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Magnesium, a vasodilator, anti-inflammatory, and pain reliever, could alter the pathophysiology of sickle cell pain crises. We hypothesized that intravenous magnesium would shorten length of stay, decrease opioid use, and improve health-related quality of life (HRQL) for pediatric patients hospitalized with sickle cell pain crises. The Magnesium for Children in Crisis (MAGiC) study was a randomized, double-blind, placebo-controlled trial of intravenous magnesium vs normal saline placebo conducted at 8 sites within the Pediatric Emergency Care Applied Research Network (PECARN). Children 4 to 21 years old with hemoglobin SS or Sβ(0) thalassemia requiring hospitalization for pain were eligible. Children received 40 mg/kg of magnesium or placebo every 8 hours for up to 6 doses plus standard therapy. The primary outcome was length of stay in hours from the time of first study drug infusion, compared using a Van Elteren test. Secondary outcomes included opioid use and HRQL. Of 208 children enrolled, 204 received the study drug (101 magnesium, 103 placebo). Between-group demographics and prerandomization treatment were similar. The median interquartile range (IQR) length of stay was 56.0 (27.0-109.0) hours for magnesium vs 47.0 (24.0-99.0) hours for placebo (P = .24). Magnesium patients received 1.46 mg/kg morphine equivalents vs 1.28 mg/kg for placebo (P = .12). Changes in HRQL before discharge and 1 week after discharge were similar (P > .05 for all comparisons). The addition of intravenous magnesium did not shorten length of stay, reduce opioid use, or improve quality of life in children hospitalized for sickle cell pain crisis. This trial was registered at www.clinicaltrials.gov as #NCT01197417.

Introduction: Pediatric and young adult oncology patients treated with intense chemotherapy and stem cell transplant regimens have a high incidence of transfusional iron overload.(Acquazzino and Schloemer et al, ASH 2015). Iron deposition can lead to significant complications including heart failure, arrhythmias, liver abnormalities, endocrine dysfunction and ineffective erythropoiesis, as well as increased cancer and mortality risk. However there is a paucity of data regarding recommendations for management of transfusionial iron overload in childhood cancer survivors. Consequently, long-term complications of transfusional iron overload specific to pediatric cancer survivors have not been assessed. We have instituted screening and phlebotomy based treatment algorithms for pediatric and young adult oncology patients with transfusional iron overload. Methods: We conducted a retrospective chart review of pediatric and young adults who after having completed oncology management, were diagnosed with transfusional iron overload through a screening algorithm, and then initiated phlebotomy treatment. Tiered screening occurred in patients who received at least 5 packed red blood cell (pRBC) transfusions. Patients were recommended for further evaluation and discussion of possible phlebotomy treatment if they had: (1) liver iron concentration (LIC) greater than 5 mg of iron/gram dry weight liver tissue as measured by ferriscan and/or (2) cardiac MRI T2* < 20 ms. Management of phlebotomy treatment was conducted according to Table 1. During phlebotomy patient iron status was assessed at least quarterly and phlebotomy discontinued with achievement of LIC Results: Patient Demographics: We identified 25 pediatric and young adult childhood cancer survivors who underwent phlebotomy for transfusional iron overload. The mean age was 11.6 years (SD 6.1) and 10 (40%) were female. Oncologic diagnoses included ALL (36%), AML (8%), NHL (12%), Ewing sarcoma (16%), Osteosarcoma (4%), Neuroblastoma (12%) and CNS malignancies (12%). Transfusions/Phlebotomy/Diagnostic Tests: (Table 2) Patients received a median of 25.0 (IQR 17 - 34) pRBC transfusions. No patient had fewer than 10 transfusions. Median number of phlebotomy sessions was 6 (IQR 4-8) occurring over a median period of 0.36 years (IQR 0.28 - 0.59). Prior to phlebotomy, the median LIC was 7.5 mg/g (IQR 5.6-9.0) and median ferritin was 1110.0 ng/mL (IQR 700 - 2030). No patients demonstrated cardiac transfusional iron overload on T2* MRI (n=18). 23 (92%) patients have completed phlebotomy. One patient discontinued phlebotomy due to inability to safely obtain vascular access and no patients developed iron deficiency anemia. LIC was reduced by a median of 2.4 mg/g (IQR 1.1 - 3.6) and ferritin was reduced by median of 586 ng/mL (IQR 366-875). Correlation between number of transfusions received and phlebotomy sessions administered was poor (R2=0.017). Conclusions: Management guidelines are absent for transfusional iron overload in pediatric and young adult survivors of cancer. We demonstrate a phlebotomy algorithm that is an effective and well tolerated treatment for pediatric and young adult oncology patients with therapy related transfusional iron overload. Correlation between number of transfusions received and phlebotomy treatments required to remove deposited iron was poor necessitating serial assessments of iron status. Using this management algorithm, prospective studies can evaluate the effect of excess iron removal on iron overload complications in pediatric and young adult cancer survivors. Disclosures No relevant conflicts of interest to declare.

