Sickle Red Blood Cell Adhesion Research Articles

Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner.

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca2+ ) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1-10 μM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca2+ influx. Yoda1 induced Ca2+ -dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo-/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.

Sickle red blood cell-derived extracellular vesicles activate endothelial cells and enhance sickle red cell adhesion mediated by von Willebrand factor.

Endothelial activation and sickle red blood cell (RBC) adhesion are central to the pathogenesis of sickle cell disease (SCD). Quantitatively, RBC-derived extracellular vesicles (REVs) are more abundant from SS RBCs compared with healthy RBCs (AA RBCs). Sickle RBC-derived REVs (SS REVs) are known to promote endothelial cell (EC) activation through cell signalling and transcriptional regulation at longer terms. However, the SS REV-mediated short-term non-transcriptional response of EC is unclear. Here, we examined the impact of SS REVs on acute microvascular EC activation and RBC adhesion at 2 h. Compared with AA REVs, SS REVs promoted human pulmonary microvascular ECs (HPMEC) activation indicated by increased von Willebrand factor (VWF) expression. Under microfluidic conditions, we found abnormal SS RBC adhesion to HPMECs exposed to SS REVs. This enhanced SS RBC adhesion was reduced by haeme binding protein haemopexin or VWF cleaving protease ADAMTS13 to a level similar to HPMECs treated with AA REVs. Consistent with these observations, haemin- or SS REV-induced microvascular stasis in SS mice with implanted dorsal skin-fold chambers that was inhibited by ADAMTS13. The adhesion induced by SS REVs was variable and was higher with SS RBCs from patients with increased markers of haemolysis (lactate dehydrogenase and reticulocyte count) or a concomitant clinical diagnosis of deep vein thrombosis. Our results emphasise the critical contribution made by REVs to the pathophysiology of SCD by triggering acute microvascular EC activation and abnormal RBC adhesion. These findings may help to better understand acute pathophysiological mechanism of SCD and thereby the development of new treatment strategies using VWF as a potential target.

Antithrombin-III mitigates thrombin-mediated endothelial cell contraction and sickle red blood cell adhesion in microscale flow.

SummaryIndividuals with sickle cell disease (SCD) have persistently elevated thrombin generation that results in a state of systemic hypercoagulability. Antithrombin‐III (ATIII), an endogenous serine protease inhibitor, inhibits several enzymes in the coagulation cascade, including thrombin. Here, we utilize a biomimetic microfluidic device to model the morphology and adhesive properties of endothelial cells (ECs) activated by thrombin and examine the efficacy of ATIII in mitigating the adhesion of SCD patient‐derived red blood cells (RBCs) and EC retraction. Microfluidic devices were fabricated, seeded with ECs, and incubated under physiological shear stress. Cells were then activated with thrombin with or without an ATIII pretreatment. Blood samples from subjects with normal haemoglobin (HbAA) and subjects with homozygous SCD (HbSS) were used to examine RBC adhesion to ECs. Endothelial cell surface adhesion molecule expression and confluency in response to thrombin and ATIII treatments were also evaluated. We found that ATIII pretreatment of ECs reduced HbSS RBC adhesion to thrombin‐activated endothelium. Furthermore, ATIII mitigated cellular contraction and reduced surface expression of von Willebrand factor and vascular cell adhesion molecule‐1 (VCAM‐1) mediated by thrombin. Our findings suggest that, by attenuating thrombin‐mediated EC damage and RBC adhesion to endothelium, ATIII may alleviate the thromboinflammatory manifestations of SCD.

S107: P-SELECTIN INHIBITOR INCLACUMAB REDUCES CELL ADHESION IN AN IN-VITRO ASSAYS SHOWING POTENTIAL FOR PREVENTION OF VASO-OCCLUSION EVENTS IN SICKLE CELL DISEASE

Introduction: Sickle cell disease (SCD) is a genetic disorder that leads to serious clinical complications including vaso-occlusive events (VOE). Development of VOEs had been linked to translocation of endothelial P-Selectin from Weibel–Palade bodies to the cell surface, a process promoting sickle Red Blood Cell (SS RBC) adhesion to vessel walls. Up-regulation of P-selectin contributes to cell–cell interactions, as activated platelets bind to neutrophils to form aggregates in a P-selectin–dependent manner. Previous studies have shown that anti-P-selectin agents decrease SS RBC adhesion in vivo, increase microvascular flow, and reduce adhesion of the leukocyte to endothelium, showing the potential of P-selectin as a therapeutic target. In November 2019, the FDA approved the first P-selectin inhibitor, crizanlizumab-tmca (Adakveo, Novartis), to reduce the frequency of vaso-occlusive crises (VOCs) in adults and pediatric patients with SCD. Different types of P-selectin inhibitors can offer physicians and patients new treatment options enhancing the arsenal of drugs to combat SCD. Inclacumab (Global Blood Therapeutics) is a fully human monoclonal antibody designed to bind to and selectively inhibit P-selectin. Differences in static cell adhesion to P-selectin substrate due to inclucumab were shown previously. The present study aimed to validate the dose-dependentability of inclacumab in blocking P-selectin cell adhesion in samples from SCD patients using in-vitro Whole Blood (WB) and Isolated White Blood Cell (I-WBC) assays. Methods: Five deidentified samples from SCD patients undergoing phlebotomy prior to blood exchange had been collected at the Children’s Hospital of Michigan with Flow Adhesion on P-selectin measured using proprietary assays in both Whole Blood (WB) and in Isolated WhiteBlood Cells (I-WBC), with and without Inclacumab after 5 minutes incubation at room temperature. Microfluidic channels were coated with P-selectin, and I-WBC (5x106/mL) suspension or WB sample diluted 1:1 with phosphate buffer was passed through the channel for 6 (I-WBC) or 10 (WB) minutes at a shear rate of 1 dyne/cm and pulsatility of 1.67 Hz. The channel was washed for 5 minutes with the same buffer, and the number of cells adherring to the channel surface was quantified. Results: Incubation of either WB or I-WBC with inclacumab significantly reduced flow adhesion to P-Selectin with the overall inhibition being stronger when assessed on isolated white cells (ab. 35% inhibition on WB vs. ab. 55% on I-WBC after incubation with 40 mM Inclacumab as compared to baseline Flow Adhesion values, Fig. 1). Adhesion on P-selectin showed a pronounced dose response that followed logarithmic response curve, with changes in adhesion with I-WBC better correlated with inclacumab dose, than those measured using WB. Decrease in adhesion on P-selectin was reliably detected at 2 μg/mL treatment dose for 3 out of 5 samples, with dose-depemdent decrease in adhesion in all samples at 10 and 40 μg/mL treatment doses (for I-WBC; and similar for WB). For flow adhesion with both WB and I-WBC elevated adhesion at lower doses was observed in some samples. Conclusions: Both WB and I-WBC adhesion to P-Sselectin show a dose dependent inhibition by inclacumab, with clinically relevant doses likely demonstrating greater inhibition. P-selectin adhesion assays described in this study may serve as potential surrogate biomarkers of clinical response tp p-selectin inhibitors, like inclacumab.

