Blood viscosity is an important determinant of local flow characteristics, which exhibits shear thinning behavior: it decreases exponentially with increasing shear rates. Both hematocrit and plasma viscosity influence blood viscosity. The shear thinning property of blood is mainly attributed to red blood cell (RBC) rheological properties. RBC aggregation occurs at low shear rates, and increases blood viscosity and depends on both cellular (RBC aggregability) and plasma factors. Blood flow in the microcirculation is highly dependent on the ability of RBC to deform, but RBC deformability also affects blood flow in the macrocirculation since a loss of deformability causes a rise in blood viscosity. Indeed, any changes in one or several of these parameters may affect blood viscosity differently. Poiseuille’s Law predicts that any increase in blood viscosity should cause a rise in vascular resistance. However, blood viscosity, through its effects on wall shear stress, is a key modulator of nitric oxide (NO) production by the endothelial NO-synthase. Indeed, any increase in blood viscosity should promote vasodilation. This is the case in healthy individuals when vascular function is intact and able to adapt to blood rheological strains. However, in sickle cell disease (SCD) vascular function is impaired. In this context, any increase in blood viscosity can promote vaso-occlusive like events. We previously showed that sickle cell patients with high blood viscosity usually have more frequent vaso-occlusive crises than those with low blood viscosity. However, while the deformability of RBC decreases during acute vaso-occlusive events in SCD, patients with the highest RBC deformability at steady-state have a higher risk of developing frequent painful vaso-occlusive crises. This paradox seems to be due to the fact that in SCD RBC with the highest deformability are also the most adherent, which would trigger vaso-occlusion. While acute, intense exercise may increase blood viscosity in healthy individuals, recent works conducted in sickle cell patients have shown that light cycling exercise did not cause dramatic changes in blood rheology. Moreover, regular physical exercise has been shown to decrease blood viscosity in sickle cell mice, which could be beneficial for adequate blood flow and tissue perfusion.

SummaryAlthough pulmonary hypertension, leg ulcers, priapism, stroke and glomerulopathy in sickle cell anaemia (SCA) result from the adverse effects of chronic haemolysis on vascular function (haemolytic phenotype), osteoneocrosis, acute chest syndrome and painful vaso-occlusive crises are caused by abnormal vascular cell adhesion and increased blood viscosity (viscosityvaso-occlusion phenotype). However, this model with two sub-phenotypes does not take into account the haemorheological dimension. We tested the relationships between the biological parameters reflecting the haemolytic rate (haemolytic component) and red blood cell (RBC) rheological characteristics in 97 adults with SCA. No significant difference in the proportion of patients with low or high haemolytic component in the low and high blood viscosity groups was observed. The RBC elongation index (i.e. deformability) was negatively correlated with the haemolytic component. The RBC aggregates strength (i.e. RBC aggregates robustness) was negatively correlated with RBC elongation index. Sickle RBCs with high density had lower elongation index and higher aggregates strength. In conclusion, (i) the 'haemolytic' phenotype is characterized by decreased RBC deformability and increased RBC aggregates strength and (ii) the viscosityvaso-occlusive phenotype is characterized by increased RBC deformability but not always by increased blood viscosity. a-thalassaemia modulates the haemorheological properties but other factors seem to be involved.

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Abstract Microparticles (MPs) are submicron extracellular vesicles exposing phosphatidylserine (PS), detected at high concentration in the circulation of sickle cell anemia (SS) patients. Several groups studied the biological effects of MPs generated ex vivo. Here, we analyzed for the first time the impact of circulating MPs on endothelial cells (ECs) from 60 sickle cell disease (SCD) patients. MPs were collected from SCD patients and compared with MPs isolated from healthy individuals (AA). Other plasma MPs were purified from SS patients before and 2 years after the onset of hydroxyurea (HU) treatment or during a vaso-occlusive crisis and at steady-state. Compared with AA MPs, SS MPs increased EC ICAM-1 messenger RNA and protein levels, as well as neutrophil adhesion. We showed that ICAM-1 overexpression was primarily caused by MPs derived from erythrocytes, rather than from platelets, and that it was abolished by MP PS capping using annexin V. MPs from SS patients treated with HU were less efficient to induce a proinflammatory phenotype in ECs compared with MPs collected before therapy. In contrast, MPs released during crisis increased ICAM-1 and neutrophil adhesion levels, in a PS-dependent manner, compared with MPs collected at steady-state. Furthermore, neutrophil adhesion was abolished by a blocking anti–ICAM-1 antibody. Our study provides evidence that MPs play a key role in SCD pathophysiology by triggering a proinflammatory phenotype of ECs. We also uncover a new mode of action for HU and identify potential therapeutics: annexin V and anti–ICAM-1 antibodies.