Genetic differences in endothelial biology could underlie development of phenotypic heterogeneity among persons afflicted with vascular diseases. We obtained blood outgrowth endothelial cells from 20 subjects with sickle cell anemia (age, 4-19 years) shown to be either at-risk (n = 11) or not-at-risk (n = 9) for ischemic stroke because of, respectively, having or not having occlusive disease at the circle of Willis. Gene expression profiling identified no significant single gene differences between the 2 groups, as expected. However, analysis of Biological Systems Scores, using gene sets that were predetermined to survey each of 9 biologic systems, showed that only changes in inflammation signaling are characteristic of the at-risk subjects, as supported by multiple statistical approaches. Correspondingly, subsequent biologic testing showed significantly exaggerated RelA activation on the part of blood outgrowth endothelial cells from the at-risk subjects in response to stimulation with interleukin-1β/tumor necrosis factorα. We conclude that the pathobiology of circle of Willis disease in the child with sickle cell anemia predominantly involves inflammation biology, which could reflect differences in genetically determined endothelial biology that account for differing host responses to inflammation.

Sickle red blood cells (SS-RBCs) have enhanced adhesion to the plasma and subendothelial matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. TSP has at least four domains that mediate cell adhesion. The goal of this study was to map the site(s) on TSP that binds SS-RBCs. Purified TSP proteolytic fragments containing either the N-terminal heparin-binding domain, or the type 1, 2, or 3 repeats, failed to sustain SS-RBC adhesion (

von Willebrand disease (vWD) is a common, autosomally inherited, bleeding disorder caused by quantitative and/or qualitative deficiency of von Willebrand factor (vWF). We describe two families with a variant form of vWD where affected members of both families have borderline or low vWF antigen levels, normal vWF multimer patterns, disproportionately low ristocetin cofactor activity, and significant bleeding symptoms. Whereas ristocetin-induced binding of plasma vWF from affected members of both families to fixed platelets was reduced, botrocetin-induced platelet binding was normal. The sequencing of genomic DNA identified unique missense mutations in each family in the vWF exon 28. In Family A, a missense mutation at nucleotide 4105T → A resulted in a Phe606Ile amino acid substitution (F606I) and in Family B, a missense mutation at nucleotide 4273A → T resulted in an Ile662Phe amino acid substitution (I662F). Both mutations are within the large disulfide loop between Cys509 and Cys695 in the A1 domain that mediates vWF interaction with platelet glycoprotein Ib. Expression of recombinant vWF containing either F606I or I662F mutations resulted in mutant recombinant vWF with decreased ristocetin-induced platelet binding, but normal multimer structure, botrocetin-induced platelet binding, collagen binding, and binding to the conformation-sensitive monoclonal antibody, AvW-3. Both mutations are phenotypically distinct from the previously reported variant type 2MMilwaukee-1 because of the presence of normal botrocetin-induced platelet binding, collagen binding, and AvW-3 binding, as well as the greater frequency and intensity of clinical bleeding. When the reported type 2M mutations are mapped on the predicted three-dimensional structure of the A1 loop of vWF, the mutations cluster in one region that is distinct from the region in which the type 2B mutations cluster.

