Sickle RBC with low level external PS (Type I PS+) are present in cells of all densities and include some reticulocytes. Sickle RBC with high external PS (Type II PS+) are primarily found in the dense fraction. Type II cells are thought to be more important because: the high level of external PS should have greater consequence; high level external PS occurs primarily in pathologically dehydrated sickle RBC; and low level external PS appears to be physiological in immature RBC. We have previously shown that dense, dehydrated sickle RBC, including the small number of dense transferrin receptor positive (TfR+) reticulocytes, have markedly inhibited APLT. In the current studies, we examined the relationships among external PS, APLT, PLSCR, and density in mature RBC and TfR+ reticulocytes using 3-color flow cytometry. APLT and PLSCR activities were assayed using fluorescent PL analogues (NBD-PS and NBD-PC, respectively), and expressed as the fraction of probe internalized. External PS was measured with Annexin V-PE and TfR+ reticulocytes were identified with anti-TfR-PE/Cy5. PS+ cells had lower APLT activity compared to PS- cells that did not reach significance for n=3 (NBD-PS internalization fraction for PS-: 0.586±0.053; Type I PS+: 0.517±0.158, Type II PS+: 0.523±0.033). PS- sickle RBC had a uniformly low PLSCR activity similar to normal RBC (NBD-PC internalization fractions ∼ 0.1). In mature sickle RBC, PLSCR was more active in PS+ cells (PS-: 0.097±0.096; Type I PS+: 0.163±0.070, Type II PS+: 0.248±0.043; n=3; PS- vs Type I PS+: p=0.06; PS- vs Type II PS+: p=0.04; Type I versus Type II: p=0.03). TfR+ reticulocytes had increased APLT and PLSCR activity compared to mature sickle RBC, but there was no apparent relationship between PLSCR and external PS. Since dense sickle RBC had markedly inhibited APLT, we evaluated the relationship between dehydration and APLT activity. Dehydration of AA RBC from an MCHC of 35.6±2.2 to 49.2±2.0 g/dL inhibited APLT (from 0.484±0.068 to 0.301±0.076; n=7, p= 0.01). Dehydration of SS RBC from an MCHC of 34.8±3.5 to 50.1±3.9 g/dL also inhibited APLT (from 0.460±0.060 to 0.361±0.047; n=3, p=0.006), but not as low as in SS RBC dehydrated in vivo (0.222±0.036 at 44.7±5.6 g/dL; n=4, p=0.007 vs. SS RBC dehydrated in vitro). Rehydration of AA and SS RBC that had been dehydrated in vitro reversed APLT inhibition. However, APLT activity was not reversed upon rehydration of sickle RBC dehydrated in vivo. In summary, our data show that: many dense sickle RBC with significantly inhibited APLT are PS-, indicating that APLT inhibition alone does not result in PS externalization; dehydration contributes to, but is not entirely responsible for, the APLT inhibition seen in dense sickle RBC; and PS+ sickle RBC have increased PLSCR activity.

Description: Sickle red blood cells (RBCs) become depleted of potassium, leading to dehydration and abnormally elevated cellular density. The increased sickling that results is important for both hemolysis and vasocclusion. In this study, sickle cells were subjected to high-speed centrifugation, and the bottom 15% were isolated. This procedure removed light cells and to a variable degree enriched cells that were denser than normal to produce a high-density–enriched (HDE) population of sickle cells. Autologous HDE cells from 3 subjects were labeled with biotin and re-infused. The following determinations were performed: (1) the survival and density changes of HDE cells; (2) the amount of fetal hemoglobin (HbF) in labeled cells after magnetic isolation; (3) the percentage of labeled F cells; (4) the percentage of labeled cells displaying external phosphatidylserine (PS). For patients with 3.5%, 4.5%, and 24% HbF in the HDE RBCs, the circulation half-time was 40, 80, and 180 hours, respectively. The percentage of HbF (measured in all 3 subjects) and of F cells (measured in 2 subjects) in labeled RBCs increased with time after re-infusion, indicating that HDE F cells have longer in vivo survival than HDE non-F cells. The percentage of PS+, biotin-labeled HDE cells showed no consistent increase or decrease with time after re-infusion. These data provide evidence that HDE sickle cells, especially those that do not contain HbF, have a very short in vivo survival, and that the percentage of PS+ cells in a re-infused HDE population does not change in a consistent manner as these cells age in the circulation.