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

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.

Influence of rs1042713 and rs1042714 polymorphisms of β2-adrenergic receptor gene with erythrocyte cAMP in sickle cell disease patients from Odisha State, India.

The vaso-occlusive crisis (VOCs) in sickle cell disease (SCD) is often associated with stress. Epinephrine released during stress acts via beta 2-adrenergic receptors (β2-AR or ADRB2) to stimulate the synthesis of cyclic adenosine monophosphate (cAMP) in the red blood cells (RBCs). Higher cAMP levels promote adhesion of sickled RBCs to vascular endothelium, a major contributor for VOCs. Several single-nucleotide polymorphisms (SNPs) of the β2-AR gene have been reported; two of them at codon 16 (rs1042713) and codon 27 (rs1042714) have been extensively studied for their clinical relevance. Therefore, we assessed the influence of polymorphism at these two sites of the β2-AR gene on the RBC cAMP concentrations with and without epinephrine stimulation in SCD subjects. We determined the frequency distribution of different genotypes of codon 16 and codon 27 of the β2-AR gene using the Sanger sequencing method in the SCD subjects. We measured the RBC-cAMP levels at baseline and after stimulation with epinephrine, to ascertain the influence of different genotypes in determining cAMP levels. There was no difference in the socio-demographic and hematological indicators in different genotypes of both codon 16 and 27. In the sham-treated erythrocytes, the cAMP levels were significantly different with three genotypes of codon 16 (F = 3.39, P = 0.036; one way ANOVA) but not with different genotypes of codon 27. A significant increase in cAMP levels was noticed with epinephrine treatment in all genotypes of codons 16 and 27 (P = 0.001; Wilcoxon signed-rank test). However, the extent of increase in the epinephrine-treated cAMP values from the sham-treated (baseline) cAMP values was significantly different between the three genotypes of codon 16 (H = 8.74; P = 0.012; Kruskal-Wallis test) but not in codon 27 genotypes. Polymorphism in codon 16 (rs1042713) of the β2-AR gene influences cAMP concentrations in the RBC both before and after epinephrine treatment. Higher cAMP levels may lead to increased adhesion of sickle cell RBCs to vascular endothelium and may increase the frequency of VOCs.

Red blood cell adhesion to ICAM-1 is mediated by fibrinogen and is associated with right-to-left shunts in sickle cell disease

AbstractSickle cell disease (SCD), which afflicts 100 000 Americans, as well as millions worldwide, is associated with anemia, lifelong morbidity, and early mortality. Abnormal adhesion of sickle red blood cells (RBCs) to activated vascular endothelium may contribute acutely to the initiation of painful vaso-occlusive crises and chronically to endothelial damage in SCD. Sickle RBCs adhere to activated endothelium through several adhesion mechanisms. In this study, using whole blood from 17 people with heterozygous SCD (HbS variant) and 55 people with homozygous SCD (HbSS) analyzed in an in vitro microfluidic assay, we present evidence for the adhesion of sickle RBCs to immobilized recombinant intercellular adhesion molecule 1 (ICAM-1). We show that sickle RBC adhesion to ICAM-1 in vitro is associated with evidence of hemolysis in vivo, marked by elevated lactate dehydrogenase levels, reticulocytosis, and lower fetal hemoglobin levels. Further, RBC adhesion to ICAM-1 correlates with a history of intracardiac or intrapulmonary right-to-left shunts. Studies of potential ICAM-1 ligands on RBC membranes revealed that RBC–ICAM-1 interactions were mediated by fibrinogen bound to the RBC membrane. We describe, for the first time, RBC rolling behavior on ICAM-1 under high shear rates. Our results suggest that firm adhesion of sickle RBCs to ICAM-1 most likely occurs in postcapillary venules at low physiological shear rates, which is facilitated by initial rolling in high shear regions (eg, capillaries). Inhibition of RBC and ICAM-1 interactions may constitute a novel therapeutic target in SCD.

First report of 68Ga-PRGD2 PET/MRI molecular imaging of vaso-occlusion in a patient with sickle cell disease

Increased vascular cell adhesion (hyperadhesion) to the endothelium is responsible for the hallmark acute pain episodes, or vaso-occlusive crises (VOC), of sickle cell disease. The integrin αvβ3 plays an important role in VOC since it mediates sickle red blood cell adhesion to the endothelium, a process that leads to ischemia and painful bone infarction. In the pilot study presented herein, we hypothesized that real-time imaging of hyperadhesion could quantify VOC severity and identify the most vulnerable anatomical sites. We also hypothesized that harnessing hyperadhesion as a proximate event in VOC would provide sensitive, objective evidence of VOC before pain has developed. Specifically, we tested whether positron emission tomography (PET) imaging of integrin αvβ3 using the PET tracer 68Ga-PRGD2 would successfully image hyperadhesion associated with VOC in a patient with sickle cell disease. We observed persistently higher tracer uptake in the femurs during VOC compared to baseline. In the vessel, after an initial and transient increase during VOC, blood pool activity was similar between baseline and VOC. These findings suggest that PET imaging of integrin αvβ3 may be a valuable strategy for imaging of VOC.

