SS RBC Adhesion Research Articles

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.

Longitudinal assessment of adhesion to vascular cell adhesion molecule-1 at steady state and during vaso-occlusive crises in sickle cell disease.

SummarySickle cell disease (SCD) is characterized by frequent and unpredictable vaso‐occlusive crises (VOCs). Sickle erythrocytes (SSRBCs) contribute to VOCs by participating in a series of adhesive events with blood cells and the vascular endothelium. Adhesion assays have been used to evaluate the relationship between SSRBC adhesion and SCD severity. We developed a standardized, clinical flow adhesion assay of whole blood to vascular cell adhesion molecule (FA‐WB‐VCAM). The objective of this study was to assess the variability and clinical predictive value of FA‐WB‐VCAM in a six‐month longitudinal, observational study (ELIPSIS) in SCD subjects during at‐home, steady‐state and self‐reported VOCs, and following VOC resolution. We observed a strong relationship between FA‐WB‐VCAM and SCD severity. Adhesion indices were significantly lower in SCD subjects on hydroxycarbamide and increased during VOCs; at‐home VOCs had significantly higher FA‐WB‐VCAM than steady‐state and contact VOCs. SCD subjects with a high frequency of self‐reported VOCs had a pro‐adhesive phenotype at steady state and were stratified into a high‐adhesive phenotype cohort; two years prospectively we observed a higher frequency of VOCs in the high‐adhesion cohort. This study supports stratifying SCD subjects based on steady‐state FA‐WB‐VCAM and suggests that FA‐WB‐VCAM may be a plausible surrogate end‐point for SCD severity.

Restoration of Normal Blood Flow in Mouse Models of Sickle Cell Vaso-Occlusion Following Intravenous or Subcutaneous Administration of a Highly Potent E-Selectin Specific Inhibitor

Background: In sickle cell disease (SCD), recurrent episodes of vaso-occlusive crisis (VOC) contribute to morbidity and accelerated mortality. Analysis of the RESET Phase 3 study demonstrated that administration of the pan-selectin inhibitor, rivipansel, early in the course of VOC was associated with efficacy outcomes in favor of rivipansel, suggesting that reversal of vaso-occlusion may be achieved by treatment early in VOC. While both P- and L-selectin likely contribute to the underlying inflammatory cascade leading to VOC, previous studies have shown that E-selectin expression on the vascular luminal surface appears to be crucial for adhesion of sickled (SS)-RBC and leukocytes to the endothelium. GMI-1687 is an innovative potent E-selectin specific antagonist that demonstrates high bioavailability following subcutaneous (SC) administration. In the current investigation, we report on in vitro and in vivo studies evaluating the activity of GMI-1687 in SCD mouse models of VOC. Methods: Townes transgenic mice and nude mice transfused with human SS-RBC were used as in vivo models of SCD. VOC was initiated by TNF injection in both models. Adhesion to inflamed venules and development of VOC was recorded using intravital microscopy at sites of window chamber implants. The in vivo activity of E-selectin inhibition with GMI-1687 was assessed following IV injection in the nude mouse and Townes transgenic models, and SC injection in the nude mouse model of SCD. Results: In an initial study, IV treatment with saline or GMI-1687 was initiated 30 min post infusion of SS-RBC (t30) into TNF-treated mice, a time during which sickle cells are adherent to inflamed venules and VOC has initiated, and repeated 30 minutes later (t60). As expected, mice treated with saline alone showed marked adhesion of human SS-RBC to venules (61% occlusion of the total vessels recorded at t30) with evident blood stasis. Under these conditions, GMI-1687 at 40 μg/kg significantly reversed SS-RBC adhesion and VOC at both t30 and t60 (p=0.002 and 0.0073, respectively, compared to saline treatment). This led to restoration of blood flow in 85% of the venules in GMI-1687 treated mice vs. 18% of vessels with normal blood flow in the control saline treated group. Because of reduced sickle cell adhesion and VOC, the number of circulating SS-RBC increased by approximately 6-fold in mice treated with GMI-1687. Similar anti-adhesive and anti-VOC activities of GMI-1687 treatment were obtained in the Townes transgenic model of SCD where following IV administration of the E-selectin antagonist, adhesion of sickled RBCs was significantly diminished and as a result, the percentage of the total occluded vessel segments was reduced by 80-90%. We next assessed the activity of SC administration with 40 μg/kg GMI-1687 on human SS-RBC adhesion and development of VOC in the nude mouse model. Similar to the IV experiments, GMI-1687 initiated at t30 and t60 post SS-RBC infusion significantly reduced SS-RBC adhesion in inflamed vessels (69% reduction at t30, and 63% at t60, p=0.0001). The reduction in SS-RBC adhesion led to a lower incidence of VOC (2% compared to 30% of vessels in mice injected with saline), and a 5.1-fold improvement (p=0.0315) in the number of circulating human SS-RBC. It is noteworthy that the effective dose of GMI-1687 used in these IV and SC studies resulting in the restoration of normal blood flow was approximately 500-fold less than that reported for rivipansel. Conclusions: These data demonstrate that the administration of the potent and E-selectin specific compound GMI-1687 was effective in restoring blood flow in two mouse models of SCD, and support the development of GMI-1687 formulated for SC use and self-administration with the potential for early intervention of VOC. Disclosures Thamilarasan: GlycoMimetics: Research Funding. Fogler:GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zennadi:GlycoMimetics: Research Funding.

Nitric oxide loading reduces sickle red cell adhesion and vaso-occlusion in vivo.