Description: 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. (Blood. 2003;102: 365-370)

Description: Abstract 3198 Background: Red blood cell (RBC) extravasation contributes to the growth of atherosclerotic plaque, and is associated with plaque rupture. High Fat Diet (HFD) fuels systemic inflammation by promoting monocyte activation and their recruitment/transendothelial migration. RBC bind chemokines such as monocyte chemotactic protein-1 (MCP-1) via Duffy Antigen Receptor for Chemokines (DARC). While it was previously reported that HFD may impact RBC cholesterol content, very little is known about the impact – if any – of HFD on RBC function(s). Hence, we studied whether HFD affects biochemical and functional properties of RBC in ways potentially relevant to the progression of atherosclerosis. Methods and Results: Wild type (WT) C56BL6/J mice were fed either chow (10% total fat) or HFD (60% total fat) for 312 weeks. MCP-1 levels were measured in plasma before and after the release of DARC-bound MCP-1 by heparin. Released MCP-1 was 1.5 fold higher in HFD RBC (12 weeks on diet, n=3) compared to chow RBC. Levels of intracellular reactive oxygen species (ROS) were measured by DCFH fluorescence using flow cytometry, and were increased in HFD RBC (17 weeks on diet, n=3) by ∼1.2 fold compared to chow RBC (p

Description: 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

Description: 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.

Description: Percent fetal hemoglobin (HbF) is an important determinant of clinical severity in sickle cells disease (SCD). There is a dichotomous distribution of HbF in sickle cells, with one population containing 20–25% HbF (F cells) and another in which HbF is not detectable (nonF cells). Increased HbF in SCD is due to two factors: 1) Increased HbF synthesis, which also occurs in other conditions having markedly increased erythropoiesis; 2) Longer survival of F cells in the circulation compared to nonF cells, which appears to be of great importance in SCD. We previously showed the lifespan of biotin-labeled F cells in the circulation to be about three times longer than nonF cells. We now examine these differences in greater detail, focusing on two issues. The first is whether the range of HbF content (HbF per F cell, pg) that is presumably present in F cells influences cell survival. The second is whether the survival of F and/or nonF cells is dependent upon the fraction of F cells in the circulation. To address these questions, we used the previously described biotin label for RBC. Up to 10 ml of autologous RBC were labeled and reinfused, and overall RBC survival was defined by the time-dependent disappearance of labeled RBC from the circulation. At selected time points after reinfusion, HbF was evaluated in two ways: 1) The percentage of biotin-labeled cells that were F cells was determined by flow cytometry; 2) The biotin-labeled cells were isolated with streptavidin-coated magnetic beads and the percent HbF determined by HPLC. These two assays can be used to determine the individual survival of F and nonF cells and to calculate the HbF per F cell of labeled RBC as a function of time after reinfusion. There were 12 studies in 10 patients, including 2 who were studied before and after hydroxyurea (HU). A total of 4 patients were taking HU at the time of study. F cells ranged from 4 to 90%, including 2 patients with HU and one without who had greater than 88%. There was a time-dependent linear increase in HbF per F cell with a slope of 0.09 ± 0.07 pg/day (n = 11). This is consistent with longer survival of the F cells with higher HbF content. NonF cell survival was lower in patients with a higher percentage of F cells. Subjects with 〈 50% F cells had an S30 (time until 30% of the labeled RBC remain in the circulation, days) for nonF cells of 14.6 ± 2.9 days (1 SD, n = 5), whereas subjects with 〉 50% F cells had an S30 for nonF cells of 7.7 ± 2.9 days (P 〈 0.005). The range of S30 values was from 5 to 17 days, and there was a linear correlation between the S30 of nonF cells and % F cells with R2 = 0.65. For a given % F cells, there appeared to be no dependence on HU. These data indicate that the survival of nonF cells (but not F cells) is dependent on the percentage of F cells. Possible reasons for this include 1) the presumed higher oxygen affinity of F cells, leading to lower venous PO2 and thus increased sickling and decreased survival of the nonF cells, and 2) a more sensitive detection of damaged RBC by the RES when RBC turnover is lower due to a high percentage of F cells, again leading to decreased survival of the nonF cells. This could have important implications in chronic transfusion therapy, in which overall RBC turnover is decreased by the presence of donor cells with relatively high oxygen affinity.

