Aldosterone Promotes Inflammation and Endothelial Cell-Sickle Cell Adhesion: Potential for a New Therapeutic Target in Sickle Cell Disease

Abstract Sickle cell disease is fundamentally an inflammatory state, and endothelial activation and dysfunction have significant roles in the pathophysiology of this disease. In the last decade, research in the cardiovascular field has proven that the hormone aldosterone, canonically viewed as a regulator of renal electrolyte handling and blood pressure, also has direct, pro-inflammatory effects on the vascular endothelium that are independent of its classical effects. Excessive aldosterone is now known to cause microvascular damage, vascular inflammation, oxidative stress and endothelial dysfunction although the molecular mechanisms remain poorly understood (Brown, Hypertension 2008). In addition, aldosterone decreases endothelial cell production of nitric oxide and upregulates VCAM-1 and ICAM-1 production, leading to increased leukocyte-endothelial cell adhesion (Oberleithner, PNAS, 2007; Krug, Hypertension 2007). In animal models, aldosterone-mediated vascular injury in the brain, heart, and kidneys leads to stroke, myocardial injury, and renal damage (Marney, Clin Sci 2007). In addition, several large clinical trials have shown that aldosterone-antagonizing medications decrease mortality in patients with renal and heart failure, due in part to the blocking of the inflammatory vascular effects of this hormone (Pitt, N Engl J Med, 2003). Although the vascular effects of aldosterone are similar to those that occur in sickle cell disease, no published studies to date have investigated the possible interactions between aldosterone and sickle cell disease. Furthermore, the efficacy of aldosterone-antagonists as a potential therapy/prophylaxis for sickle cell complications has not been evaluated. We found that patients with Hemoglobin SS (n=21) have abnormally elevated aldosterone plasma levels, as measured with ELISA, that range from 1.5–40 times (median: 8.6 times) higher than normal levels, similar in range to those of patients with heart failure (Struthers, Eur J of Heart Failure 2004). In addition, aldosterone levels in sickle cell patients positively correlated with secretory phospholipase A2 levels (R=0.43, p<0.05), a known biomarker for predicting acute chest syndrome. To determine how aldosterone affects endothelialsickle cell adhesion, we exposed human umbilical vein endothelial cells (HUVECs) and sickle erythrocytes and leukocytes isolated from patient samples to varying physiologic concentrations (1.0–100 nM) of aldosterone ex vivo for 2 hours and then utilized static and dynamic flow adhesion assays. We found that aldosterone increases sickle erythrocyte (but not normal erythrocytes), neutrophil and mononuclear cell (monocytes + lymphocytes) adhesion to endothelial cells in a dose-dependent manner (compared to controls, p<0.05 for all concentrations between 1–10 nM, p<0.001 for all concentrations >10nM) in static conditions. Compared to controls, endothelial-sickle blood cell adhesion increased up to 100 times with aldosterone exposure. Similarly, under physiologic flow conditions (shear stress: 1 dyne/cm2), endothelial cell exposure to aldosterone increased capture of sickle erythrocytes and leukocytes in a dose dependent manner (compared to controls, p<0.05 for all concentrations >10 nM). Furthermore, measurements with atomic force microscopy (AFM), a highly sensitive tool used to measure and track cell adhesion and deformability at the single cell level, revealed that the adhesive force between single sickle cell erythrocytes and HUVECs increases over time with aldosterone exposure. With the addition of spironolactone, an aldosterone antagonist, all adhesive interactions decreased to near baseline levels/controls (p>0.3 for all comparisons with baseline levels/controls) as measured with static and dynamic flow adhesion assays and AFM. To investigate the underlying mechanisms of these phenomena, fluorescence imaging revealed increased reactive oxygen species production and expression of VCAM-1 and ICAM-1 in HUVECs exposed to aldosterone for only 2 hr when compared to controls. Aldosterone exposure did not affect sickle erythrocyte or leukocyte deformability as measured with ektacytometry and AFM, respectively. Taken together, these results suggest that aldosterone may play an important role in sickle cell vasculopathy and the high levels of this hormone may provide an effective therapeutic target for this disease.