Sickle red blood cells (SSRBCs) are adherent to the endothelium, activate leukocyte adhesion, and are deficient in bioactive nitric oxide (NO) adducts such as S-nitrosothiols (SNOs), with reduced ability to induce vasodilation in response to hypoxia. All these pathophysiologic characteristics promote vascular occlusion, the hallmark of sickle cell disease (SCD). Loading hypoxic SSRBCs in vitro with NO followed by reoxygenation significantly decreased epinephrine-activated SSRBC adhesion to the endothelium, the ability of activated SSRBCs to mediate leukocyte adhesion in vitro, and vessel obstruction in vivo. Because transfusion is frequently used in SCD, we also determined the effects of banked (SNO-depleted) red blood cells (RBCs) on vaso-occlusion in vivo. Fresh or 14-day-old normal RBCs (AARBCs) reduced epinephrine-activated SSRBC adhesion to the vascular endothelium and prevented vaso-occlusion. In contrast, AARBCs stored for 30 days failed to decrease activated SSRBC adhesivity or vaso-occlusion, unless these RBCs were loaded with NO. Furthermore, NO loading of SSRBCs increased S-nitrosohemoglobin and modulated epinephrine's effect by upregulating phosphorylation of membrane proteins, including pyruvate kinase, E3 ubiquitin ligase, and the cytoskeletal protein 4.1. Thus, abnormal SSRBC NO/SNO content both contributes to the vaso-occlusive pathophysiology of SCD, potentially by affecting at least protein phosphorylation, and is potentially amenable to correction by (S)NO repletion or by RBC transfusion.

Disrupting the vicious cycle created by NOX activation in sickle erythrocytes exposed to hypoxia/reoxygenation prevents adhesion and vasoocclusion

In sickle cell disease (SCD), recurrent painful vasoocclusive crisis are likely caused by repeated episodes of hypoxia and reoxygenation. The sickle erythrocyte (SSRBC) adhesion plays an active role in vasoocclusion. However, the effect of prolonged reoxygenation after hypoxic stress on the molecular mechanisms in SSRBCs involved in onset of episodic vasoocclusion remain unclear. Exposure of human SSRBCs to hypoxia followed by 2 h reoxygenation, increased reactive oxygen species (ROS) production. Using specific pharmacological inhibitors, we show that excess ROS production in both reticulocytes and mature SSRBCs is regulated by NADPH oxidases (NOXs), the mitogen-activated protein kinase (ERK1/2), and G-protein coupled-receptor kinase 2 (GRK2). Consequently, SSRBC ROS create an intracellular positive feedback loop with ERK1/2 and GRK2 to mediate SSRBC adhesion to endothelium in vitro, and vasoocclusion in a mouse model of vasoocclusion in vivo. Importantly, reducing ROS levels in SSRBCs with redox-active manganese (Mn) porphyrins, commonly known as mimics of superoxide dismutase (SOD), disrupted the cycle created by ROS by affecting NOX and GRK2 activities and ERK1/2 phosphorylation, thus abrogating RBC-endothelial interactions. Inhibition adhesion assays show that LW (ICAM-4, CD242) blood group glycoprotein and CD44 are the RBC adhesion molecules mediating endothelial binding. Conversely, hypoxia/reoxygenation of normal RBCs failed to activate this feedback loop, and adhesion. These findings provide novel insights into the pathophysiological significance of the deleterious cycle created by NOX-dependent ROS, GRK2 and ERK1/2 within SSRBCs activated by hypoxia/reoxygenation, and involved in SSRBC adhesion and vasoocclusion. Thus, this loop in SSRBCs, which can be disrupted by Mn porphyrins, likely drives the profound SCD vasculopathy, and may point to new therapeutic targets to prevent chronic vasoocclusive events.

Microfluidic experimental setup for adhesion and recovery measurements of red blood cells in sickle cell disease

Current microfluidic assays, which aim at quantifying mechanical properties of sickle cell red blood cells (SS-RBCs), suffer from a number of drawbacks in functionalization and flow control. Specifically, physical adsorption functionalization techniques produce inconsistent functional surfaces, and common volumetric flow pumps cannot be used to adjust the flow inside microchannels with minimal delay. We have designed an experimental setup that alleviates these complications by implementing aspiration for microchannel assembly that enables the use of most functionalization techniques and a pressure controller that allows instant and precise changes in the microchannel flow. Utilizing this setup, we have quantified SS-RBC adhesion to the integrin αvβ3, a specific adhesion protein expressed on the endothelium, as well as measured the shear modulus and viscosity of the SS-RBC plasma membrane.

Increased erythrocyte adhesion to VCAM-1 during pulsatile flow: Application of a microfluidic flow adhesion bioassay.

Sickle cell disease (SCD) is characterized by microvascular occlusion mediated by adhesive interactions of sickle erythrocytes (SSRBCs) to the endothelium. Most in vitro flow adhesion assays measure SSRBC adhesion during continuous flow, although in vivo SSRBC adhesive interactions occur during pulsatile flow. Using a well-plate microfluidic flow adhesion system, we demonstrate that isolated SSRBCs adhere to vascular cell adhesion molecule (VCAM-1) at greater levels during pulsatile versus continuous flow. A significant increase in adhesive interactions was observed between all pulse frequencies 1 Hz to 2 Hz (60–120 beats/min) when compared to non-pulsatile flow. Adhesion of isolated SSRBCs and whole blood during pulsatile flow was unaffected by protein kinase A (PKA) inhibition, and exposure of SSRBCs to pulsatile flow did not affect the intrinsic adhesive properties of SSRBCs. The cell type responsible for increased adhesion of whole blood varied from patient to patient. We conclude that low flow periods of the pulse cycle allow more adhesive interactions between sickle erythrocytes and VCAM-1, and sickle erythrocyte adhesion in the context of whole blood may better reflect physiologic cellular interactions. The microfluidic flow adhesion bioassay used in this study may have applications for clinical assessment of sickle erythrocyte adhesion during pulsatile flow.

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.

