ALBERT

Description: Abstract 1515 Poster Board I-538 BACKGROUND Acute chest syndrome (ACS) is a common cause of morbidity and mortality for individuals with sickle cell disease (SCD). The treatment of ACS is mainly supportive. Prior studies have shown that intravenous pulse-dose corticosteroids, such as dexamethasone, can decrease the duration and morbidity of ACS. However, such treatment may also precipitate ‘rebound‘ episodes of vaso-occlusive pain in some individuals that might negate the overall benefit of corticosteroids. Tapered corticosteroid therapy might decrease the rebound effect while maintaining therapeutic benefit. Therefore, we designed a prospective study to begin to test this hypothesis and to assess biomarkers that might clarify the therapeutic and toxic mechanisms of corticosteroids in SCD, predict outcome, and guide therapy. METHODS We conducted a multi-center, placebo-controlled pilot study to test the feasibility and safety of high-dose oral dexamethasone followed by a taper for ACS. Children and adults with SCD and ACS of any severity were randomized to dexamethasone (0.3 mg/kg q12h x 2, 0.3 mg/kg q24h x 2, 0.2 mg/kg q24h x 2, 0.1 mg/kg q24h x2, then stop) or placebo to start within 24 hours of diagnosis. All subjects received standard, protocol-directed supportive care for ACS. We defined the duration of ACS using a novel, objective tool that assessed rate and effort of breathing, oxygen saturation in room air, thoracic pain, and use of supplemental oxygen and ventilatory support. The primary outcomes were the duration of ACS and the duration of hospitalization for ACS. We also measured a panel of biomarkers before, during and after therapy. Inflammatory biomarkers included high sensitivity C-reactive protein and secretory phospholipase A2 (sPLA2). White cell and endothelial activation markers included sVCAM-1, sICAM-1, vWF:Ag and vWF:RCoF, sE-selectin, sP-selectin, sL-selectin, nitric oxide metabolites (NO), and whole blood tissue factor. We used generalized linear mixed models controlling for age to test for differences between treatment groups. RESULTS We enrolled 12 subjects (9 children, 3 adults; mean age 17.3 years, range 5 - 45) with homozygous sickle cell anemia at 4 centers and randomized 11 (1 drop-out) to either dexamethasone (N=5) or placebo (N=6). The objective ACS assessment tool was completed on all subjects without difficulty. In this pilot study, dexamethasone reduced the duration of hospitalization (41.5 vs 62.3 hrs; P=0.024), but not the duration of ACS (log of duration 2.4 vs 3.5 hrs; P=0.127), supplemental oxygen (17.5 vs 41.2 hrs; P=0.876), hypoxemia (13.8 vs 34.3 hrs; P=0.770), or total opioid usage in morphine equivalents (54.4 vs 68.8 mg; P=0.885). There were no statistically significant differences in adverse events between arms. However, 3 patients treated with dexamethasone had a painful event in the 2 weeks after hospital discharge (1 required re-hospitalization), compared to 1 patient in the placebo group (0 re-hospitalizations). No marked leukocytosis occurred in either treatment group, but the leukocyte count at 1-week follow-up had decreased less from baseline in the dexamethasone group compared to placebo (-17.7 vs -37.6%; P=0.028). The baseline sPLA2 concentration was 3 100 ng/mL in 4/5 and 4/6 patients in the dexamethasone and placebo arms. The white cell activation marker, sL-selectin was significantly decreased at 1-week follow-up in the dexamethasone group compared to placebo (573.8 ng/mL vs 742.8; change from baseline -121.2 vs 57.2; P

Description: Abstract 2587 Poster Board II-563 Background: SCD is considered to be a hypercoagulable state. Because of the great complexity of the hemostatic system it has not been easy to establish an etiological link between hypercoagulability and SCD vascular pathology. In this study we evaluate hemostatic perturbations in adult SCD patients by employing the computerized automated thrombogram (CAT), a novel thrombin generation assay that provides a global measure of coagulation potential and a direct assessment of the coagulation phenotype (Hemker HC et al.,Thromb Haemost. 2006; 96:553–61). We aim to characterize the coagulation phenotype of the SCD patient by a panel of CAT assay parameters. Methods: A total of 23 SCD patients (HbSS and HbSbeta0Thalassemia; 18 –58 years) were evaluated. Control group consisted of 6 age-matched controls (males=3, females=3). SCD plasma samples were obtained at baseline during routine clinic visits. Platelet poor plasma (PPP) ± corn trypsin inhibitor (CTI) - to minimize variability from activation of contact pathway during sample collection- was analyzed by CAT using 2 different triggers for initiation of thrombin generation: i) High trigger –5pM Tissue factor (TF) with 4uM phospholipid (PL) and ii) Low trigger- 1pM TF with 4uM PL. Five CAT assay parameters were studied – Lag time, Endogenous thrombin potential (ETP), Peak thrombin (Peak), Time to peak (ttPeak) and Start Tail. Students t-test was used to compare means of CAT assay parameters between SCD patients and controls. Lactate dehydrogenase (a biomarker of hemolysis-associated nitric oxide resistance and endothelial dysfunction) correlated with CAT assay parameters including lagtime (r= −0.35, p=0.05); ttPeak (r=−0.45, p