Mn Porphyrin-Based Redox Active Drugs As Novel Anti-Adhesive and Anti-Inflammatory Agents Targeting Sickle Red Blood Cell Oxidative Damage In Vivo

In sickle cell disease (SCD), painful vaso-occlusive crises and end-organ damage are caused by occlusion of the vessels due largely to sickle red blood cell (RBC) adhesion to both the endothelium and adherent leukocytes. RBC oxidative damage caused by continuous endogenous and exogenous oxidative stress may participate in the occurrence of vaso-occlusive crises.We have evaluated the effects of scavenging reactive oxygen species (ROS) in sickle RBCs on cell adhesion and vaso-occlusion in a humanized mouse model of vaso-occlusion in vivo analyzed by intravital microscopy. To scavenge RBC ROS, we used our manganese porphyrin-based superoxide dismutase (SOD) mimics MnTnBuOE-2-PyP5+ (MnBuOE) and MnTE-2-PyP5+ (MnE), powerful catalysts of superoxide dismutation, and reductants of peroxynitrite, peroxide and hypochlorite. Intravital microscopy observations of enflamed vessels visible through dorsal skin-fold window chamber implants was performed after the inflammatory trigger of tumor necrosis factor alpha (TNFα) to induce vaso-occlusion in transgenic sickle mice followed by subcutaneous injection of MnBuOE at 0.1, 0.2 or 2 mg/kg, or MnE at 0.5 or 2 mg/kg.Treatment of sickle mice with only one dose of 0.1, 0.2 and 2 mg/kg MnBuOE decreased dose-dependently adhesion of both sickle cells and leukocytes in enflamed vessels by 68±4% (p<0.01), 85±2.3% (p<0.01) and 89±4.3% (p<0.01), respectively, compared with vehicle-treated sickle mice. MnBuOE at 0.1, 0.2 and 2 mg/kg also caused significant and dose-dependent reduction in leukocyte rolling flux (p<0.05). Similar inhibitory benefits were obtained when MnE was administered to TNFa-treated sickle mice. MnE at 0.5 and 2 mg/kg significantly decreased the number of adherent sickle cells and leukocytes by 76±8.6% (p<0.01) and 92±2.5% (p<0.01), respectively, and leukocyte rolling flux (p<0.01) compared to vehicle-treated animals. The effect of these two SOD mimics on sickle RBCs and leukocyte adhesion, and leukocyte rolling flux was rapid, because a decline in cell adhesion and leukocyte rolling flux were already detectable within the first 15 minutes after injection of the compounds. In contrast, cell adhesion and leukocyte rolling flux were already pronounced 15 minutes following vehicle injection. Reduced cell adhesion to the endothelium by the SOD mimics resulted in improved microcirculatory blood flow in sickle mice. These favorable effects on cell adhesion and vaso-occlusion following SOD mimic treatment were indeed due at least to the significant decrease in sickle RBC ROS levels compared to vehicle-treated mice (p<0.001).The long-term anti-adhesive and anti-inflammatory effects of MnBuOE and MnE in sickle mice were next examined. Subcutaneous administration for 28 days of MnBuOE at 0.1 and 0.5 mg/kg inhibited significantly adhesion of RBCs and leukocytes in enflamed venules by 34±13% (p<0.05) and 69±3.5% (p<0.001), respectively, and leukocyte rolling flux (p<0.001) compared to vehicle-treated sickle mice. Subcutaneous injection of MnE at 0.5 and 1 mg/kg for 28 days also had significant effect on sickle cell and leukocyte adhesion (p<0.01), and leukocyte rolling flux (p<0.01). In addition, venous blood gases were significantly improved by the SOD mimics. The levels of partial pressure of Carbon dioxide (pCO2), partial pressure of oxygen (pO2), base excess of the extracellular fluid (BEecf), bicarbonate (HCO3-) concentration, total CO2 (TCO2) concentration, and the indicators of hypoxia, hemoglobin saturation of oxygen (sO2) and lactate, became close to or within the normal ranges (p<0.05) in sickle mice treated with 1 mg/kg MnE. MnBuOE at 0.1 mg/kg showed only a trend toward an increase in venous blood gases, with a significant decrease in lactate (p<0.05). Leukocytosis in sickle mice treated with the SOD mimics was also alleviated. A significant drop in leukocyte (p<0.05), neutrophil (p<0.01), lymphocyte (p<0.05) and monocyte (p<0.05) counts was detected in sickle mice treated with either 0.1 mg/kg MnBuOE or 1 mg/kg MnE. These beneficial therapeutic outcomes induced by the SOD mimics were due at least in part to a decline in RBC ROS levels (p<0.001) and RBC phosphatidylserine surface exposure (p<0.05), an eryptosis marker.These results suggest that our SOD mimics may represent a valuable novel therapeutic intervention for not only vaso-occlusive crises, but inflammation as well, that should be further evaluated in patients with SCD. DisclosuresNo relevant conflicts of interest to declare.