MEK Inhibitors, Novel Anti-Adhesive Molecules, Reverse Sickle Red Blood Cell Adhesion and Vaso-Occlusion in Vivo

In sickle cell disease (SCD), painful vaso-occlusive crises and end-organ damage are caused by obstruction of the vessels due in large part to sickle erythrocyte (SSRBC) adherence to both the endothelium and leukocytes. Despite the contribution of SSRBC adhesiveness to vaso-occlusion, we lack treatments that specifically target intracellular signaling mechanisms critical for SSRBC cell-cell interactions. We have previously shown that the mitogen-activated protein (MAP) kinase ERK1/2 is aberrantly activated in SSRBCs. ERK1/2 signaling activation is critical for SSRBC interactions with the vascular endothelium and leukocytes. We have now evaluated the anti-adhesive properties of RDEA119, an inhibitor of MEK1/2, the kinase that activates the MAP kinase ERK1/2, in in vitro adhesion assays and in vivo in both nude mouse and humanized models of vaso-occlusion analyzed by intravital microscopy. We used RDEA119 because this dryg has previously been developed for human use. We found that RDEA119 caused a dose-dependent inhibition of human SSRBC adhesion to cultured TNFα-activated human micro-vascular endothelial cells (HMVECs) in vitro with a half-maximum inhibitory concentration (IC50) < 2 nmol/L. This suggests that RDEA119 would be effective at very low doses in reducing SSRBC adhesion and vaso-occlusion in vivo. To determine if RDEA119 prevents vaso-occlusion in nude mice, animals were injected with TNFα followed by infusion of RDEA119 (or placebo control), and then human SSRBCs. Intravital microscopy observations of enflamed vessels visible through dorsal skin-fold window chamber implants immediately after human SSRBC infusion showed persistent human SSRBC adhesion to both small and large vessels, with mean diameter of 29±3.5 μm, and vaso-occlusion in placebo-treated nude mice for at least 2 h, promoting blood stasis. However, treatment of nude mice with either 0.2 mg/kg or 2 mg/kg RDEA119 markedly reduced human SSRBC adhesion and mitigated vaso-occlusion. While human SSRBC adhered to 68.5±4.6% of total vessels recorded in placebo-treated animals, SSRBCs occupied only 33 ± 6.6% (p<0.0001) and 25 ± 3.7% (p<0.0001) of vessels in 0.2 and 2 mg/kg RDEA119-treated nude mice, respectively. As a result, normal blood flow was restored in 95±3.5% and 76±6.2% of vessels in 0.2 and 2 mg/kg RDEA119-treated animals, respectively, compared to 43±5.8% of vessels with normal blood flow in placebo-treated mice (p<0.001). Nude mice treated with 0.2 mg/kg RDEA119 also exhibited reduced human SSRBC trapping in the lungs, liver and spleen (p<0.0001), and an increased number of human SSRBCs remaining in circulation. RDEA119 was also administered to nude mice after onset of vaso-occlusion for a more clinically relevant outcome. Infusion of a single dose of 1 mg/kg RDEA119 to nude mice reduced human SSRBC adhesion and vaso-occlusion within 5 min of RDEA119 administration, and adhesion progressively decreased thereafter compared to SSRBC adhesion and vaso-occlusion in these same vessel segments prior to drug administration (p<0.0001). RDEA119 led to a 72% reduction in the percentage of vessels occupied by adherent human SSRBCs (p<0.001) and improvement in blood flow in 89±2.4% of vessels (p<0.001). Similar therapeutic benefits were obtained when RDEA119 was administered after the inflammatory trigger of TNFα to induce vaso-occlusion in transgenic sickle mice. RDEA119 resulted in a significant decrease in adherent sickle cells in enflamed vessels of sickle mice treated with only one dose of RDEA119 at 0.1, 0.05 or 0.025 mg/kg (p<0.001). RDEA119 at 0.1, 0.05 or 0.025 mg/kg also significantly reduced adhesion of leukocytes to the vascular endothelium compared with placebo-treated sickle mice (p<0.001). The effect of RDEA119 on both sickle RBCs and leukocyte adhesion was rapid, because a reduction in adhesion of the cells was already detectable within the first 15 minutes after drug infusion, and adhesion further decreased thereafter. In contrast, adherence of leukocytes increased progressively over time in placebo-treated sickle mice. Treatment of sickle mice with RDEA119 led to improved microcirculatory blood flow in enflamed vessels visible through the dorsal skin-fold window chamber and improved survival. These results suggest that RDEA119 may represent a valuable novel therapeutic intervention for acute pain crises that should be further evaluated in humans with SCD. Disclosures No relevant conflicts of interest to declare.

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.

Sevuparin Reduces Adhesion Of Both Sickle Red Cells and Leukocytes To Endothelial Cells In Vitro and Inhibits Vaso-Occlusion In Vivo