Description: Sickle red blood cells (RBC) become dehydrated as a consequence of potassium loss. This process depends at least partly on deoxygenation and may be influenced by the presence of oxygenation/deoxygenation cycles and the frequency of cycling. In this study, sickle RBC were subjected to approximately 180 oxygenation/deoxygenation cycles during 4 hours to evaluate RBC dehydration with cycle periods more similar to in vivo cycles than those in previous studies. A continuous-flow, steady-state apparatus circulated a dilute RBC suspension through gas-permeable silicone tubing with segments that were exposed to either nitrogen or ambient oxygen. The percentage of sickling and partial pressure of oxygen were measured by means of sampling ports in the deoxygenation and oxygenation regions. The density increase (dehydration) of young (transferrin receptor-positive) and mature (transferrin receptor-negative) RBC and the requirements for calcium and chloride were evaluated. Density increase correlated with the percentage of sickled cells at the deoxygenation sampling port and was observed only in the presence of calcium, thereby implicating the calcium-dependent potassium channel (Gardos pathway). Density increase was not dependent on the presence of chloride, making it unlikely that KCl cotransport was an important pathway under these conditions.

Description: Red blood cell (RBC) membrane phosphatidylserine (PS) is normally confined to the inner leaflet. In sickle RBC, however, PS externalization has been observed and may contribute to thrombogenesis, endothelial adhesion, and shortened RBC lifespan. Increased calcium leads to PS externalization through inhibition of aminophospholipid translocase (APLT), which normally returns external PS to the inner leaflet, and activation of phospholipid scramblase, which allows nonspecific phospholipid equilibration between the leaflets. Sickle RBC in the light and dense (dehydrated) fractions exhibit increased PS externalization compared to sickle RBC in the normal density fractions. Sickle RBC with modest PS exposure (Type I PS+) occur in both light and dense fractions and, especially in the light fractions, include many reticulocytes. Sickle Cells with high levels of PS exposure (Type II PS+) occur predominantly in the dense fraction. Type II PS+ sickle cells have low levels of HbF, are more adherent than Type I PS+ cells, and tend to increase in number as sickle cells age in the circulation. Dense sickle cells have decreased APLT activity and this has been considered necessary (but probably not sufficient) for PS externalization. In the current studies we have examined the relationship between PS externalization and translocase activity in sickle cell density fractions. APLT activity and PS externalization were determined flow cytometrically using NBD-PS internalization and annexin V-phycoerythrin binding respectively. Sickle RBC density fractions were isolated using a discontinuous Optiprep® gradient, and two fractions were selected for further study, Fx 4 with essentially normal hydration (1.093〈 g/cc 1.120 g/cc). As expected, Fx 6 sickle RBC had a decreased percentage of APLT positive cells (29.9±10.8, N=4) compared to normal RBC (94.5±3.6, N=3) and Fx 4 sickle RBC (92.9±2.3, N=4). Fx 4 contained 5.7±2.8% Type I and 0.3±0.2% Type II PS+ cells. Fx 6 had 9.1±2.5% Type I and 3.2±2.3% Type II PS+ cells. Figure 1 shows the percentage of APLT positive cells in the PS negative (PS-), Type I PS+, and Type II PS+ groups for Fx 4 and Fx 6. Type I PS+ cells in Fx 4 are positive for APLT, whereas those in Fx 6 are mostly negative. In Fx 4, the Type I PS+ group most likely contains many reticulocytes, and in a separate experiment TfR+ reticulocytes had a uniform, slightly elevated APLT activity. The Type II PS+ cells in both Fx 4 and Fx 6 had decreased percentages of APLT positive cells. It thus appears that high levels of PS externalization, regardless of hydration state, are associated with low APLT activity. We therefore conclude: In normally hydrated sickle cells, Type II PS+ cells, but not Type I PS+ cells, are associated with decreased APLT; In dense sickle RBC, both Type I and Type II PS+ cells are associated with decreased APLT. These results further emphasize the pathologic nature of the Type II PS+ sickle cells. Fig 1. APLT Activity is PS Groups form F × 4 and F × 6 Fig 1. APLT Activity is PS Groups form F × 4 and F × 6