Heterogeneous Hypoxia-Mediated Neutrophil and Red Blood Cell Adhesion to E-Selectin in Microscale Flow

Hypoxia promotes red blood cell (RBC) sickling, oxidative stress, systemic endothelium activation, vascular inflammation, and activation of coagulation in sickle cell disease (SCD), which results in a malicious cycle contributing to the progression of vaso-occlusion and other consequent clinical manifestations, such as acute chest syndrome and ischemic injuries. In the multistep and multicellular paradigm of vaso-occlusion, recruitment of neutrophils and adhesion of sickle RBCs to activated endothelial cells are critical in initiating this cascade of events. To better understand the role of hypoxia in this pathophysiological process, we assessed the adhesion profiles of RBCs and neutrophils to immobilized e-selectin utilizing blood samples from a clinically diverse patient population with SCD.Blood samples were collected from 11 subjects with homozygous SCD (HbSS) and 5 normal subjects (HbAA). Prior to the experiments, whole blood samples were mixed with Hank's balanced salt buffer solution modified with calcium and magnesium (1:1 v/v). A total volume of 25 µl blood sample was perfused through each e-selectin immobilized microchannel under both normoxic and hypoxic (7.5% oxygen level) conditions using SCD Biochip microfluidic adhesion assay [1, 2]. Blood perfusion was followed by a rinse with Hank's buffer solution at 1 dyne/cm2 corresponding to the typical shear stress levels observed in post-capillary venules. Thereafter, neutrophil adhesion, neutrophil rolling, neutrophil-platelet aggregation, and RBC adhesion data were obtained and analyzed.E-selectin functionalized microchannels supported neutrophil adhesion as well as neutrophil rolling when flowing normal blood samples, where we observed higher adhesion and rolling rates in hypoxia (Fig. 1A). SCD subjects were categorized into two distinct groups based on their adhesion profiles: Group 1: hypoxia-enhanced neutrophil adhesion without significant RBC adhesion (N=7), and Group 2: hypoxia-reduced RBC adhesion with marginal neutrophil adhesion (N=4) (Fig. 1B). We find that both normal and SCD neutrophil adhesion to e-selectin is significantly enhanced under hypoxic conditions (Fig. 1C, p<0.05, paired t-test). Moreover, we observed significantly increased neutrophil-platelet aggregates and an increase in the percentage of adhered neutrophils involved in neutrophil-platelet aggregation induced by hypoxia (Fig. 1D&E), suggesting that hypoxia is strongly associated with neutrophil-platelet aggregation driven vaso-occlusive events in Group 1. Furthermore, rolling velocity of 20 neutrophils from each SCD subjects under shear stress was measured, and hypoxia-mediated neutrophil rolling behavior was determined (Fig. 1F). A unique adhesion profile was observed in Group 2, in which the number of adhered RBCs was significantly reduced in response to hypoxia (Fig. 1G, p<0.05, Mann-Whitney non-parametric analysis).Here, we report two different adhesion profiles among SCD sub-populations using an e-selectin functionalized microfluidic model. We observed elevated numbers of adherent neutrophils, decreased neutrophil rolling velocities, and enhanced neutrophil-platelet aggregation induced by hypoxia in one group, while lowered number of adhered RBCs mediated by hypoxia in the other group. We speculate that there may be two distinct mechanisms that initiate vaso-occlusive events in SCD: in Group 1, neutrophils are responsive to endothelial activation. In this group, neutrophil recruitment and the resulting neutrophil-platelet aggregates and other complexes may precipitate vaso-ooclusion, which is strongly susceptible to hypoxia. In Group 2, in whom neutrophil recruitment is less effective, vaso-occlusion may be induced by vascular RBC adhesion, in which hypoxia may be a less proximate trigger.

Amino Acids: Riddles from A to Y.

The recent Food and Drug Administration approval of the use of L-glutamine for the reduction of pain episodes in patients with sickle cell disease (SCD)2 highlights the importance of familiar amino acids in new therapeutics. For example, lysine analogs are used as antifibrinolytics to stop bleeds, whereas an arginine analog is used as an antithrombotic agent. What is the mechanism of action of L-glutamine in SCD? L-glutamine has a favorable impact on the ratio of reduced to total NAD and redox potential in sickle red blood cells (RBCs) exposed to oxidative stress (1). RBCs from individuals with SCD who were treated with L-glutamine showed reduced sickling on the peripheral blood smear and were less sickled and less adherent to vascular cells than RBCs from untreated individuals (2). Oral L-glutamine also reduced endothelial adhesion of sickle RBCs to human umbilical vein endothelial cells (3). Improved redox biology might make sickle RBCs less adhesive. Both tranexamic acid and e-aminocaproic acid, which are analogs of lysine, competitively block the binding of fibrinogen and fibrin to their lysine binding sites on plasminogen, thus reducing the rate of fibrinolysis. Aprotinin, a serine protease inhibitor, has been removed from the market due …

Extracellular fluid tonicity impacts sickle red blood cell deformability and adhesion

AbstractAbnormal sickle red blood cell (sRBC) biomechanics, including pathological deformability and adhesion, correlate with clinical severity in sickle cell disease (SCD). Clinical intravenous fluids (IVFs) of various tonicities are often used during treatment of vaso-occlusive pain episodes (VOE), the major cause of morbidity in SCD. However, evidence-based guidelines are lacking, and there is no consensus regarding which IVFs to use during VOE. Further, it is unknown how altering extracellular fluid tonicity with IVFs affects sRBC biomechanics in the microcirculation, where vaso-occlusion takes place. Here, we report how altering extracellular fluid tonicity with admixtures of clinical IVFs affects sRBC biomechanical properties by leveraging novel in vitro microfluidic models of the microcirculation, including 1 capable of deoxygenating the sRBC environment to monitor changes in microchannel occlusion risk and an “endothelialized” microvascular model that measures alterations in sRBC/endothelium adhesion under postcapillary venular conditions. Admixtures with higher tonicities (sodium = 141 mEq/L) affected sRBC biomechanics by decreasing sRBC deformability, increasing sRBC occlusion under normoxic and hypoxic conditions, and increasing sRBC adhesion in our microfluidic human microvasculature models. Admixtures with excessive hypotonicity (sodium = 103 mEq/L), in contrast, decreased sRBC adhesion, but overswelling prolonged sRBC transit times in capillary-sized microchannels. Admixtures with intermediate tonicities (sodium = 111-122 mEq/L) resulted in optimal changes in sRBC biomechanics, thereby reducing the risk for vaso-occlusion in our models. These results have significant translational implications for patients with SCD and warrant a large-scale prospective clinical study addressing optimal IVF management during VOE in SCD.