IntroductionSevuparin is a new chemically modified heparin with low anticoagulant activity currently being studied as an adjuvant therapy for severe malaria. Heparins generally have multiple biological properties, including antithrombin III-dependent inhibition of thrombin as well as blockade of P-selectin-mediated adhesion. Selectins have been shown to contribute to both sickle red cell (SS RBC) and neutrophil (PMN) adhesion in vitro and in mice with sickle cell disease (SCD). Sickle mice lacking both P- and E-selectins are relatively resistant to tumor necrosis factor-α (TNFα)-induced vaso-occlusion. We therefore theorized that sevuparin would show activity in inhibiting the selectin-dependent adhesion of red cells and leukocytes seen in the context of SCD, with the potential to decrease vaso-occlusion at a low level of anti-coagulation. MethodsWe studied both RBC and PMN adhesion to human umbilical vein endothelial cells (ECs) in an in vitro flow chamber. For studies of SS RBC adhesion, we exposed confluent cultures of ECs on gelatin-coated glass slides to interleukin (IL)-13 (50 ng/ml) for 48 h, followed by histamine (100 μM) for 10 min at 37°C immediately prior to performance of adhesion assays. Upregulation of P-selectin expression was confirmed by FACS of mechanically dislodged ECs. RBC adhesion was quantified during continuous flow. For assays of neutrophil adhesion, ECs were treated with human TNFα (10 μg/ml) overnight, followed by incubation for 10 min at 37°C with 100 µM histamine. All slides bearing ECs were washed after incubation with histamine and immediately mounted in the flow chamber for adhesion assays. Inhibition by sevuparin was tested by pre-incubating ECs with different concentrations of sevuparin for 20 min at 37°C prior to adhesion assays. PMNs from SCD patients were tested for adhesion to treated ECs with and without preceding exposure of ECs to sevuparin. In addition, PMNs from normal donors were first exposed to SS RBCs, as previously described, and then tested for adhesion to ECs with and without pre-exposure to sevuparin. PMN adhesion was quantitated after 10 min of no flow, followed by wash out at various shear stresses. The ability of sevuparin to prevent vaso-occlusion in vivo was studied by infusing red fluorescent SS RBCs into nude mice to effect in vivo adhesion and vaso-occlusion visible in implanted window chambers (Zennadi et al. 2007); 500 ng of murine TNFα in 100 μl was injected intraperitoneally (IP) 4 h before infusion of SS RBCs to increase endothelial expression of selectins. Sevuparin or saline control solution was injected SQ at various dose levels prior to infusion of SS RBCs into nude mice. ResultsSS RBC adhesion to ECs stimulated with IL13 and histamine was greater than adhesion to similarly stimulated ECs pretreated with sevuparin at 100, 200, 400 and 600 µg/ml prior to exposure to flowing SS RBCs (p = 0.047, 0.031, 0.094, 0.065, respectively, using a paired t test, in which each patient sample was only compared to itself), with a robust dose-response (p < 0.001) (Fig 1A). In a similar analysis, 7.5 µg/ml of function blocking monoclonal antibody to CD62P (9E10) also significantly reduced RBC adhesion (p = 0.038). Sevuparin significantly inhibited adhesion of SCD PMNs to ECs treated with TNFα and histamine, and this inhibition exhibited a modest dose-response relationship (Fig 1B). When normal PMNs stimulated by SS RBCs were studied, the differences between adhesion in the absence of sevuparin and in the presence of either 600 or 800 µg/mL sevuparin were also highly significant at both 1 and 2 dyne/cm2 (p = 0.019 and p = 0.011 at 1 dyne/cm2 and p = 0.013 and p = 0.008 at 2 dynes/cm2, respectively) (Fig 1C). In vivo, injection of sevuparin prior to infusion of SS RBCs significantly decreased SS RBC adhesion to vessel walls, as measured by the percent of vessel lengths occupied by adherent cells (Fig 2A). Sevuparin treatment also significantly increased the percent of venules that maintained normal blood flow (Fig 2B). [Display omitted] [Display omitted] ConclusionsSevuparin is an effective inhibitor of SS RBC adhesion and both SCD and normal PMN adhesion to endothelial cells in vitro. In vivo, sevuparin effectively decreased vaso-occlusion and improved blood flow after TNFα treatment. Therefore, we consider sevuparin a promising anti-adhesion agent with potential to reduce vaso-occlusion in SCD, via reducing RBC adhesion and leukocyte adhesion, possibly through its effect on selectins. Disclosures:Batchvarova:Dilaforette, AB: Research Funding. Shan:Dilaforette, AB: Research Funding. Zennadi:Dilaforette, AB: Research Funding. Lindgren:Dilaforette, AB: Employment. Leitgeb:Dilaforette, AB: Employment. Sulila Tamsen:Dilaforette, AB: Employment. Telen:GlycoMimetics, Inc.: Research Funding; Dilaforette, NA: Research Funding; Pfizer, Inc.: Consultancy.

Application of Microfluidic Technology to Evaluate the Influence of Pulsatile Blood Flow On Adhesion of Pediatric Sickle Erytrhocytes