The etiology of stroke in sickle cell disease is unclear, but may involve abnormal red blood cell (RBC) adhesion to the vascular endothelium and altered vasomotor tone regulation. Therefore, we examined both the adhesion of sickle (SS)-RBCs to cerebral microvessels and the effect of SS-RBCs on cerebral blood flow when the nitric oxide (NO) pathway was inhibited. The effect of SS-RBCs was studied in the rat cerebral microcirculation using either a cranial window for direct visualization of infused RBCs or laser Doppler flowmetry (LDF ) to measure RBC flow. When fluorescently labeled human RBCs were infused into rats, SS-RBCs had increased adhesion to rat cerebral microvessels compared with control AA-RBCs (P = .01). Next, washed SS-RBCs or AA-RBCs were infused into rats prepared with LDF probes after pretreatment (40 mg/kg intravenously) with the NO synthase inhibitor, N-ω-nitro-L-arginine methyl ester (L-NAME), or the control isomer, D-NAME. In 9 rats treated with systemic L-NAME and SS-RBCs, 5 of 9 experienced a significant decrease in LDF and died within 30 minutes after the RBC infusion (P = .0012). In contrast, all control groups completed the experiment with stable LDF and hemodynamics. Four rats received a localized superfusion of L-NAME (1 mmol/L) through the cranial window followed by infusion of SS-RBCs. Total cessation of flow in all observed cerebral microvessels occurred in 3 of 4 rats within 15 minutes after infusion of SS-RBCs. We conclude that the NO pathway is critical in maintaining cerebral blood flow in the presence of SS-RBCs in this rat model. In addition, the enhanced adhesion of SS-RBCs to rat brain microvessels may contribute to cerebral vaso-occlusion either directly, by disrupting blood flow, or indirectly, by disturbing the vascular endothelium.

Introduction: Pediatric and young adult oncology patients often require supportive red blood cell transfusions throughout their therapy course, which may result in iron overload. Despite this, screening recommendations to identify transfusional iron overload in childhood cancer survivors are lacking. Iron deposition can lead to end-organ damage and complications including heart failure, arrhythmias, liver abnormalities, endocrine dysfunction, ineffective erythropoiesis, and increased cancer and mortality risk. Little is known about long-term complications of iron overload in pediatric cancer survivors and treatment guidelines for iron overload in this patient population have not been developed. We instituted an algorithm to identify pediatric and young adult oncology patients with iron overload and refer them for treatment. Methods: This quality improvement project identified pediatric and young adult oncology patients for screening of transfusional iron overload, recommended diagnostic testing, and referred those with iron overload (as defined by liver T2* Results: Thirty patients were screened for iron overload. The mean age was 10.3 years (SD 7.5), 12 (40%) were female and 17 (57%) had a diagnosis of leukemia or lymphoma. The median number of transfusions received was 15 (10-47) and the median volume of blood received was 129 mL/kg (33-572). Thirty-six pediatric and young adult oncology patients were identified for iron overload screening, of whom only 2 (6%) had already received appropriate screening, 4 (11%) had received partial screening and 30 (83%) had received no screening. After the initial 6 months, 30 of the 34 patients who had undergone no or partial screening completed the recommended algorithm (88% compliance, see Figure 1). Nineteen (95%) of 20 patients with positive screening labs had iron overload by MRI. Fourteen (74%) of 19 patients with iron overload were seen by Hematology and 9/14 (64%) started treatment for iron overload (8-phlebotomy and 1-oral chelation). Nineteen patients (63%) were diagnosed with iron overload. The mean age was 11.8 years (SD 7.5), 6 (32%) were female and 12 (63%) had leukemia or lymphoma. The median number of transfusions received was 18 (10-47) and the median volume of blood received was 139 mL/kg (33-572). The median ferritin was 942 ng/mL (316-2340), median liver T2* was 6.6 (2.6-12.9) and median cardiac T2* was 26.7 (19.6-36). Number of transfusions (r=-0.79, p Conclusion: Targeted screening of pediatric and young adult oncology patients identified a high percentage of transfusional iron overload. Provider recognition of the need for iron overload screening in this high risk group was poor prior to institution of this quality improvement initiative. Prospective studies are required to determine if identification and subsequent treatment of transfusional iron overload can alter long-term complications of iron deposition and decrease morbidity of oncology treatment in pediatric and young adult survivors. Disclosures No relevant conflicts of interest to declare.