Patient-Specific Adhesion of Sickle Red Blood Cells in Shear-Gradient Microscale Flow

The pathophysiology of sickle cell disease (SCD) involves altered biophysical properties of red blood cells (RBCs) and increased cellular adhesion, which can synergistically trigger recurrent and painful vaso-occlusive events in the microcirculatory network. RBC adhesion to the endothelial wall is heterogeneous and may initiate such occlusions by disrupting the local flow thus activating platelets and promoting subsequent cell-cell interactions. Moreover, these episodic events take place within a wide range of dynamically changing shear rates at the microscale. In order to better understand the role of shear rate on this process, we quantified shear-dependent RBC adhesion to endothelial proteins fibronectin (FN) and laminin (LN) utilizing a microfluidic system that can simulate physiologically relevant shear gradients of microcirculatory blood flow at a single flow rate.Whole blood samples were collected from 20 patients (10 males and 10 females) with homozygous SCD (HbSS). Samples were perfused through FN and LN immobilized shear-gradient microchannels (Fig. 1A) in which the shear rate continuously changes along flow direction. Computational simulations characterized the flow dynamics near the adherent RBCs (Fig. 1B). Based on the numerical results, a rectangular “field of interest (FOI)”, along which the shear rate dropped approximately three-fold, was chosen for quantification of shear-dependent RBC adhesion.We observed changes in RBC adhesion to LN and FN in the shear gradient flow. Figure 1C and 1D show typical adhesion curves of surface adherent RBCs for an individual SCD sample within the FOI. To assess patient specific shear-dependent adhesion, we defined a parameter, “shear dependent adhesion rate (SDAR)”, which is the slope of the adhesion curves based on normalized RBC adhesion numbers.A higher SDAR value was indicative of marked numbers of adherent RBCs that detach at higher shear rates whereas the effect of shear rate on RBC detachment was less for a lower SDAR. We observed an inverse relationship between SDAR and number of persistently adherent RBCs at high shear rates. Shear-dependent RBC adhesion to LN was heterogeneous among SCD patients. Patients with higher WBC counts constituted the low SDAR population with a threshold SDAR value of 60 (Fig. 1E, p=0.005, ANOVA). WBCs from patients with higher SDARs (and fewer persistently adhered cells) were all within the normal range. Patients in the low SDAR group also had significantly elevated absolute neutrophil counts (Fig. 1F, p=0.006, ANOVA), and ferritin levels (Fig. 1G, p=0.007, ANOVA). The mean ferritin level of those with low SDAR was nearly ten times greater than normal (mean= [3272.3 ± 791.9] μg/L vs. [784.5±219.6] μg/L). No white blood cell (WBC) adhesion was observed in the experiments.Here, we report a novel shear dependent adhesion ratio of sickle RBCs utilizing LN and FN functionalized microchannels. The approach presented here enabled us to create a shear gradient throughout the channel which may simulate the physiological flow conditions in the post-capillary venules. We further analyzed shear-dependent RBC adhesion in a patient specific manner and identified patient groups with low and high SDAR. The findings also suggested a link between lower shear dependent sickle RBC adhesion to LN and patient clinical phenotypes including inflammation and iron overload.Acknowledgments: This work was supported by grant #2013126 from the Doris Duke Charitable Foundation, National Heart Lung and Blood Institute R01HL133574, and National Science Foundation CAREER Award 1552782.Figure 1: Shear-dependent sickle RBC adhesion in microscale flow. (A) Macroscopic image of the shear-gradient microchannel with the arrow indicating flow direction. (B) Velocity and shear rate contours on a 2D plane above the bottom surface. The dashed rectangular area indicates the field of interest (FOI) where the experimental data were obtained. (C, D) Typical distribution of adherent deformable and non-deformable RBCs in LN and FN functionalized microchannels with the shear gradient. Dashed lines represent the adhesion curves and the corresponding equations were used to quantify shear dependent adhesion data. Shear-dependent RBC adhesion was lower (nSDAR<60) in patients with elevated white blood cell counts (E), absolute neutrophil counts (F), and serum ferritin levels (G). The dashed rectangles indicate the normal clinical values. [Display omitted] DisclosuresLittle:Hemex Health: Equity Ownership. Gurkan:Hemex Health: Employment, Equity Ownership.

Adhesion of Sickle RBCs to Heme-Activated Endothelial Cells Correlates with Patient Clinical Phenotypes