Description: Plasma levels of heme in the 20 to 600 μM range are found in clinical conditions associated with intravascular hemolysis including paroxysmal nocturnal hemoglobinuria and sickle cell disease, conditions also associated with a thrombotic tendency. Objectives: To investigate whether heme, an inflammatory mediator and a product of intravascular hemolysis in patients with hemolytic anemia including sickle cell disease (SCD), could modulate hemostasis by an effect on endothelial tissue factor (TF) expression. Additionally, in SCD patient-related studies, we assessed whether any association existed between whole blood TF activity (WBTF) and levels of surrogate markers of intra-vascular hemolysis including lactate dehydrogenase (LDH) and reticulocyte counts. Methods: Following incubation of human endothelial cells (from umbilical vein and/or lung microvasculature) with heme (1 to 100 μM) for various times (30 minutes to 8 hours), levels of TF protein were assessed using ELISA, flow cytometry and/or Western blotting; and TF mRNA by a semi-quantitative RT-PCR. An assay for TF functional activity was performed using a chromogenic tenase activity kit where specificity of TF activity was tested in antibody-blocking experiments. Three TF-specific antibodies including a rabbit polyclonal and two mouse monoclonal (clones hTF-1 and TF9-10H10) antibodies were used in assays involving TF protein analysis. All experiments were performed in media containing polymyxin B to neutralize any potential endotoxin contamination. In patient-related studies, 81 subjects with SCD (1 to 21 years) were evaluated for levels of WBTF, LDH, and reticulocyte counts and data analyzed for potential relationships. Results: Heme induced TF protein expression on the surface of both macro- and micro-vascular endothelial cells in a concentration-dependent manner with 12- to 50-fold induction noted (ELISA assays) between 1 and 100 μM heme (P

Description: Evaluation of fluid phase markers reveals activated blood coagulation in sickle cell disease (SCD), with enhanced in vivo thrombin generation. Studies have shown that whole blood tissue factor (WTBF) activity is elevated, and that both monocytes and endothelial cells (ECs) contribute to WBTF. In a recent preliminary study, we have found an unexpected and interesting positive correlation between WBTF and the levels of both reticulocytes (R=0.6) and phosphatidylserine (PS)-positive red cells (R=0.4) in SCD and an inverse correlation with hemoglobin (R=-0.4). We have therefore evaluated potential mechanisms for erythroid modulation of WBTF. Reticulocytes being PS-positive, we initially evaluated the effects of PS-positive red cells on endothelial TF using human lung microvascular ECs and ionophore-treated PS-exposing control erythrocytes, since sickle erythrocytes also express other adhesion markers. Following incubation with appropriate treatments for 24 hours, ECs were analyzed for TF expression (with E-selectin as a positive control for EC activation). Co-incubation of PS-positive red cells (15% ±) with ECs increased endothelial expression of TF in a hematocrit-dependent manner. PS-positive cells increased TF expression by 140, 200, 260 and 320% at hematocrits of 2, 5, 10 and 20%, respectively, compared to media controls. Results similar to those observed with TF were also noted with E-selectin. PS-positive sickle erythrocytes have been classified as either type I or type II cells. Type I express low levels of PS (reticulocytes), while type II are highly PS-positive and include sickle dense cells. We next evaluated whether both type I and type II PS-positive red cells triggered endothelial TF expression. Type II cells were prepared by incubating control erythrocytes with calcium ionophore, and type I cells were generated by incubating type II cells with predetermined amounts of annexin V (100 μg annexin V per 25X106 PS-positive cells). FACS analyses demonstrated appropriate PS levels in each PS positive category. We show that type I cells had no effect. TF expression occurred following incubation of ECs with type II erythrocytes (0.21±0.06 units TF with type II vs 0.10±0.06 with PS-negative cells at a 2% hematocrit, n=5, P