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Endothelin-1 Receptor Antagonists Regulate Cell Surface-Associated Protein Disulfide Isomerase In Sickle Cell Disease

Blood ◽

10.1182/blood.v116.21.265.265 ◽

2010 ◽

Vol 116 (21) ◽

pp. 265-265 ◽

Cited By ~ 1

Author(s):

Gregory N Prado ◽

Jessica Alves ◽

Anna J Hernandez ◽

Enrique R Maldonado ◽

Rodeler Youte ◽

...

Keyword(s):

Sickle Cell Disease ◽

Cell Surface ◽

Sickle Cell ◽

Hydration Status ◽

Channel Activity ◽

Cell Disease ◽

Receptor Antagonists ◽

Sickle Erythrocyte ◽

Gardos Channel ◽

Sickle Erythrocytes

Abstract Abstract 265 Erythrocyte hydration status and endothelial cell activation have been proposed as important contributors to vaso-occlusion and impaired blood flow in the pathophysiology of sickle cell disease (SCD). However, the physiological mechanism(s) that mediate the interplay between erythrocytes hydration status and the endothelium in SCD are unclear. We have recently reported a role for dual endothelin-1 receptor antagonists in improving sickle erythrocyte hydration status and K+ transport in vivo via modulation of Gardos channel activity (Rivera A., 2008, Amer J Physiol). The Gardos channel is an important contributor to sickle erythrocyte dehydration that maybe modulated by protein disulfide isomerase (PDI). PDI in leukocytes has been reported to catalyze disulfide interchange reactions, mediate redox modifications and has been observed to be up-regulated under hypoxic conditions. We report the detection of PDI by western blot analyses in membranes from both human and mouse sickle erythrocytes. We observed greater levels of cell surface associated PDI in sickle vs Hb A-containing erythrocytes. We also quantified PDI activity and observed a significant correlation between Gardos channel activity and cell surface associated PDI activity in human sickle erythrocytes and Hb A-containing cells (n=40, r2=0.3046, p=0.0002). In fact, closer examination revealed that sickle erythrocyte membranes had higher PDI activity than Hb A-containing erythrocyte membranes (5.07±0.4 vs 1.30±0.1%, n=22 and 18, respectively p<0.0001). Similar results were observed in membrane preparations of erythrocytes isolated from the BERK sickle transgenic mouse model when compared with wild-type controls. Consistent with a functional role for PDI in Gardos channel activation, we also observed that sickle erythrocytes incubated in cycles of oxygenation/de-oxygenation for 3 hr in the presence of PDI antibodies were associated with reduced sickle dense cell formation. Similar results were observed with bacitracin, another PDI inhibitor. We then treated BERK mice with dual ET-1 receptor antagonists (BQ123/BQ788) for 14 days and measured erythrocyte surface associated PDI activity. We observed that as with Gardos channel activity, cell surface associated PDI activity was significantly reduced following treatment with BQ123/BQ788 (8.80±0.5 to 6.4±0.6%, n=3 P<0.02). These changes were associated with an increase in erythrocyte MCV (31.3±1.63 to 40.4±0.35 fL, n=3, p<0.002) and a decrease in MCHC (40.4±0.8 to 27.4±3 g/dL, n=3, p<0.003). We then studied the direct effects of ET-1 on the human endothelial cell line, EA.hy926 (EA), as well as in primary cultures of BERK mouse aortic endothelial cells (BMAEC). Using quantitative RT-PCR with Taqman chemistries and GAPDH and beta-actin as endogenous controls, we observed that stimulation of EA cells with 100nM ET-1 for 4 hr was associated with increased mRNA expression of PDI levels that was 1.89 fold greater than vehicle treated cells (n=6, P<0.04). Similar results were observed on PDI mRNA expression in BMAEC isolated and cultured for 10 days then incubated with 100 nM ET-1 for 4 hr. Thus, our results strongly implicate cell surface associated PDI in cellular hydration status and its regulation by ET-1. We posit that aberrant regulation of PDI activity and/or its expression and secretion from either erythrocytes or endothelial cells represent a novel target aimed at ameliorating the complications associated with the pathophysiology of Sickle Cell Disease. Supported by NIH R01HL090632 to AR. Disclosures: No relevant conflicts of interest to declare.