Severe hemolysis and associated high levels of hemolytic biomarkers, including LDH and heme, are among major constituents of the pathophysiology of sickle cell disease (SCD). Elevated extracellular heme due to hemolysis overwhelms endogenous detoxification mechanisms and leads to oxidative stress, triggering systemic endothelium activation, vascular dysfunction, and end organ damage. To understand the role of red blood cells (RBC) in this process, we assessed sickle RBC adhesion to heme-activated endothelial cells utilizing an endothelialized microfluidic platform in a clinically diverse patient population.Human umbilical vein endothelial cells (HUVECs) were seeded into fibronectin (FN) functionalized microfluidic channels and incubated for 4 hours in a 37°C and 5% CO2 environment. Next, the confluent monolayers were loaded and incubated for 45 minutes with fresh culture medium or at one of two concentrations of heme solutions: (1) 20 μM that corresponds to physiological heme levels in SCD patients, and (2) 40 μM. SCD blood samples, collected from 8 patients (7 HbSS and 1 HbS/β0 thal; 3 males and 5 females), were centrifuged to remove the plasma and washed with PBS thrice prior to flow experiments. RBCs were re-suspended in culture medium at a hematocrit of 25%. A total volume of 15 µl RBC suspension was perfused into the microchannels followed by rinsing with fresh culture medium at 1 dyne/cm2, which corresponds to the typical shear stress value observed in post-capillary venules. The fully closed and hermetically sealed microfluidic system design ensured the stability of gas composition in the culture medium during the experiments.Endothelialized microchannels supported sickle RBC adhesion to non-treated, 20 µM heme-activated, and 40 µM heme-activated HUVECs (Fig. 1A, B, C). Adhesion results suggested that activation of HUVECs by heme mediated RBC adhesion in a concentration-dependent manner, with a significant difference observed at 40 µM (Fig. 1D, paired t-test, p<0.05). Notably, a heterogeneous heme-mediated adhesion profile was seen among patients. Sickle RBC adhesion to 20 μM heme-activated HUVECs was significantly increased in patients with higher LDH levels (Fig. 1E, p=0.024, ANOVA), higher absolute reticulocyte counts (Fig. 1F, p=0.002, ANOVA), and lower total hemoglobin (Fig. 1G, p=0.016, ANOVA), which are indicative of elevated hemolysis. All patients in the high adhesion group (>200 adherent RBCs) had elevations in serum LDH levels and in absolute reticulocyte counts. Moreover, patients with a recent transfusion had higher RBC adhesion to 40 µM heme-activated HUVECs compared to patients with no transfusion in the last 3 months (Fig. 1H, p<0.05, ANOVA).Here, we report a direct link between heme-driven endothelial activation and RBC adhesion in a patient-specific manner. In patients with a more severe clinical phenotype, as reflected in LDH, total hemoglobin, and absolute reticulocyte or recent blood transfusions, we found greater RBC adhesion to heme-activated HUVECs. These findings suggest that RBCs from those patients most likely to experience hemolysis in vivo may also be those RBCs most likely to adhere to heme-activated endothelium.Acknowledgments: This work was supported by grants #2013126 and #2015191 from the Doris Duke Charitable Foundation, National Heart Lung and Blood Institute R01HL133574, and National Science Foundation CAREER Award 1552782.Figure 1: Sickle RBC adhesion to heme-activated HUVECs and clinical associations. Sickle RBCs adherent to immobilized HUVECs in (A) non-activated, (B) 20 µM heme-activated, and (C) 40 µM heme-activated microchannels. The microscope images illustrate a small portion of the full microchannel surface. (D) RBC adhesion to HUVECs increased depending on the heme concentration, with a significant difference at the 40 µM level (paired t test, p<0.05). Patients with higher LDH (E) as well as absolute reticulocyte counts (F), and lower total hemoglobin (G) showed significantly greater RBC adhesion to 20 µM heme-activated HUVECs (ANOVA). (H) Furthermore, patients with a recent transfusion history displayed elevated RBC adhesion to 40 µM heme-activated HUVECs compared to non-transfused patients (ANOVA). The dashed rectangles indicate the normal clinical values for healthy individuals, while all patients had total hemoglobin levels lower than normal. Scale bars represent 30 µm. [Display omitted] DisclosuresLittle:Hemex Health: Equity Ownership. Gurkan:Hemex Health: Employment, Equity Ownership.

Dynamic deformability of sickle red blood cells in microphysiological flow.

In sickle cell disease (SCD), hemoglobin molecules polymerize intracellularly and lead to a cascade of events resulting in decreased deformability and increased adhesion of red blood cells (RBCs). Decreased deformability and increased adhesion of sickle RBCs lead to blood vessel occlusion (vaso-occlusion) in SCD patients. Here, we present a microfluidic approach integrated with a cell dimensioning algorithm to analyze dynamic deformability of adhered RBC at the single-cell level in controlled microphysiological flow. We measured and compared dynamic deformability and adhesion of healthy hemoglobin A (HbA) and homozygous sickle hemoglobin (HbS) containing RBCs in blood samples obtained from 24 subjects. We introduce a new parameter to assess deformability of RBCs: the dynamic deformability index (DDI), which is defined as the time-dependent change of the cell's aspect ratio in response to fluid flow shear stress. Our results show that DDI of HbS-containing RBCs were significantly lower compared to that of HbA-containing RBCs. Moreover, we observed subpopulations of HbS containing RBCs in terms of their dynamic deformability characteristics: deformable and non-deformable RBCs. Then, we tested blood samples from SCD patients and analyzed RBC adhesion and deformability at physiological and above physiological flow shear stresses. We observed significantly greater number of adhered non-deformable sickle RBCs than deformable sickle RBCs at flow shear stresses well above the physiological range, suggesting an interplay between dynamic deformability and increased adhesion of RBCs in vaso-occlusive events.

Commonly Used Clinical Intravenous Fluid Formulations Differentially Affect Sickle Red Blood Cell Stiffness and Transit Time