Chronic hemolysis, enhanced oxidative stress, and decreased nitric oxide (NO) bioavailability promote vasculopathy in sickle cell anemia (SCA). Oxidative stress and NO are known to modulate eryptosis in healthy red blood cells (RBCs); however, their role in SCA eryptosis and their impact on the genesis of RBC-derived microparticles (RBC-MPs) remains poorly described. RBC-MPs could play a role in vascular dysfunction in SCA. The aims of this study were to evaluate the roles of oxidative stress and NO in eryptosis and RBC-MPs release, and to determine whether RBC-MPs could be involved in vascular dysfunction in SCA. Markers of eryptosis and oxidative stress, plasma RBC-MPs concentration and arterial stiffness were compared between SCA and healthy (AA) individuals . In-vitro experiments were performed to test: 1) the effects of oxidative stress (antioxidant: n-acetylcysteine (NAC); pro-oxidant: cumene hydroperoxide) and NO (NO donor: sodium nitroprusside (SNP); NO-synthase inhibitor (L-NIO)) on eryptosis, RBC deformability and RBC-MP genesis; 2) the effects of SCA/AA-RBC-MPs on human aortic endothelial cell (HAEC) inflammatory phenotype and TLR4 pathway. Eryptosis, RBC-MPs, oxidative stress and arterial stiffness were increased in SCA. NAC increased RBC deformability and decreased eryptosis and RBC-MPs release, while cumene did the opposite. SNP increased RBC deformability and limited eryptosis, but had no effect on RBC-MPs. L-NIO did not affect these parameters. Arterial stiffness was correlated with RBC-MPs concentration in SCA. RBC-MPs isolated directly from SCA blood increased adhesion molecules expression and the production of cytokines by HAEC compared to those isolated from AA blood. TLR4 inhibition alleviated these effects. Our data show that oxidative stress could promote eryptosis and the release of RBC-MPs that are potentially involved in macrovascular dysfunction in SCA.

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Leg ulcer is a disabling complication in patients with sickle cell anemia (SCA) but the exact pathophysiological mechanisms are unknown. The aim of this study was to identify the hematological and hemorheological alterations associated with recurrent leg ulcers. Sixty-two SCA patients who never experienced leg ulcers (ULC-) and 13 SCA patients with a positive history of recurrent leg ulcers (ULC+) - but with no leg ulcers at the time of the study – were recruited. All patients were in steady state condition. Blood was sampled to perform hematological, biochemical (hemolytic markers) and hemorheological analyses (blood viscosity, red blood cell deformability and aggregation properties). The hematocrit-to-viscosity ratio (HVR), which reflects the red blood cell oxygen transport efficiency, was calculated for each subject. Patients from the ULC+ group were older than patients from the ULC- group. Anemia (red blood cell count, hematocrit and hemoglobin levels) was more pronounced in the ULC+ group. Lactate dehydrogenase level was higher in the ULC+ group than in the ULC- group. Neither blood viscosity, nor RBC aggregation properties differed between the two groups. HVR was lower and RBC deformability tended to be reduced in the ULC+ group. Our study confirmed increased hemolytic rate and anemia in SCA patients with leg ulcers recurrence. Furthermore, our data suggest that although systemic blood viscosity is not a major factor involved in the pathophysiology of this complication, decreased red blood cell oxygen transport efficiency (i.e., low hematocrit/viscosity ratio) may play a role.

Sickle cell anaemia (SS) and sickle cell-haemoglobin C disease (SC) patients exhibit severe red blood cell (RBC) rheological alterations involved in the development of several complications. The contribution of oxidative stress in these haemorheological abnormalities is still unknown. We compared RBC reactive oxygen species (ROS) and glutathione (GSH) content, and the haemorheological profile of SS (n = 11), SC (n = 11) and healthy subjects (n = 12) at baseline and after in-vitro treatment with t-butyl hydroperoxide (TBHP). We showed: (i) higher RBC ROS content in SS and SC patients, with the highest level observed in SS patients; (ii) lower RBC GSH content in sickle syndrome patients, especially in SS patients; (iii) TBHP increased RBC ROS production and decreased RBC GSH content in all groups; (iv) TBHP decreased RBC aggregation and increased the strength of RBC aggregates in all groups but the increase in RBC aggregates strength was greater in sickle cell patients; (v) TBHP decreased RBC deformability in the three groups but with a higher magnitude in sickle cell patients. These data suggest that RBCs from sickle cell patients have an exaggerated response to oxidative stress, which is accompanied by a profound abnormal haemorheological profile, with greater alterations in SS than in SC patients.