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Measuring the Direct Effects of Sickle Cell Vaso-Occlusion on Endothelial Cells Using Microfluidic Technology

Blood ◽

10.1182/blood.v118.21.897.897 ◽

2011 ◽

Vol 118 (21) ◽

pp. 897-897

Author(s):

David R Myers ◽

Yumiko Sakurai ◽

Prasanthi Chappa ◽

Gilda Barabino ◽

David R. Archer ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Endothelial Cell ◽

Sickle Cell Disease ◽

Endothelial Function ◽

Sickle Cell ◽

Endothelial Cell Dysfunction ◽

Cell Disease ◽

Cell Dysfunction ◽

Sickle Erythrocytes

Abstract Abstract 897 Sickle cell disease is a complex process involving biophysical and biological phenomenon such as microvascular occlusion due to rigid sickle erythrocytes, hemolysis, and aberrant cellular interactions involving endothelial cells and sickle erythrocytes and leukocytes. Indeed, a key aspect of sickle cell pathophysiology is endothelial cell dysfunction. Cardiovascular research in recent years has shown that endothelial cells biologically respond to the local mechanical environment, particularly to the changes in the applied shear stresses (Chiu and Chien, Physiological Reviews, 2011). Interestingly, no studies investigating how the biophysical alterations in sickle cell disease may directly affect endothelial function have been published. The classic view has been that vaso-occlusion is simply due to sickled erythrocytes becoming stuck in microvasculature at low oxygen tensions leading to decreased blood flow and tissue ischemia. However, the mechanical aspects of sickle cell vaso-occlusion themselves, that is, the physical phenomenon of sickling erythrocytes tightly packed in an occluded blood vessel, may directly affect endothelial biology and lead to dysfunction. We hypothesize that these pathologic forces induced by sickling erythrocytes directly lead to dysfunction of endothelial cells, which are mechanosensitive, and contribute to sickle cell pathophysiology. However, these sickling-induced forces and their effects on endothelial cells have been difficult to measure, in part due to a lack of available tools. To that end, we have developed two microfluidic tools to assess the role of sickle-cell vaso-occlusion on endothelial cells. The first device is an in vitro microfluidic platform featuring microchannels the size of post-capillary venules (30 μm) with human endothelial cells cultured within and completely lining the entire inner surface of those microchannels (Figure 1A). This “microvasculature-on-a-chip” enables the visualization of blood cell-endothelial cell interactions during vaso-occlusion under a controlled hemodynamic environment and provides a platform to study the effect of vaso-occlusion on endothelial cells. To date we have characterized this “endothelialized” microfluidic device, showing that endothelial cells are confluent using anti-VE-cadherin immunostaining and adequately generate nitric oxide. Furthermore, we have flowed blood samples from patients with sickle cell disease and found that hydroxyurea treatment both reduces the number of occlusions and increases the mean velocity of the blood traveling through the device, as expected (Figure 1B–E). To decouple whether it is a biochemical or biophysical phenomenon that causes endothelial cell dysfunction during vaso-occlusion, a second micromechanical device was created to quantitatively measure the forces generated by sickling events. The device captures whole blood and will deform outward when forces are applied by the sickle erythrocytes as shown in Figure 2. The membrane above the sickle cells has been coated with 2 μm fluorescent beads which will change focus during deflection. Deflections of one or two beads indicates that a single sickle cell is locally applying force, whereas deflections of large numbers of beads indicates that the cells are collectively applying a pressure to the membrane. The device has been fully fabricated and loaded with blood cells. An accompanying experimental setup enabling the deoxygenation of the device coupled with microscopy has also been created and preliminary tests show successful deoxygenation of sickle erythrocytes from patients with hemoglobin SS disease and the Berkeley sickle cell mouse model. By combining insights gained from each device, future work will determine how the mechanical process of sickling and vaso-occlusion directly affect endothelial function and will lead to a new understanding of sickle cell pathophysiology. Sickle cell vaso-occlusion will be induced in the “endothelialized” microfluidic device while monitoring nitric oxide production and the upregulation of inflammatory markers, such as adhesion molecules and free radicals. The second device will provide quantitative numbers of forces produced by sickling erythrocytes, leading to experiments in which these forces are applied to endothelial cells while monitoring the same metrics. Disclosures: No relevant conflicts of interest to declare.