Background:Disruption of red blood cell (RBC) volume regulation and water homeostasis is a major component of sickle cell pathophysiology. As such, hydration is a mainstay of prevention and treatment of vaso-occlusive crises (VOC) in patients with sickle cell disease (SCD). However, evidence for guiding clinicians' choice for intravenous (IV) fluid hydration in the acute setting is lacking (Okomo, Coch Data Syst Rev, 2012). Pediatric hematologists typically discourage rapid infusion of hypotonic fluid given the risk of hyponatremia, although such fluids as 5% dextrose with 34meq/L or 77meq/L sodium are often used after stabilizing various acute clinical situations relevant to SCD. Although altered sodium concentration and osmolarity have been shown to affect erythrocyte swelling and rheology, dextrose-containing fluids used clinically such as D5 ¼ and D5 ½ normal saline (NS), have not been studied (Reinhart et al., Mic Res, 2015; Hijiya et al., J Lab Clin Med, 1991). To those ends, we sought to investigate the effect of different clinically relevant IV fluid formulations on normal and sickle RBC stiffness and deformability.Methods:Fresh blood was obtained from healthy volunteers and patients with sickle cell anemia (SS) on hydroxyurea and not transfused for at least 100 days. Sterile, clinical grade fluids stored at room temperature were used for the experiments (Baxter, Figure 1A). Our laboratory has previously published a description of a microfluidic device comprised of multiple parallel microchannels 5μm wide recapitulating the in vivo geometry of capillaries (Rosenbluth et al., Lab Chip, 2008). This microvasculature-on-a-chip enables measurements of single-RBC transit times, which correlate with cell stiffness (Figure 1B,C). For the transit time experiments, a master silicon wafer was used to mold the microfluidic channels out of polydimethylsiloxane (PDMS) silicone. Centrifuged RBCs were washed with phosphate-buffered saline (PBS) and then diluted to 0.5% hematocrit (HCT) in the various fluids prior to flow. Cell suspensions were perfused into a microfluidic device pre-coated with 2% bovine serum albumin (BSA) at an average linear flow rate of 0.50 mm s-1 in the smallest channels with a syringe pump (Harvard Apparatus) and then imaged at 20x at 20 frames per second. Images were recorded for future analysis. For the experiments assessing RBC shape, washed RBCs were diluted with PBS to 0.5% HCT and imaged at 40x in plastic wells at time 0. PBS was removed and 100 μL of each clinical fluid formulation was added to the wells and images were then obtained over time. RBC circularity was calculated using custom-written scripts in Matlab (Figure 1D).Results:RBC transit times in both healthy and sickle blood were affected by osmolarity and the various solute concentrations (Figures 2A,B). Transit times of sickle RBCs in all IV fluid formulations were significantly higher, over 10 times, than that of RBCs from healthy donors. Transit times for both healthy and sickle donors were least in the D5 ¼ NS solution. Of note, sickle RBC transit time was greatest in the NS solution. Sickle RBC circularity also changed with solute concentration and osmolarity with statistical significance (Figure 2C). Cells with the highest change in circularity from baseline were also those exposed to the D5 ¼ NS solution.Conclusion:Our results suggest the stiffness of sickle RBCs is affected by different formulations of clinical IV fluids. Increased transit time of sickle RBCs in NS through our device may in part be explained by the decreased circularity, indicating that RBCs adopt more irregular shapes in this fluid. This, in turn, could lead to increased propensity of microchannel obstruction. Although the exact mechanisms are unclear, this begs the question of whether NS is an appropriate choice for initial fluid resuscitation for VOC and other SCD-related complications as it could exacerbate the already high stiffness and shape irregularity of sickle RBCs, further increasing microvascular occlusion. As these in vitro results have significant clinical implications, ongoing experiments involve investigating how these fluid-dependent effects may alter sickle RBC adhesion in 'endothelialized' microfluidic devices, how different oxygen tensions affect these fluid-mediated effects, potential differences on and off of hydroxyurea, and the underlying mechanisms of this IV fluid formulation-dependent effect. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gαs proteins activate p72Syk and p60-c-Src tyrosine kinases to mediate sickle red blood cell adhesion to endothelium via LW-αvβ3 and CD44–CD44 interactions

G protein-coupled receptors (GPCRs) have been suggested as new drug targets to treat a variety of diseases. In sickle cell disease (SCD), the LW erythrocyte adhesion receptor can be activated by stimulation of β2 adrenergic receptors (β2ARs), to mediate sickle erythrocyte (SSRBC) adhesion to endothelium. However, the involvement of tyrosine protein kinases in β2AR signaling to activate SSRBC adhesion to endothelium has not been thoroughly elucidated. Either direct activation with Cholera toxin of Gαs protein, which acts downstream of β2ARs, or inhibition with Pertussis toxin of Gαi, mediating suppression of adenylyl cyclase, increased SSRBC adhesion to endothelium over baseline adhesion. This effect involved the non-receptor tyrosine kinases, p72Syk and p60-c-Src, which were more abundant in SSRBCs than in normal erythrocytes. In contrast, Pertussis toxin and Cholera toxin failed to increase adhesion of normal erythrocytes. SSRBC Gαi inhibition also increased phosphorylation of p72Syk and p60-c-Src. Further, we investigated the relevance of activation of p72Syk and p60-c-Src, and identified LW (ICAM-4, CD242) and CD44 as the erythroid adhesion molecules both physically interacting with activated p60-c-Src. As a result, SSRBC LW underwent increased tyrosine phosphorylation, leading to SSRBC LW and CD44 binding to endothelial αvβ3 integrin and CD44, respectively. These data provide in vitro mechanistic evidence that p60-c-Src, which could act downstream of Gαs/p72Syk, associates with LW and CD44 on SSRBCs leading to their interactions with endothelial αvβ3 and CD44, respectively. Thus, increased activation of these signaling mechanisms in SSRBCs could initiate or exacerbate vascular occlusion, the hallmark of SCD.

Modulation of Sickle Red Blood Cell Adhesion and its Associated Changes in Biomarkers by Sulfated Nonanticoagulant Heparin Derivative.

Abnormal cellular adhesion is one of the primary causes of vaso-occlusive crisis in sickle cell disease (SCD). Levels of intercellular adhesion molecule 1 (ICAM-1) and P-selectin are upregulated, resulting in increased adhesion of leukocytes and sickle red blood cells (RBCs) to endothelium. This study compares the inhibitory effect of a sulfated nonanticoagulant heparin (S-NACH) derivative with a low-molecular-weight heparin, tinzaparin, on the adhesion of sickle RBCs to endothelium. The S-NACH exhibits minimum effects on hemostasis and bleeding and interferes with the binding of pancreatic cancer cells to endothelial cells via P-selectin. We show by static binding assay that pretreatment of both erythrocytes and endothelial cells with S-NACH significantly inhibits the increased adhesion of sickle RBCs to endothelial cells. The S-NACH treatment also decreases the higher plasma levels of (adhesion biomarkers) ICAM-1 and P-selectin in SCD mice. This investigation signals further research into the potential use of S-NACH in treating vaso-occlusions with minimal bleeding events in patients with SCD.