There is currently no standardized approach to emergency department (ED) and inpatient treatment of an acute pain event for patients with sickle cell disease (SCD). In addition, how the initial treatment of a pain event influences the outcome of that pain event is not well described. In surgical patients, aggressive pre-operative and peri-operative pain management is associated with improved pain outcomes post-operatively. Furthermore, in post-operative patients, using oral opioids alone or in combination with intravenous (IV) opioids is shown to provide better pain control, decreased IV opioid consumption, less side effects and higher patient satisfaction. In SCD, it is not known whether more aggressive opioid treatment at presentation to ED care or earlier initiation of oral opioids after hospitalization is associated with improved outcomes. Thus, we sought to determine the impact of the following treatment-related aspects of acute pain events on length of hospital stay: 1) total opioid dose at presentation, 2) time to initiation of first IV opioid at presentation, and 3) time to initiation of first oral opioid after hospitalization. We hypothesized that increased total initial dose of opioids, more rapid initiation of IV opioids in the ED, and earlier initiation of oral opioids after hospitalization would be associated with shorter length of hospital stay. We conducted a secondary analysis of data from the randomized controlled Magnesium for children in Crisis (MAGiC) trial. The MAGiC trial was a double-blind randomized controlled trial of IV magnesium versus saline placebo added to standard treatment for pediatric pain events. All patients had HbSS or HbSβ0 thalassemia to be eligible. The primary outcome, length of stay, was a composite endpoint of actual discharge or 12 hours after administration of last IV opioid, whichever came first. Patients were defined as having severe disease if a history of acute chest syndrome and/or 3 or more pain hospitalizations in the prior 3 years existed. A priori, all pain events were considered severe as the need for hospitalization indicated failure of outpatient therapy. Our primary variables of interest were: 1) Total initial opioid dose: total mg/kg/hr of morphine equivalents administered between first opioid in ED through start of study drug, 2) Time to first IV opioid: total hours between ED arrival and first IV opioid, 3) Time to first oral opioid: total hours between ED arrival and start of first oral opioid after hospitalization. Spearman correlations were used to determine the association between the above variables and length of stay and between length of stay and rehospitalization within 7 days to determine the consequence of earlier discharge. A total of 204 patients were enrolled at 8 sites. Mean (SD) age was 13.6 (4.7) years, 51.5% were female, 88.2% had severe disease and 60.3% were taking hydroxyurea. The primary MAGiC trial analysis found IV magnesium had no impact on length of stay. The mean total initial dose of opioid was 0.043 (0.029) mg/kg/hr that was administered over a median of 8.5 (IQR 6.4-13.1) hours. Higher total initial opioid dose was correlated with shorter length of stay (r=0.34, p Earlier initiation of oral opioids was strongly associated with shorter length of stay without associated increased rehospitalizations. Higher doses of opioids during the initial management of a pain event also contributed to shorter length of stay, however, more rapid initiation of IV opioids did not alter length of stay. These data suggest that higher initial doses of opioids in the ED and earlier introduction of oral opioids after hospitalization may improve outcomes of pain events. Prospective trials designed to evaluate the impact of initial ED care on outcomes of hospitalizations for pain events and trials aimed at standardizing inpatient treatment for pain events are needed and could ultimately lead to improved outcomes for pain in children with SCD. Disclosures Brandow: NIH, ASH: Research Funding. Panepinto:NKT Therapeutics, Inc: Consultancy; HRSA, NIH: Research Funding. Brousseau:NIH: Research Funding.