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Increased Erythrocyte Rigidity Is Sufficient to Cause Endothelial Dysfunction in Sickle Cell Disease

Blood ◽

10.1182/blood.v120.21.818.818 ◽

2012 ◽

Vol 120 (21) ◽

pp. 818-818 ◽

Cited By ~ 2

Author(s):

Robert Mannino ◽

David R Myers ◽

Yumiko Sakurai ◽

Russell E. Ware ◽

Gilda Barabino ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Endothelial Dysfunction ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Adhesion Molecule ◽

Endothelial Cell Dysfunction ◽

Cell Disease ◽

Cell Dysfunction ◽

Sickle Erythrocytes

Abstract Abstract 818 Endothelial dysfunction is a major component of sickle cell disease (SCD) pathophysiology. Interestingly, previous cardiovascular research has definitively shown that endothelial cells biologically respond to mechanical forces and aberrations in these forces cause endothelial dysfunction via pro-inflammatory pathways that are also involved in SCD. While endothelial dysfunction in SCD has been well characterized biologically, little research has focused on the direct biophysical effects of SCD blood on endothelium. As endothelial cells are in constant contact with flowing “stiffened” sickle erythrocytes, we propose that the direct mechanical interactions between the physically altered sickle erythrocytes and endothelial cells are an additional cause of endothelial dysfunction in SCD (Figure 1A). Endothelial dysfunction in SCD is thought to be caused by the downstream effects of vaso-occlusion and/or hemolysis. Our laboratory has recently developed and published a description of an in vitro microvasculature model comprised of endothelial cells that are cultured throughout the entire 3D inner surface of a microfluidic system designed for investigating cellular interactions in hematologic diseases (Tsai, et al, JCI, 2012), (Figure 1B-D). This microvasculature-on-a-chip recapitulates an ensemble of physiological processes and biophysical properties, including adhesion molecule expression, blood cell-endothelial cell interactions, cell deformability, cell size/shape, microvascular geometry, hemodynamics, and oxygen levels (Myers et al. JoVE, 2012), all of which may contribute to endothelial dysfunction in SCD. We hypothesize that the mechanical interactions between sickle erythrocytes and endothelial cells alone are sufficientto cause endothelial dysfunction in our microvasculature-on-a-chip. To test our hypothesis, we flowed different suspensions of healthy red blood cells (RBCs), and stiffened RBCs, through our microvasculature on a chip cultured with HUVECs. We suspended fresh human RBCs in media at a low hematocrit recapitulating the anemic conditions typically seen in SCD patients as a control. The experimental conditions used the same solution as the control, but also contained glutaraldehyde-stiffened RBCs, which are of the same stiffness as irreversibly sickled cells (ISCs), at approximately the same concentrations as ISCs in SCD patients. The stiffened RBC suspension was washed multiple times to eliminate all traces of glutaraldehyde and to ensure that any endothelial cell dysfunction in our system was due to mechanical effects between the endothelium and RBCs. After 4 hours of perfusion, the number of occlusions in our microsystem was counted and the cells were fixed and stained for Vascular Cell Adhesion Molecule 1 (VCAM-1). VCAM-1 been shown to be a marker of endothelial cell dysfunction and is a biomarker for severe vasculopathy in SCD (Dworkis, Am J Hematol, 2011). Immunofluorescence staining in our microsystem confirmed that VCAM1 is upregulated (Figure 2) in HUVECs when exposed to flowing stiffened RBCs compared to control RBCs. VCAM-1 upregulation appears to be diffuse throughout the length of the device. After experimentation, endothelial cells in our system can be isolated for further RT-PCR or microarray analysis. As such, ongoing work involves investigating and quantifying the expression of other pro-inflammatory molecules to elucidate the underlying mechanisms of this biomechanical process involving RBCs and endothelial cells. Additional experiments complementary experiments using endothelial cells from other anatomic areas, SCD patient samples, and murine SCD models are also underway. Our data indicates that purely physical interactions between endothelial cells and stiffened RBCs are sufficient to cause some degree of endothelial dysfunction, even in the absence of vaso-occlusion, ischemia, or oxidative stress due to hemolysis. As sickle RBCs and ISCs are constantly circulating in the blood of SCD patients, our results have profound implications for SCD pathophysiology and may help explain why SCD patients develop chronic diffuse vasculopathy over time. Disclosures: No relevant conflicts of interest to declare.