Description: Phosphatidylserine (PS)-dependent erythrocyte adhesion to both cultured endothelial cells and the components of sub-endothelial matrix appears to be mediated in part via thrombospondin-1 (TSP). While TSP exhibits multiple cell-binding domains, the PS-binding site on TSP has not been identified. Since a cell-binding domain for anionic heparin is located at the amino-terminal domain of TSP, we hypothesized that anionic PS-positive (PS+ve) red cells bind to this domain. In a recent preliminary study, using a flow adhesion system and PS+ve erythrocytes (prepared by treating control AA red cells with A23187), we have demonstrated that heparin inhibited PS+ve erythrocyte adhesion to immobilized TSP in a concentration-dependent manner with 58 to 77% inhibition noted at concentrations between 1 and 50 U/ml (n=9, P

Description: Abstract 2566 Poster Board II-543 The polyunsaturated fatty acids (PUFAs): arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are structural and functional components of normal cell membranes. These fatty acids are precursors of several bioactive lipid mediators including eicosanoids, resolvins and protectins that regulate many cellular functions including inflammation (Annu Rev Pathol Mech Dis 3:279; 2008). We investigated whether the red cell membrane PUFA composition was abnormal in children with homozygous HbSS disease (HbSS) in an attempt to identify whether any of the pathobiologic processes such as inflammation and/or hemolysis noted in HbSS are related to PUFA deficiency. The study population included 18 children with HbSS (ages 2 to 16 years) and 10 age- and race- matched controls. Fatty acid content of membrane phospholipids (PLPs) was measured by capillary gas chromatography following isolation of total red cell lipids and transmethylation of resolved PLPs. PLPs analyzed included phosphatidylcholine or PC (an outer leaflet membrane PLP), phosphatidylserine or PS and phosphatidylethanolamine or PE (two inner leaflet membrane PLPs). Since the n-6 PUFA, AA, is a precursor of pro-inflammatory eicosanoids, and since the n-3 PUFA, DHA, contributes to the generation of anti-inflammatory resolvins and protectins, we also determined AA/DHA ratios to assess for imbalances in AA to DHA in each of these PLP fractions. As shown in the Table, the AA/DHA ratios were increased in all three PLP fractions. In the PC and PS fractions, this change in ratio was due to significant decreases in membrane DHA. In the PE fraction the abnormal AA/DHA ratio was due to an elevation in AA content. We next assessed whether DHA deficiency in children with HbSS disease was associated with abnormal inflammatory and/or hemolytic biomarkers. Hemolytic markers evaluated were plasma LDH, hemoglobin, hematocrit, and reticulocyte count. Inflammatory markers assessed included WBC count, absolute polys, and plasma levels of high sensitivity C-reactive protein or hs-CRP (Nat Clin Pract Cardiovasc Med 2:29; 2005). Correlations with these biomarkers were tested for both membrane DHA content and the AA/DHA ratio. Hs-CRP was the only biomarker that demonstrated significant correlations. Hs-CRP correlated positively with AA/DHA ratios from all three PLP fractions (r=0.51 to 0.58, P=0.01 to 0.03) and negatively with DHA content (r=−0.43 to −0.52, P=0.03 to 0.05). Recent evidence suggest that inflammation plays a role in sickle cell disease (SCD) pathophysiology including microvessel occlusion. A previous pilot study in adult patients with SCD has also demonstrated that oral EPA and DHA supplementation (as in fish oil) decreased the number of vaso-occlusive or painful episodes (Thromb Haemost 85:966; 2001). Such previous reports together with our current data which demonstrate an imbalance in membrane AA and DHA (the precursors of potent pro- and anti-inflammatory mediators) suggest that the n-3 and n-6 PUFAs may play a role in SCD microvessel pathophysiology. Disclosures: No relevant conflicts of interest to declare.