While there is strong evidence that chronic red cell transfusion is effective in preventing primary stroke and reducing the risk of recurrent stroke in sickle cell disease (SCD), it is less clear whether chronic transfusions will prevent admissions for other acute vaso-occlusive complications, including pain, priapism and/or acute chest syndrome. To our knowledge, no study to date has investigated the effect of chronic transfusion on the frequency of admissions for acute vaso-occlusive complications in children with all diagnoses of SCD and treated with chronic transfusion for a variety of indications. In addition, this study included a special focus on the effect of chronic transfusion on children who were transfused specifically for recurrent vaso-occlusive episodes. We performed a single-site retrospective chart review. We selected subjects from all children aged 0 to 19 years who were treated (lived in the Milwaukee area) and followed by the Wisconsin Sickle Cell Center at Children’s Hospital of Wisconsin from 1984 to May 2014 (n=695 subjects). Data was extracted from any individual who was enrolled in a chronic transfusion program for a minimum of six months. Data on admissions for painful vaso-occlusive crises, acute chest syndrome (ACS), other SCD complications as well as sickle diagnosis, age at time of transfusion, CBC, reticulocyte count, and percent sickle hemoglobin (HbS%) were collected for 24 months prior to onset of transfusion and for all months during transfusion until the age of 19 yrs. Unless otherwise indicated, all statistical analyses on extracted data were done by paired Student’s t-Test. We extracted data from 103 unique subjects for 108 chronic transfusion programs (as defined above); 5 subjects were chronically transfused twice, separated by at least 4 years without chronic transfusion. 55% were female; average age was 8.6 ± 5.6 (mean ± SD) years and the sickle diagnosis included 94% SS, 3% SC, 2% Sβ°-Thalassemia and 1% SD. The indication for transfusion included pain (n=31), priapism (n=6), ACS (n=5), central nervous system complications (n=37, including stroke, TIA, and abnormal TCD), splenic sequestration (n=25), pulmonary hypertension (n=2), retinopathy (n=1) and osteomyelitis (n=1). The hemoglobin level increased from a baseline of 7.6 ± 2.2 gm/dL to 9.6 ± 0.8 gm/dL during transfusion (p Subanalyses were performed on specific indications for transfusion. For children transfused due to frequent acute vaso-occlusive complications (pain, priapism and ACS were arbitrarily included in this group), the average age at initiation of transfusion was 11.9 ± 4.4 yr, and admissions for acute painful episodes dropped from 4.0 ± 3.2 admits/yr during the 24 months pre-transfusion to 2.1 ± 2.6 admits/yr during transfusion (p=0.003). When the indication for transfusion was splenic sequestration (age 2.3±2.7 yr), the admission rate for acute painful episodes did not change (0.8±1.7 vs 0.3±0.5 admits/yr, p= 0.14). For children transfused for CNS complications (age 8.5±4.6 yr), the admission rate for pain improved from 0.9±1.3 to 0.2±0.5 admissions/yr (p=0.007). In agreement with previous studies, our data also showed an increase in the rate of admissions for pain (nontransfused) as subjects aged (r2=0.19, p In summary, our data suggest that chronic transfusion reduces hospital admissions for pain and acute chest syndrome in children with SCD. Our data also support the notion that chronic transfusion is an effective treatment to prevent not only stroke, but also other painful, life-threatening and life-limiting complications of sickle cell disease. Disclosures No relevant conflicts of interest to declare.

Introduction: There are approximately 18,000 hospitalizations and 75,000 hospitalization days annually in the United States for children experiencing sickle cell vasoocclusive crises. Despite advances in the management of other comorbidities of sickle cell disease, little has changed in the management of sickle cell pain crises. Magnesium, a known vasodilator, anti-inflammatory and pain reliever, has the potential to alter the pathophysiology of pain crises, shortening length of stay and decreasing opioid use. A previous pilot study showed IV magnesium shortened length of stay compared to historical controls. A randomized trial conducted in Canada showed no decrease in length of stay with the use of intravenous magnesium. In the MAGiC (MAGnesium for children in Crisis) study, we hypothesized that the addition of intravenous (IV) magnesium to standard therapy would shorten hospital length of stay, result in decreased opioid use and improve quality of life for pediatric patients hospitalized with sickle cell pain crises. Methods: The MAGiC study was a multi-center, randomized, double-blind, placebo-controlled trial of IV magnesium versus normal saline for the