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Elevated Adenosine Signaling Via Adenosine A2B Receptor Induces Normal and Sickle Erythrocyte Sphingosine Kinase 1 Activity

Blood ◽

10.1182/blood.v124.21.4067.4067 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4067-4067 ◽

Cited By ~ 1

Author(s):

Kaiqi Sun ◽

Yujin Zhang ◽

Mikhail Bogdanov ◽

William Dowhan ◽

Modupe Idowu ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Adenosine Receptor ◽

Sphingosine Kinase ◽

Dependent Manner ◽

Sphingosine Kinase 1 ◽

Cell Disease ◽

Primary Mouse ◽

Sickle Erythrocytes ◽

Deficient Mice

Abstract Sickle Cell Disease (SCD) is one of the most devastating hemolytic genetic disorders affecting millions worldwide. Erythrocytes possess high sphingosine kinase 1 (Sphk1) activity and are considered to be the major cell type for supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is up-regulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte Sphk1 activity is regulated in SCD remains unknown. In an effort to identify specific factors and signaling pathways involved in regulation of erythrocyte SphK1 activities in SCD, we first chose to screen the effects of molecules known to induce SphK1 activities in other cell types and/or reported to be elevated in the circulation of SCD including tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), endothelin 1 (ET-1), vascular endothelial growth factor (VEGF), S1P and adenosine, on the activities of SphK1 in cultured primary mouse normal erythrocytes. Among all of those molecules tested, we found that adenosine is a previously unidentified hypoxia inducible molecule directly inducing SphK1 activity in vitro in a time and dosage-dependent manner. Next, using four adenosine receptor deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in cultured primary mouse normal and sickle erythrocytes. Subsequently, we provided in vivo genetic evidence that adenosine deaminase (ADA)-deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Mechanistically, we revealed that PKA functions downstream of ADORA2B mediating ERK activation and subsequently underlying adenosine-induced SphK1 activities in cultured mouse erythrocytes. Finally, we conducted human translational studies and reported that adenosine signaling via ADORA2B directly increases SphK1 activity in cultured primary human normal and sickle erythrocytes in a PKA/ERK-dependent manner. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms to control SphK1 activity in normal and sickle setting. Disclosures No relevant conflicts of interest to declare.