Description: Phosphatidylserine (PS)-positive erythrocytes adhere to both endothelial cells and the sub-endothelial matrix components. While thrombospondin mediates these interactions, it is not known whether PS-associated erythrocyte-endothelial adhesion occurs in the absence of plasma ligands. Using ionophore-treated PS-expressing control erythrocytes, we demonstrate that PS-positive erythrocytes adhered directly to human lung micro-endothelial cells in the absence of plasma ligands, that this adhesion was further enhanced following endothelial activation with IL-1α, TNF-α, LPS, and hypoxia (2.5- to 8-fold increase), and that this adhesive interaction was selective to erythrocyte-PS. We next explored whether micro-endothelial cells express an adhesion receptor that recognizes cell surface expressed PS (PSR) similar to that expressed on activated macrophages. Using RT-PCR and Western blotting, we demonstrate constitutive expression of both PSR mRNA and protein which were up-regulated in a time-dependent manner following endothelial activation (with maximal increases of 3-fold in mRNA and 2-fold in protein noted at 4 and 6 hour, respectively). While minimal PSR expression was noted on un-stimulated cell surface (8% positivity), endothelial activation up-regulated surface expression of this receptor (35–40% positivity in IL-1α and TNF-α activated cultures). In antibody blocking studies, using both artificially generated PS-positive erythrocytes and also using PS-positive erythrocytes from patient with sickle cell disease, we demonstrate that PSR was functionally active supporting PS-mediated erythrocyte adhesion to activated endothelial cells. Our results demonstrate the existence of a novel functional adhesion receptor for PS on the micro-endothelium which is up-regulated by such pathologically relevant agonists as hypoxia and cytokines.

Description: Abstract 4210 Several lines of evidence suggest that hemostasis is activated in patients with chronic hemolytic anemias, including sickle cell disease (SCD), with documented laboratory evidence for increased thrombin generation, fibrin formation, and elevated circulating levels of tissue factor (TF). TF, a cell surface receptor for factor-VII/VIIa, is the physiologic cellular activator of hemostasis. Previous studies have identified a circulating pool of TF that is associated with both hematopoietic cells and microparticles. We investigated: (i) whether cell-associated or microparticle-associated TF contributes to thrombin generation in SCD; and (ii) whether any of the SCD-related pathobiologic processes such as hemolysis, inflammation and/or endothelial activation contribute to hemostatic perturbations. The study cohort included 40 subjects with SCD in steady state (ages 2 to 21 years), 25 with SS and 15 with the SC genotype. Whole blood TF (WBTF) and microparticle-associated TF (MPTF) were measured using a two-stage functional clotting assay. Besides the early stage markers (WBTF and MPTF), we also assessed end-stage markers of thrombin generation and fibrin formation [thrombin-antithrombin (TAT) complexes, and D-dimer]. Surrogate biomarkers of hemolysis [lactate dehydrogenase (LDH), reticulocyte count, hemoglobin, and % phosphatidylserine-positive-RBCs]; inflammation [high sensitivity C-reactive protein (hs-CRP), white blood cell (WBC), neutrophil and monocyte counts]; and endothelial activation (soluble VCAM-1, E-selectin, and P-selectin) were assessed. Data were analyzed using Spearman correlation and multiple regression models. While correlations were observed in univariate analyses between WBTF and markers of hemolysis, inflammation, and endothelial activation (p

Description: Introduction: Type-2 phosphatidylserine (PS)-positive red cells are a subpopulation of erythrocytes that are highly positive for PS, contain low levels of fetal hemoglobin, are specific for sickle cell disease (SCD) and have been identified in the dense red cell fraction. Studies have implicated PS-positive red cells in enhancing anemia due to phagocytosis and hemolysis. Shielding of red cell PS by diannexin, a synthetic homodimer of human annexin-V, has been demonstrated to provide protection against hemolysis and prevent activation of prothrombinase. Methods: Using flow cytometry, we measured the levels of type-1 (red cells with low PS positivity) and type-2 PS-positive red cells in 50 children with SCD (31 with HbSS and 19 with HbSC), and assessed their association with various markers of hemolysis and hemostatic activation. Markers of hemolysis evaluated included plasma lactate dehydrogenase (LDH), reticulocyte count, and hemoglobin. Whole blood tissue factor (WBTF), pro-thrombin fragment F1+2, and D-dimer were evaluated as markers of hemostatic activation. Results: We demonstrate that the levels of type-2 PS-positive red cells are significantly increased in HbSS patients (1.37 ± 0.97%, p

Description: To determine whether fetal hemoglobin (HbF) protects against activation of hemostasis, endotheliium, and circulating blood elements, infants, children and young adults with homozygous SS disease (n=35) were studied. F-cell numbers, biomarkers of endothelial activation (including sVCAM-1, sE- and sP-selectin), white cell and platelet activation (sL-selectin and sP-selectin respectively) and markers of hemostatic activation, including D-Dimer and prothrombin fragment F1.2 were analyzed. Subjects with HbSS demonstrated significant increases (P