AbstractAbstract 3238 Introduction:Sickle cell disease (SCD) is characterized by microvascular occlusion mediated in part by adhesion of sickle erythrocytes (SS RBCs) to the vasculature. Advanced flow adhesion (FA) technology facilitates SS RBC adhesion studies in conditions that simulate in vivo microvascular physiology. Most currently available FA systems measure SS RBC adhesion in non-pulsatile flow conditions, versus pulsatile blood flow conditions generated by the cardiac cycle. The influence of pulsatile blood flow on SS RBC adhesion may be particularly important in pediatric SS RBC adhesion, as children have a broad range of heart rates. This study compares SS RBC adhesion in non-pulsatile and pulsatile flow conditions, utilizing a commercially available, microfluidic FA system. Methods:Peripheral blood was obtained from patients with homozygous SCD (n=7) in steady state (5–18 years) from the Sickle Cell Center at the Children’s Hospital of Michigan. FA assays were performed in non-pulsatile and pulsatile flow conditions, at a shear stress of 1.0 dyne/cm2, and increasing shear stress up to 20 dyne/cm2to assess avidity. A programmable control unit regulated pulse frequency, shear stress, and temperature. Adhesion was measured to immobilized human laminin and vascular cell adhesion molecule-1 (VCAM-1). A static adhesion assay was used to assess initrinsic adhesive properties of SS RBCs independent of flow dynamics. Results:Standard assays were performed with 30 mL of isolated SS RBCs (1× 107 cells/mL), and SS RBC adhesion was comparable to levels previously reported in parallel plate flow adhesion assays. FA assays showed that adhesion to both laminin and VCAM-1 was significantly increased in the context of pulsatile blood flow (1.67Hz) compared to non-pulsatile blood flow by 8-fold and 56-fold, respectively. The relationship of SS RBC adhesion to increasing pulse frequencies was variable from patient to patient, although adhesion to both laminin and VCAM-1 was uniformly greater in all pulse frequencies tested (1.0, 1.5, 1.67, and 2.0 Hz) compared to non-pulsatile blood flow. When avidity of adhesion was tested 78% of SS RBCs remained adhered to VCAM-1 at the maximum 20dyne/cm2 shear stress, whereas 6% of SS RBCs remained adhered to laminin at a shear stress of 20 dynes/cm2. Pulsatile adhesion to VCAM-1 and laminin was unaffected by protein kinase A (PKA) inhibition, although adhesion to laminin decreased by 31% in one of three patients. To determine if increased adhesion under pulse-flow conditions was due to increased contact time with the immobilized substrate versus a change in the SS RBC’s intrinsic adhesive state, we measured SS RBC adhesion in a static adhesion assay following exposure to pulsatile versus non-pulsatile conditions. There was no significant difference in static adhesion to VCAM-1, however adhesion of pulse-exposed SS RBCs to laminin was more variable. Static adhesion of pulse-exposed SS RBCs to laminin was reduced by 60% in the presence of a PKA inhibitor. Conclusions:Our data demonstrate the application of a commercially available microfluidic flow adhesion assay system for efficient assessment of SS RBC adhesive properties. In the future, such advances may allow SS RBC adhesive properties to be evaluated clinically as a predictive tool for future vaso-occlusive events, and to predict individual patient response to anti-adhesive therapy. The small volume of blood required makes this system particularly attractive for studying pediatric samples. Additionally, our data demonstrate that adhesion to both an endothelial cell substrate (VCAM-1) and a subendothelial matrix substrate (laminin) is significantly influenced by the presence of pulsatile blood flow. Although PKA may play a minor role in pulsatile adhesion to laminin, increased contact time with immobilized laminin and VCAM-1 may be a greater contributor to increased adhesion under pulsatile conditions versus non-pulsatile conditions. Pediatric SS RBCs adhered to VCAM at higher levels and with more avidity compared to laminin. The pulsatile flow conditions described in this study more closely approximate in vivo microvascular conditions compared to non-pulsatile conditions commonly used to study SS RBC adhesion. Based on these differences in adhesion under pulsatile versus non-pulsatile flow, incorporating pulsatile flow in future adhesion studies may be more representative of in vivo conditions. Disclosures:No relevant conflicts of interest to declare.

P60src and p72Syk Tyrosine Kinases Activate Both Lw and CD44 to Mediate Sickle Red Blood Cell Adhesion to Endothelium

Abstract 3223The vaso-occlusive process in patients with sickle cell disease is complex and involves interactions between hemoglobin S red blood cells (SSRBCs) and vascular endothelial cells (ECs). However, the pathophysiologic triggers inducing SSRBC adhesion and vaso-occlusion are poorly understood. Elucidation of these mechanisms at the molecular level would allow development of new preventive and treatment strategies to abrogate vaso-occlusive events. Because non-receptor tyrosine kinases in non-erythroid cells are known to mediate cell-cell interactions, we have now investigated the role of non-receptor tyrosine kinases in modulating SSRBC adherence to cultured ECs, identified the kinases involved, and defined both the receptors on activated SSRBCs and the ligands on ECs involved in these interactions. Less than 20% of non-treated SSRBCs were able to adhere to non-stimulated ECs in vitro in intermittent flow conditions at a shear stress of 2 dynes/cm2. However, treatment of SSRBCs with sodium orthovanadate (Na3VO4), a potent broad spectrum inhibitor of protein tyrosine phosphatases, was able to significantly up-regulate RBC adhesion to cultured ECs by 4.6±1-fold over baseline adhesion in vitro. Na3VO4 in contrast, completely failed to increase adhesion of normal RBCs to cultured ECs. The increased SSRBC adhesion induced by Na3VO4 was significantly inhibited with piceatannol (p<0.001), which inhibits non-receptor tyrosine kinases p72Syk and p56Lck, and PP1 (p<0.001), an src-selective tyrosine p56Lck, p59Fyn and p60src kinase inhibitor. However, genistein, a broad range inhibitor of tyrosine kinases, and damnacanthal, a highly potent and selective inhibitor of p56Lck, completely failed to inhibit the effect of Na3VO4 on SSRBC adhesion. In addition, phenylarsine oxide, which specifically activates p56Lck tyrosine kinase, did not affect SSRBC adhesion to ECs. Together, these data suggest that SSRBC adhesion to ECs can be upregulated via activation of at least p72Syk and p60src tyrosine kinases, but not via the src-tyrosine kinase p56Lck. We further confirmed that increased SSRBC adhesion by Na3VO4 treatment is indeed a result of the activation of p72Syk and p60src tyrosine kinases. Slight basal phosphorylation of p72Syk and p60src was detected in all SSRBC samples tested. However, Na3VO4-treatment of SSRBCs significantly enhanced phosphorylation of both p60src and p72Syk kinases over basal phosphorylation (p<0.05), and this effect induced by Na3VO4 was completely blocked with the src-selective inhibitor PP1 or PP2, and piceatannol, respectively, suggesting that p72Syk and p60src undergo enhanced activation and are involved in up-regulation of SSRBC adhesion to endothelium. Moreover, at a shear stress of 2 dynes/cm2, both anti-LW (ICAM-4) and anti-CD44 antibodies individually inhibited adhesion of activated SSRBCs to ECs, by up to 83% and 78%, respectively. Recombinant soluble LW (srLW) and CD44 (srCD44) proteins also completely abolished adhesion of activated SSRBCs to ECs, identifying LW and CD44 as the RBC receptors involved in this interaction. The EC ligands for activated SSRBCs were also identified using antibody inhibition studies, as the αvβ3 integrin, a ligand previously shown to be important to SSRBC adhesion to activated ECs in vivo, and the endothelial CD44. These data demonstrate that activation of p72Syk and p60src-dependent pathways can act to activate LW- and CD44-mediated SSRBC adhesion to endothelial αvβ3 integrin and CD44, respectively, suggesting that this mechanism may initiate or exacerbate vaso-occlusion by increasing SSRBC adhesion to the endothelium. RBC CD44 is also the first adhesion molecule shown to be involved in SSRBC adhesion to endothelium. Disclosures:No relevant conflicts of interest to declare.