Natalizumab Blocks VLA-4 Mediated Red Blood Cell Adhesion and Is a Potential Therapy for Sickle Cell Disease

Sickle cell disease (SCD) is caused by a point mutation in the beta-chain of hemoglobin, which triggers a complex pathophysiology resulting in recurrent, painful vaso-occlusive events (VOCs) and chronic hemolytic anemia. Among other abnormalities, sickle red blood cells (RBCs) are more adhesive than normal RBCs. Sickle RBC adhesion is an important pathway leading to both VOC and hemolysis: adhesive interactions promote the formation of blood flow-obstructing heterocellular aggregates that induce ischemic tissue damage and slow the transit of RBCs through the vasculature, promoting sickle hemoglobin polymerization and hemolysis. The capacity of sickle RBCs to adhere to endothelium positively correlates with disease severity. Interventions that reduce sickle RBC adhesion, in particular that of reticulocytes, by blocking specific molecular targets may limit or prevent disease sequelae. Very Late Antigen – 4 (VLA-4 or α4β1 integrin) is an important integrin on reticulocytes that mediates adhesive interactions to endothelial and plasma vascular cell adhesion molecule - 1 (VCAM-1), plasma fibrinogen, and other ligands. VLA-4 is expressed on the surface of sickle reticulocytes, with levels decreasing during maturation such that mature RBCs do not express surface VLA-4. Natalizumab is a recombinant humanized antibody that binds to the α4 subunit of VLA-4 and is used to treat multiple sclerosis (MS) and Crohn’s Disease (CD) by preventing leukocyte trafficking into tissues at sites of inflammation. We investigated the ability of natalizumab to block VLA-4 in the context of sickle whole blood, and evaluated the effect on sickle reticulocyte, mature erythrocyte, and leukocyte adhesion during physiologic flow conditions. Whole blood samples obtained from SCD donors were analyzed for saturation binding of natalizumab to surface VLA-4 on leukocytes and reticulocytes using flow cytometry. Up to 20% of reticulocytes from SCD donors (n=13) were positive for VLA-4 surface staining, whereas VLA-4 was undetectable on the surface of reticulocytes from healthy donors (n=4). VLA-4 on SCD reticulocytes and mononuclear leukocytes was saturated by natalizumab concentrations lower than known plasma trough concentrations achieved in MS and CD patients after natalizumab treatment, with SCD reticulocyte EC50 = 0.11 ± 0.01 μg/mL and SCD leukocyte EC50 = 0.16 ± 0.01 μg/mL (n=6). This translates to a binding affinity of 0.7 to 1.3 nM, similar to that found for healthy donor leukocytes. Both whole blood cells and isolated leukocytes from SCD donors adhered to immobilized VCAM-1 during physiologic flow conditions simulating post-capillary venules (shear stress =1 dynes/cm2) using a microfluidic flow-based adhesion system. Leukocytes from SCD donors adhered more to VCAM-1 (Mean = 20.7 + 10.0 from n=7) than leukocytes from healthy donors (Mean = 5.0 + 1.4 from n=5). The adhesion of leukocytes and reticulocytes to VCAM-1 was blocked by natalizumab in a dose-dependent manner and as a function of natalizumab saturation of cell surface VLA-4. Therapeutic IgG4 antibodies, including natalizumab, undergo chain shuffling in vivo with endogenous IgG4, leading to mono-specific IgG4 molecules, with one Fab arm specific to the antigen it was raised against. Compared to divalent natalizumab, monovalent chain-shuffled natalizumab bound to SCD reticulocytes at a7-fold higher EC50 and inhibited SCD reticulocyte adhesion to VCAM-1 at higher antibody concentrations (10 and 1 µg/mL). While increased, these values are still below trough natalizumab levels achieved in natalizumab-treated patients. In summary, natalizumab bound VLA-4 on the surface of SCD reticulocytes and leukocytes similarly to healthy donor leukocytes and blocked adhesion of SCD reticulocytes and leukocytes to immobilized VCAM-1 under shear conditions. Natalizumab binding and adhesion inhibition occurred at plasma concentrations similar to those seen in natalizumab treated MS and CD patients. Based on these findings, natalizumab may have potential as an anti-adhesive therapy for SCD. Further clinical studies are needed to evaluate the safety and efficacy of natalizumab in SCD. DisclosuresHines:Biogen Idec: Research Funding. Krishnamoorthy:Biogen Idec: Employment, Equity Ownership. White:Biogen Idec: Research Funding. Gupta:Biogen Idec: Employment, Equity Ownership. van der Flier:Biogen Idec: Employment, Equity Ownership. Peters:Biogen Idec: Employment, Equity Ownership. Jiang:Biogen Idec: Employment, Equity Ownership. Hobbs:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment, Equity Ownership.

MEK inhibitors, novel anti-adhesive molecules, reduce sickle red blood cell adhesion in vitro and in vivo, and vasoocclusion in vivo.

In sickle cell disease, sickle erythrocyte (SSRBC) interacts with endothelial cells, leukocytes, and platelets, and activates coagulation and inflammation, promoting vessel obstruction, which leads to serious life-threatening complications, including acute painful crises and irreversible damage to multiple organs. The mitogen-activated protein kinase, ERK1/2, is abnormally activated in SSRBCs. However, the therapeutic potential of SSRBC ERK1/2 inactivation has never been investigated. I tested four different inhibitors of MEK1/2 (MEK), the kinase that activates ERK1/2, in a model of human SSRBC adhesion to TNFα-activated endothelial cells (ECs). SSRBC MEK inhibition abrogated adhesion to non-activated and TNFα-activated ECs to levels below baseline SSRBC adhesion to non-activated ECs in vitro. SSRBC MEK inhibition also prevented SSRBCs from activating naïve neutrophils to adhere to endothelium. To determine the effect of MEK inhibitors on SSRBC adherence in vivo, sham-treated or MEK inhibitor-treated SSRBCs were infused to nude mice previously treated with TNFα. Sham-treated SSRBCs displayed marked adhesion and occlusion of enflamed vessels, both small and large. However, SSRBC treatment with MEK inhibitors ex vivo showed poor SSRBC adhesion to enflamed vessels with no visible vasoocclusion in vivo. In addition, MEK inhibitor treatment of SSRBCs reduced SSRBC organ trapping and increased the number of SSRBCs circulating in bloodstream. Thus, these data suggest that SSRBC ERK1/2 plays potentially a critical role in sickle pathogenesis, and that MEK inhibitors may represent a valuable intervention for acute sickle cell crises.