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Erythrocyte membrane sulfatide plays a crucial role in the adhesion of sickle erythrocytes to endothelium

Thrombosis and Haemostasis ◽

10.1160/th10-11-0716 ◽

2011 ◽

Vol 105 (06) ◽

pp. 1046-1052 ◽

Cited By ~ 9

Author(s):

Zhou Zhou ◽

Perumal Thiagarajan ◽

Mark Udden ◽

José López ◽

Prasenjit Guchhait

Keyword(s):

Cell Adhesion ◽

Sickle Cell Disease ◽

Erythrocyte Membrane ◽

Sickle Cell ◽

Vascular Endothelium ◽

Vascular Occlusion ◽

Shear Stresses ◽

Cell Disease ◽

Single Chain ◽

Sickle Erythrocytes

SummaryEnhanced adhesion of sickle erythrocytes to the vascular endothelium and subendothelial matrix is fundamental to the development of vascular occlusion in sickle cell disease. Erythrocyte membrane sulfatide is implicated in the pathogenesis of vasoocclusive crises in sickle cell disease (SCD) patients. Because previous evidence linking sulfatide to cell adhesion has largely been circumstantial due to a lack of reagents that specifically target sulfatide, we used two sulfatide-specific strategies to address the role of erythrocyte membrane sulfatide in sickle cell adhesion to the vascular endothelium: a single-chain fragment variable chain (scFv) antibody against sulfatide as well as cerebroside sulfotransferase-deficient mice incapable of synthesising sulfatide. The sickle erythrocytes from mice and humans adhered at a greater extent and at higher shear stresses to activated endothelium than normal erythrocytes, and approximately 60% of the adhesion was prevented by the anti-sulfatide scFv. Similarly, the extent of adhesion of sulfatide-deficient erythrocytes was lower than normal erythrocytes. These findings suggest an important role for membrane sulfatide in sickle cell disease pathophysiology.

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Anti-Inflammatory Activities of Sulfated Non-Anticoagulant Heparin Derivative Beyond Its Anti-Sickling and Anti-Selectin for the Effective Management of Sickle Cell Disease

Blood ◽

10.1182/blood-2020-141632 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-27

Author(s):

Noureldien Darwish ◽

Osheiza Abdulmalik ◽

Shaker A Mousa

Keyword(s):

Cell Adhesion ◽

Sickle Cell Disease ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Bleeding Complications ◽

Dependent Manner ◽

Cell Disease ◽

Anti Inflammatory ◽

Heparin Derivative

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by the inheritance of a single point mutation, resulting in abnormal sickle hemoglobin (HbS). During hypoxia or dehydration, HbS polymerizes to form insoluble aggregates and induces sickling of red blood cells (RBCs). RBC sickling increases the adhesiveness of RBCs to alter the rheological properties of the blood and trigger inflammatory responses, leading to hemolysis, vasoocclusive crisis, pulmonary complications, plethora, and other pathological sequelae. Glycosaminoglycans, such as low molecular weight heparin (LMWH), have been suggested as treatments to relieve coagulation complications in SCD because of their ability to decrease thrombin generation and sickle cell adhesion. However, they are associated with bleeding complications after repeated dosing. An alternative, sulfated non-anticoagulant LMWH derivative (S-NACH) was previously reported to have none to low systemic anticoagulant activity and no bleeding side effects, and it interfered with P-selectin-dependent binding of sickle cells to endothelial cells, with concomitant decrease in the levels of adhesion biomarkers in SCD mice (1,2). S-NACH has been further engineered to possess an aldehyde moiety, which confers anti-sickling properties primarily due to specific interactions with HbS to increase its affinity for oxygen. Our in vitro sickling assay under hypoxic conditions using S-NACH at 0.5 - 2 mM demonstrated that S-NACH significantly reduced the sickling of SS cells and in a concentration-dependent manner, with comparable to that of 1 mM GBT440 (Figure 1 A-C). A similar concentration dependent effect on increasing HbS affinity for oxygen using oxygen equilibrium study was documented. In an vivo animal model using Townes' SCD animals plasma levels of pro-inflammatory cytokines IL-1β, IL-6, IFN-γ, MCP-1, TNF-α, M-CSF, and VEGF were increased in SCD untreated samples in contrast to a significant decrease (*P< 0.001) in S-NACH-treated animals, at both 2 and 6 h. In addition, S-NACH was able to increase the decreased levels of the endogenous anti-inflammatory IL-10 (Figure 1 D). The above set of novel findings about S-NACH established it to be effective for the management of SCD via the modulation of thromboinflammatory pathways, involved in thromboembolism and end organ damage, beside anti-sickling, anti-selectin and without causing any bleeding risk. Reference 1. Alshaiban A, Muralidharan-Chari V, Nepo A, Mousa SA. Modulation of Sickle Red Blood Cell Adhesion and its Associated Changes in Biomarkers by Sulfated Non-anticoagulant Heparin Derivative. Clin Appl Thromb Hemost. 2016 ;22(3):230-8. 2. Mousa SA: Compositions and method for anti-sickling of red blood cells in sickle cell disease. US Patent 9,822,190, November 2017. Disclosures Mousa: Vascular Vision Pharma Co.: Patents & Royalties.