Restoring Endogenous Nitric Oxide in Sickle or Transfused Red Cells Ameliorates Adhesion and Vaso-Occlusion in Vivo

Abstract 3251Sickle erythrocytes (SSRBCs) are adherent to the endothelium, activate leukocyte adhesion, are deficient in nitric oxide (NO) content, and are inefficient in inducing vasodilation in response to hypoxia, all of which promote vascular occlusion, the hallmark of sickle cell disease (SCD). Furthermore, normal RBCs stored for transfusion are frequently used in SCD but are also NO-deficient. We therefore tested the hypothesis that repletion of intracellular SS or normal RBC NO could improve the abnormal circulatory characteristics of SSRBCs, using previously described in vitro (Zennadi et al. 2004 & 2008) and in vivo systems (Zennadi et al. 2007, Zhu et al. 2011). While exposure of SSRBCs to epinephrine (epi) up-regulated SSRBC adhesion to cultured HUVECs by 2.7±0.2-fold over baseline (p=0.0019), as previously described, loading SSRBCs with NO under hypoxic conditions (favoring SNO-Hb formation and bioactive NO availability), followed by reoxygenation prior to epi treatment, significantly decreased epi-induced SSRBC adhesion by 89±6.4% (p<0.01). In contrast, sham-loading of SSRBCs by deoxygenation/reoxygenation failed to inhibit epi-activated SSRBC adhesion, suggesting that increasing NO content rather than deoxygenation/reoxygenation reduced SSRBC adhesion. NO-loading of SSRBCs also significantly reduced the ability of epi-activated SSRBCs to activate mononuclear leukocyte adhesion to HUVECs to a level below adhesion induced by non-activated SSRBCs (11±5.8% vs 21±5.6% adherent leukocytes, p=0.02). The effect of NO-loading of SSRBCs on RBC adhesion was not due to NO release by SSRBCs, with subsequent effect of cell-free NO on HUVECs, since addition of free Hb to NO-loaded SSRBC suspensions failed to prevent NO from decreasing epi-activated SSRBC adhesion to HUVECs. We then studied the ability of NO loading of red fluorescent SSRBCs to affect in vivo adhesion and vaso-occlusion visible in implanted window chambers (Zennadi et al. 2007). While sham-treated SSRBCs adhered minimally to endothelium, epi-stimulated human SSRBCs showed strong adhesion, with frequent postcapillary obstruction, as previously shown. However, NO-loading of SSRBCs reduced epi-stimulated SSRBC adhesion to vessel walls and vaso-obstruction in vivo and resulted in improved RBC circulatory behavior. When SSRBC adhesion and vaso-occlusion were quantified by red fluorescence intensity (RFI) in vessels, using images obtained 30 minutes after RBC infusion, mice infused with epi-activated SSRBCs showed substantially greater RFI in vessels compared to animals infused with sham-treated SSRBCs (1343±395 vs 5±3.5 RFI, p<0.05). However, RFI in vessels of animals infused with NO-loaded, epi-activated SSRBCs was significantly decreased by 93.5% compared to RFI in vessels of mice infused with sham-loaded, epi-activated SSRBCs (p =0.00001). We also studied the effect of loading SSRBCs with the low-mass S-nitrosothiol (SNO) S-nitroso-L-cysteine (SNO-Cys) on the ability of SSRBCs to affect pulmonary artery pressures in intact mice. SNO-Cys attenuated the exaggerated pulmonary hypertension seen basally and during hypoxia in SSRBC-perfused lungs (p<0.05), suggesting that SSRBC NO/SNO deficiency may also contribute to SCD pulmonary vascular hypertension. Finally, we studied the effect of stored normal RBCs, with and without NO repletion, on the circulatory behavior of SSRBCs by admixing fresh or banked normal RBCs stored for 14 or 30 days with epi-activated SSRBCs (1:4 ratio of normal:SS) prior to infusion into mice. Fresh or 14 day old normal RBCs reduced epi-activated SSRBC adhesion to vascular endothelium (90% and 99% inhibition, respectively), preventing visible vascular blockade. In contrast, normal RBCs stored for 30 days failed to decrease adherence of epi-activated SSRBC to endothelium or vascular occlusion. However, when normal RBCs that had been stored for 30 days were NO-loaded prior to admixture with activated SSRBCs, we observed significant abrogation of adhesion of epi-stimulated SSRBCs to postcapillary venules (97% inhibition) and markedly reduced small diameter vessel obstruction (p < 0.001). Taken together, our results suggest that abnormal SSRBC NO content and NO/SNO-dependent vasoactivity contribute to the vaso-occlusive pathophysiology of SCD and are potentially amenable to correction by transfusion of NO-repleted normal RBCs. Disclosures:Telen:GlycoMimetics: Research Funding.

Therapeutic Benefit of Small Molecule Inhibitors of MEK/ERK in Reducing Sickle Red Cell Adhesion and Vaso-Occlusion in Vivo

Abstract 376In sickle cell disease (SCD), vaso-occlusive crises are caused largely by the sickle erythrocyte (SSRBC), which plays a critical and active role in vascular occlusion. The current major limitation in developing therapeutics for vaso-occlusive crises is our poor understanding of the specific signaling mechanisms that lead to increased sickle cell adhesion to endothelium and their ability to activate leukocyte adhesion. We have previously shown that the mitogen-activated protein kinase ERK1/2 is constitutively active in SSRBCs, and can be inducible by agonist-stimulation. ERK1/2 signaling is involved in activation of ICAM-4, which mediates SSRBC adhesion to endothelial cells (ECs). To determine if inhibition of SSRBC ERK activation has therapeutic potential, we tested four different inhibitors of MEK (the upstream kinase of ERK)/ERK activation in a model of non-stimulated human SSRBC adhesion to TNFα activated ECs. The MEK/ERK inhibitor U0126 is used in animal models, while RDEA119, AZD6244 and GSK1120212 were selected based on their demonstrated safety in long-term human therapeutic studies. Less than 30% of untreated SSRBCs adhered to non-activated ECs in vitro. However, adherence of non-treated SSRBCs to TNFα activated ECs increased by 2.6±0.17-fold over basal adhesion of untreated SSRBCs to non-activated ECs. Treatment of SSRBCs with the U0126, RDEA119, AZD6244 and GSK1120212 decreased SSRBC adhesion to activated ECs to levels below baseline SSRBC adhesion to non-activated ECs, demonstrating that MEK-dependent ERK signaling in non-stimulated SSRBCs is required for RBC adhesive interaction with activated ECs. Our data also indicated that co-incubation of epinephrine-activated SSRBCs with naive polymorphonuclear cells (PMNs), resulted in significant increased PMN adhesion compared to adhesion of naïve PMN alone. However, blocking ERK activity with U0126, RDEA119, AZD6244 and GSK1120212 decreased the ability of SSRBCs to promote PMN adhesion, indicating that SSRBCs activate leukocyte adhesion via the ERK pathway activation. We then determined the effect of the ERK signaling activation on adherence of unstimulated human SSRBCs in the presence of inflammation and vaso-occlusion in nude mice in vivo. Sham-treated human SSRBCs showed strong adhesion to TNFα activated endothelium, with frequent postcapillary obstruction. However, RDEA119-treatement of SSRBCs reduced RBC adhesion to TNFα activated endothelium and vaso-obstruction and resulted in improved RBC circulatory behavior. When RBC adhesion was quantified as % venular length occupied by SSRBCs, the percent venular length occupied by RDEA119-treated SSRBCs was significantly decreased by 75±5.5% (p<0.0001) and 82±5.2% (p<0.05) in both small (≤25 μm) and large (>25 μm) vessels, respectively. Similar results were obtained with U0126-treated SSRBCs. Our findings suggest that ERK is a key mediator of RBC adhesion and vaso-occlusion that does not require prior activation, and therapeutic strategies targeting ERK can be viable in reducing vaso-occlusion. Furthermore, we also determined if MEK/ERK inhibitors can be used therapeutically to reduce vaso-occlusion in nude mice injected with TNFα, followed by infusion of 0.2 or 2 mg/kg U0126 (or placebo control), and then human SSRBCs. Placebo treated mice display marked SSRBC adhesion to inflamed vessels and vaso-occlusion with evident blood stasis. However, infusion of U0126 at either 0.2 or 2 mg/kg dramatically reduced SSRBC adhesion and vessel obstruction. Quantitation of SSRBC adhesion indicates that U0126 at 2 mg/kg and 0.2 mg/kg significantly reduced by 76±5.5% and 59±4%, respectively, the % venular length occupied by SSRBCs for vessels ≤ 25 μm in diameter (p<0.05 for either 0.2 or 2 mg/kg U0126 vs placebo). However, for vessel diameter > 25 μm, U0126 at only 2 mg/kg completely inhibited the % venular length occupied by SSRBCs. Our data suggest that in the presence of preexisting inflammation, MEK/ERK inhibitors can reduce vaso-occlusion, and their inhibitory effect could affect at least in part SSRBC adhesive function. Thus, this is the first report implicating atypical ERK activation in SSRBCs with the vaso-occlusive pathophysiology of SCD, and suggesting that small molecule inhibitors of MEK/ERK could be effective in ameliorating RBC adhesion and vaso-occlusion, the hallmarks of SCD. Disclosures:No relevant conflicts of interest to declare.

Natural antiband 3 antibodies in patients with sickle cell disease

Band 3 oligomers, precociously formed in the membrane of sickle red blood cells (SS RBC) as a result of oxidative damage, induce two significant changes: (1) contribution to the adhesive nature of these cells to endothelial cells; (2) production of recognition sites for natural antiband 3 antibodies (antiband 3 Nabs). The inhibition of the adhesion of SS RBC to endothelial cells by band 3 peptides suggests a participation of antiband 3 Nabsin the ethiology and prevention of vaso-occlusive crises (VOC). To address this question, we measured the levels of antiband 3 Nabsin sickle cell anaemia (SCA) patients (45 in steady state, 35 in VOC) and in controls (27 sickle trait, 30 normal AA subjects). A significant decreased of antiband 3 Nabs in the VOC group was demonstrated as compared with the steady state group, the sickle trait and healthy controls. This study provides data suggesting that Antiband 3 Nabs are likely to play a role in the SCA VOC.