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Increased Procoagulant Activity of Red Blood Cells from Patients with Homozygous Sickle Cell Disease and β-Thalassemia

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650577 ◽

1996 ◽

Vol 76 (03) ◽

pp. 322-327 ◽

Cited By ~ 73

Author(s):

Dominique Helley ◽

Amiram Eldor ◽

Robert Girot ◽

Rolande Ducrocq ◽

Marie-Claude Guillin ◽

...

Keyword(s):

Sickle Cell Disease ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Annexin V ◽

Mean Value ◽

Procoagulant Activity ◽

Dependent Manner ◽

Cell Disease ◽

Homozygous Sickle Cell Disease

SummaryIt has recently been proved that, in vitro, red blood cells (RBCs) from patients with homozygous β-thalassemia behave as procoagulant cells. The procoagulant activity of β-thalassemia RBCs might be the result of an increased exposure of procoagulant phospholipids (i. e. phosphatidylserine) in the outer leaflet of the membrane. In order to test this hypothesis, we compared the catalytic properties of RBCs of patients with β-thalassemia and homozygous sickle cell disease (SS-RBCs) with that of controls. The catalytic parameters (Km, kcat) of prothrombin activation by factor Xa were determined both in the absence and in the presence of RBCs. The turn-over number (kcat) of the reaction was not modified by normal, SS- or (3-thalassemia RBCs. The Km was lower in the presence of normal RBCs (mean value: 9.1 µM) than in the absence of cells (26 µM). The Km measured in the presence of either SS-RBCs (mean value: 1.6 µM) or β-thalassemia RBCs (mean value: 1.5 pM) was significantly lower compared to normal RBCs (p <0.001). No significant difference was observed between SS-RBCs and p-thalassemia RBCs. Annexin V, a protein with high affinity and specificity for anionic phospholipids, inhibited the procoagulant activity of both SS-RBCs and (3-thalassemia RBCs, in a dose-dependent manner. More than 95% inhibition was achieved at nanomolar concentrations of annexin V. These results indicate that the procoagulant activity of both β-thalassemia RBCs and SS-RBCs may be fully ascribed to an abnormal exposure of phosphatidylserine at the outer surface of the red cells.

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Abstract 65: Anemia-related Heart Failure in Sub-saharan African Sickle Cell Disease Patients

Circulation Research ◽

10.1161/res.121.suppl_1.65 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Adebayo C Atanda ◽

Yahya Aliyu ◽

Oluwafunmilayo Atanda ◽

Aliyu Babadoko ◽

Aisha Suleiman ◽

...