Nitric oxide deficiency in sickle red cells is associated with increased cell adhesion to endothelium and vaso-occlusion

Vascular occlusion is the hallmark of sickle cell disease (SCD) and is mediated largely through sickle red blood cell (SSRBC) adherence to the endothelium and SSRBC-induced activation of leukocyte adhesion. While normal RBCs release bioactive derivatives of nitric oxide (NO) in response to hypoxia, leading to vasodilation, SSRBCs are deficient in NO adducts and have reduced ability to induce vasodilation. We show that loading SSRBCs in vitro with NO under hypoxic conditions, followed by reoxygenation, significantly decreased epinephrine-dependent SSRBC adhesion in vitro ( p = 0.032). NO-loading of SSRBCs also reduced adhesion to vessel walls and vaso-obstruction observed in vivo through dorsal-skin window chambers in nude mice. Additionally, NO-loading markedly decreased the ability of SSRBCs to activate leukocyte adhesion to endothelial cells in vitro ( p = 0.027). Increasing SSRBC bioactive NO content by incubating with the low-mass S -nitrosothiol (SNO) S -nitroso- l -cysteine (SNO-Cys) attenuated the exaggerated pulmonary hypertension seen basally and during hypoxia in SSRBC-perfused lungs, suggesting that SSRBC NO/SNO deficiency may also contribute to SCD lung disease, such as pulmonary hypertension. Furthermore, since transfusion is frequently used in SCD, we studied the effects of stored normal RBCs (SNO-depleted) on SSRBC interactions in vivo . Infusion of stored (32 days) normal, human RBCs failed to prevent SSRBC adhesion and vaso-occlusion. In contrast, NO-loaded stored normal RBCs or fresh normal RBCs reduced SSRBC adhesion to endothelium and abolished frank vaso-occlusion. Our results suggest that abnormal SSRBC NO content and NO/SNO-dependent vasoactivity contribute to the vaso-occlusive pathophysiology of SCD and are potentially amenable to correction by RBC transfusion.

Mechanisms behind adhesive erythrocytes in sickle‐cell disease

Erythrocytes containing primarily hemoglobin S (SS RBCs) are abnormally adherent to a number of ligands, including normal constituents of the extracellular matrix as well as those present on the surfaces of other blood cells and endothelial cells. However, SS RBCs are not really very different from normal (AA) RBCs, except that they are younger. In general, they express the same range of adhesion receptors, and the levels of expression of these receptors, although increased in some cases, are not sufficient to explain the difference between the adhesivity of SS and AA RBCs. However, SS RBCs have activated adhesion receptors, and the signaling pathways responsible for this activation are also upregulated in SS RBCs. In addition, SS RBCs convey different extracellular signals, which are also likely to affect their adhesion to other blood cells and endothelial cells. Together, SS RBCs induce cell–cell interactions mediated by the B‐CAM/LU, ICAM4 (LW), CD44 (In), CD47, α4β1 integrin, and CD36 adhesion molecules on SS RBCs with target ligands on leukocytes and endothelial cells. Other, as yet unidentified cell surface moieties on SS RBCs are probably also responsible for SS RBC interactions with P‐selectin. Interestingly, at least some of these RBC adhesion receptors may also mediate adhesion and adverse physiologic events in the setting of transfusion of stored normal RBCs. Therefore, understanding the mechanisms behind SS RBC adhesion may offer opportunities for improving both therapy of sickle‐cell disease as well as transfusion practices.

Sickle mouse red cells activate endothelium by heme‐mediated peroxide generation and promote red cell and leukocyte adhesion

Sickle red blood cells (SS RBCs) are known to generate reactive oxygen species due to autoxidation of hemoglobin S (HbS). We hypothesize that HbS autoxidation and peroxide generation in SS mouse RBCs will result in activation of quiescent endothelium. We, therefore, infused BERK SS RBCs into congenic C57BL mice. BERK mice RBCs express exclusively HbS. A bolus of 150 μl of FITC‐labeled or unlabeled RBCs (Hct 30%) was infused iv into C57BL mice, and intravital observations made in the cremaster microcirculation. Infusion of FITC‐labeled C57BL RBCs into C57BL mice resulted in rare or no RBC adhesion, suggesting no endothelial activation. In contrast, infusion of BERK RBCs into C57BL mice caused marked increase in adhesion of infused RBCs in venules, accompanied by leukocyte recruitment. When the cremaster preparation was suffused with dihydrorhodamine (DHR), an H2O2‐sensitive probe, infusion of BERK RBCs, but not C57BL RBCs, resulted in increased endothelial fluorescence intensity. Infusion of BERK RBCs treated with catalase (100 U) to quench RBC‐generated peroxide inhibited DHR fluorescence, and adhesion of RBCs and leukocyte. Superoxide dismutase was relatively ineffective. These results suggest an obligatory role of heme‐mediated peroxide generation by SS RBC in endothelial activation, SS RBC adhesion and inflammatory effects in sickle cell disease. Supported by grants from American Heart Association and NIH (HL070047).

Erythrocyte plasma membrane–bound ERK1/2 activation promotes ICAM-4–mediated sickle red cell adhesion to endothelium

AbstractThe core pathology of sickle cell disease (SCD) starts with the erythrocyte (RBC). Aberration in MAPK/ERK1/2 signaling, which can regulate cell adhesion, occurs in diverse pathologies. Because RBCs contain abundant ERK1/2, we predicted that ERK1/2 is functional in sickle (SS) RBCs and promotes adherence, a hallmark of SCD. ERK1/2 remained active in SS but not normal RBCs. β2-adrenergic receptor stimulation by epinephrine can enhance ERK1/2 activity only in SS RBCs via PKA- and tyrosine kinase p72syk-dependent pathways. ERK signaling is implicated in RBC ICAM-4 phosphorylation, promoting SS RBC adhesion to the endothelium. SS RBC adhesion and phosphorylation of both ERK and ICAM-4 all decreased with continued cell exposure to epinephrine, implying that activation of ICAM-4–mediated SS RBC adhesion is temporally associated with ERK1/2 activation. Furthermore, recombinant ERK2 phosphorylated α- and β-adducins and dematin at the ERK consensus motif. Cytoskeletal protein 4.1 also showed dynamic phosphorylation but not at the ERK consensus motif. These results demonstrate that ERK activation induces phosphorylation of cytoskeletal proteins and the adhesion molecule ICAM-4, promoting SS RBC adhesion to the endothelium. Thus, blocking RBC ERK1/2 activation, such as that promoted by catecholamine stress hormones, could ameliorate SCD pathophysiology.