Keyword(s):

Heart Failure ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Systolic Pressure ◽

Left Ventricular ◽

Hemoglobin Level ◽

Sub Saharan Africa ◽

Cell Disease ◽

Jet Velocity ◽

Sub Saharan

Introduction: Anemia has been implicated in heart failure. Existing literatures, involving predominantly African-Americans, suggests that Sickle Cell Disease (SCD) maybe linked to various cardiovascular complications including pulmonary hypertension and left venticular dysfunction. Peculiarly, our study involves exclusively Sub-Saharan population. Method: We conducted a cross sectional observational study of 208 hydroxyurea-naive consecutive SCD patients aged 10-52 years at steady state and 94 healthy non-matched controls who were studied in an out patient clinic in Sub-Saharan Africa. SCD patients were required to have electrophoretic or liquid chromatography documentation of major sickling phenotypes. Control group was required to have non-sickling phenotypes. Cardiac measurements were performed with TransThoracic Echo according to American Society of Echocardiography guidelines. Hemoglobin level was also obtained. Results: Hemoglobin level in SCD group (8.5+/- 1.5) was significant (P<0.001) compared to control (13.8+/- 1.7). Although SCD group had significantly higher values of left ventricular (LV) size, there was no qualitative evidence of LV dysfunction. SCD group had higher values of Ejection Fraction but not statistically significant. There was no evidence of LV wall stiffening to impair proper filling in SCD group, with the ratio of early to late ventricular filling velocities, E/A ratio elevated (1.7+/-0.4 compared to 1.6+/- 0.4; P=0.010). Right ventricular systolic pressure was determined using the formula of 4x Tricuspid Reugurgitant jet (TRV) square as an indirect measurement of Pulmonary arterial systolic pressure. SCD patients had significantly higher mean±SD values for tricuspid regurgitant jet velocity than did the controls (2.1±0.6 vs. 1.8±0.5; p= 0.001). Within the SCD group, there was no clear pattern of worsening diastolic function with increased TRV. Furthermore, E/A had a significant positive relationship with jet velocity in bivariate analysis (R=0.20; P=0.013). Conclusions: We were unable to demonstrate existence of anemia-associated left ventricular dysfunction in Sub-Saharan African with SCD. Further studies is required to highlight the reason behind this finding.

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Tissue factor promotes activation of coagulation and inflammation in a mouse model of sickle cell disease

Blood ◽

10.1182/blood-2012-04-424143 ◽

2012 ◽

Vol 120 (3) ◽

pp. 636-646 ◽

Cited By ~ 63

Author(s):

Pichika Chantrathammachart ◽

Nigel Mackman ◽

Erica Sparkenbaugh ◽

Jian-Guo Wang ◽

Leslie V. Parise ◽

...

Keyword(s):

Endothelial Cell ◽

Sickle Cell Disease ◽

Tissue Factor ◽

Sickle Cell ◽

Plasma Levels ◽

Cell Injury ◽

Vascular Inflammation ◽

Cell Disease ◽

Endothelial Cell Injury ◽

Cell Adhesion Molecule 1

Abstract Sickle cell disease (SCD) is associated with a complex vascular pathophysiology that includes activation of coagulation and inflammation. However, the crosstalk between these 2 systems in SCD has not been investigated. Here, we examined the role of tissue factor (TF) in the activation of coagulation and inflammation in 2 different mouse models of SCD (BERK and Townes). Leukocytes isolated from BERK mice expressed TF protein and had increased TF activity compared with control mice. We found that an inhibitory anti-TF antibody abrogated the activation of coagulation but had no effect on hemolysis or anemia. Importantly, inhibition of TF also attenuated inflammation and endothelial cell injury as demonstrated by reduced plasma levels of IL-6, serum amyloid P, and soluble vascular cell adhesion molecule-1. In addition, we found decreased levels of the chemokines MCP-1 and KC, as well as myeloperoxidase in the lungs of sickle cell mice treated with the anti-TF antibody. Finally, we found that endothelial cell-specific deletion of TF had no effect on coagulation but selectively attenuated plasma levels of IL-6. Our data indicate that different cellular sources of TF contribute to activation of coagulation, vascular inflammation, and endothelial cell injury. Furthermore, it appears that TF contributes to these processes without affecting intravascular hemolysis.

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