treatment of pediatric sickle cell pain crisis conducted at 8 sites. Participating sites were members of the Pediatric Emergency Care Applied Research Network (PECARN), and collaborations between Pediatric Emergency Medicine physicians and Pediatric sickle cell experts facilitated enrollment. Children aged 4 to 21 years, with hemoglobin SS or hemoglobin SB° thalassemia were eligible if they required inpatient hospitalization after failing emergency department (ED) management for pain. Enrollment occurred at 8 sites between December 2010 and December 2013, with a total of 217 eligible site enrollment months. Children received 40 mg/kg of IV magnesium every eight hours for a total of 6 doses or normal saline placebo of equivalent volume (1 ml/kg). Randomization was stratified by site, age and hydroxyurea use. The primary outcome was length of stay from the time of first drug infusion until 12 hours after the last IV opioid dose or time of discharge, whichever occurred first. Secondary outcomes included opioid use, recorded as morphine equivalents, and quality of life, as measured using the PedsQL Sickle cell disease specific module, fatigue module and generic module. Side effects, specifically hypotension, weakness, warmth on infusion, or the development of acute chest syndrome (ACS) were documented. Using an intention-to-treat analysis, we compared length of stay using a Van Elteren test, stratified by the same factors used to stratify randomization. Results: 208 children were enrolled. Four children were excluded prior to receipt of any study drug, resulting in 101 children receiving magnesium and 103 receiving placebo. The 2 groups were similar with respect to age, sex ,genotype, weight, history of ACS or asthma, previous hospitalizations within the past three years and days of pain prior to arrival. The median time from first ED opioid to first study drug infusion was 7.4 hours, similar between the two groups. The median (interquartile range) length of stay was 56.0 (27.0 - 109.0) hours in the magnesium group compared to 47.0 (24.0 - 99.0) hours in the placebo group, p = 0.264. Patients who received magnesium received 1.46 mg/kg of morphine equivalents compared to 1.28 mg/kg in the placebo group (p=0.11). Quality of life scores were similar between the two groups after 48 hours on study drug and one week after discharge (p > 0.10 at both time points). Safety analysis revealed no differences in hypotension (3% for magnesium versus 1% for placebo) or weakness (7% versus 4%) between the 2 groups. Of those who received magnesium, 26% reported warmth on infusion compared to 2% of children who received placebo, p < .0001. Other adverse events, serious adverse events, and rehospitalizations within 7 days were similar between the groups. Conclusion: Intravenous magnesium does not shorten length of stay, lessen opioid use or improve quality of life in children who require hospitalization for sickle cell pain crisis. Close collaboration between Pediatric Emergency Medicine physicians and Pediatric Hematologists allows for the successful enrollment of large numbers of children in an acute intervention trial for children with sickle cell disease. Disclosures Off Label Use: magnesium for sickle cell pain crisis.

The long-term efficacy and toxicity of hydroxyurea for infants are undefined, and its role in preventing organ dysfunction is unknown. Short-term feasibility of hydroxyurea administration, toxicities, hematologic effects, and effect on spleen function in infants with sickle cell anemia (SCA) were reported (Hydroxyurea Safety and Organ Toxicity [HUSOFT] trial). These infants completing 2 years of hydroxyurea therapy (20 mg/kg/d) were offered study extension with dose escalation to 30 mg/kg/d. Patients were monitored with laboratory tests and biannual imaging studies. Hematologic indices were compared with predicted age-specific values and event rates compared with historic rates. All 21 subjects completing the original trial enrolled in the extension study: median age, 3.4 years old (range, 2.6 to 4.4 years); 12 females; 20 with Hb SS, 1 with Hb S/β0-thalassemia. Seventeen patients completed 4 years of hydroxyurea, and 11 completed 6 years. After 4 years, hydroxyurea was associated with increased hemoglobin concentration, percentage of fetal hemoglobin (Hb F), and mean corpuscular volume (MCV) and decreased reticulocytes, white blood cells (WBCs), and platelets (P < .01). Patients experienced 7.5 acute chest syndrome (ACS) events per 100 person-years, compared with 24.5 events per 100 person-years among historic controls (P = .001). Treated patients had better spleen function than expected and improved growth rates. Infants with SCA tolerate prolonged hydroxyurea therapy with sustained hematologic benefits, fewer ACS events, improved growth, and possibly preserved organ function.

The enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium and subendothelial matrix likely plays a significant role in the pathogenesis of vaso-occlusion in sickle cell disease. Sickle RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In this study, we sought to develop antibodies that bind TSP from a highly diverse library of human single-chain Fv fragments (scFvs) displayed on filamentous phage. Following 3 rounds of phage selection of increasing stringency 6 unique scFvs that bound purified TSP by enzyme-linked immunosorbent assay were isolated. Using an in vitro flow adhesion assay, 3 of the 6 isolated scFvs inhibited the adhesion of sickle RBCs to immobilized TSP by more than 40% compared with control scFvs (P < .001). Furthermore, scFv TSP-A10 partially inhibited sickle RBC adhesion to activated endothelial cells (P < .005). Using TSP proteolytic fragments to map the binding site, we showed that 2 of the inhibitory scFvs bound an epitope in the calcium-binding domain or proximal cell-binding domain of TSP, providing evidence for the role of these domains in the adhesion of sickle RBCs to TSP. In summary, we have isolated a panel of scFvs that specifically bind to TSP and differentially inhibit sickle RBC adhesion to surface-bound TSP under flow conditions. These scFvs will be useful reagents for investigating the role of the calcium and cell-binding domains of TSP in sickle RBC adhesion.

Von Willebrand disease is a bleeding disorder with reduced or abnormal function of VWF. While VWF has at least three functions (binding to FVIII, platelet GPIb, and collagen), the function of VWF has been quantified primarily using techniques in which ristocetin promotes the binding of VWF to platelet GPIb. It is recognized that the physiologic agonist for VWF-platelet interactions is shear but ristocetin facilitates a change in VWF structure to induce binding to platelet GPIb. For most individuals, this assessment of VWF function is valid, but over the past several years our group, the Zimmerman Program for the Molecular and Clinical Biology of VWD, has identified “polymorphisms” that do not affect VWF function but interfere with ristocetin binding to VWF in those laboratory tests of VWF function that utilize ristocetin. The two “polymorphisms” are P1467S and D1472H. P1467S is a rare sequence variant not seen in 12,900 alleles in the 1000-genome project. In contrast, the D1472H is a polymorphism seen in 63% of African Americans and in 17% of Caucasians. Since these two sequence variations are physically located in close proximity just C-terminal from the A1 loop, we initiated a systematic study of the adjacent N- and C-terminal sides of the A1 domain by alanine scanning mutagenesis. We concentrated on the sequence between p.C1237 – p.C1272 and p.C1458 – p.K1491 and replaced each amino acid with an alanine. These sequences were then transferred into a full-length expression construct. The individual altered VWF proteins were studied by GPIb-binding in two solid phase assays. The first assay, VWF:RCo, is an ELISA using an antibody-captured recombinant GPIb-alpha in which ristocetin-induced bound VWF is detected using an anti-VWF monoclonal antibody. The second assay, VWF:IbCo, is a similar assay, but the expressed GPIb contained two mutations, D235Y and M239V, that confer spontaneous binding to VWF without the addition of ristocetin as our laboratory has previously published. Where possible all assays were done at similar concentrations of VWF antigen and expressed as a ratio of VWF:IbCo/VWF:RCo compared to VWF:Ag concentration. An increased ratio indicates a reduction in ristocetin-induced binding. A pronounced difference was identified at several amino acids between p.D1459 and p.P1471. Interestingly, the D1472H polymorphism was not abnormal by this approach (D1472A) – thereby indicating that alanine substitution for the aspartic acid does not confer the same conformation that the histidine does in African Americans. Although earlier studies by Mohri and coworkers (JBC 1988) suggest a second binding site, only a minimal signal was identified for the region between p.C1237 –p.P1251. Hot spots were identified with VWF:IbCo/VWF:RCo scores 7- to 30-fold higher than background. To date, natural sequence variations have not yet been identified in these positions. Studies by Scott and coworkers (JBC 1991), suggested that ristocetin binding to proteins was proportional to the presence of X-P-G-X’ beta-turns. The region of VWF that we are studying contains one of these sequences and is associated with a major effect on the VWF:IbCo/VWF:RCo signal. Since ristocetin dimerizes at a concentration of 1 mg/ml and the dose used in VWF:RCo is usually 1 mg/ml, these studies support the concept that alterations in ristocetin binding brought about by sequence variations may give the false impression that VWF function is profoundly affected when it is only the in vitro assay that is abnormal. Alanine scanning mutagenesis provides further substantiation of the role of the VWF-A1-loop in the binding of ristocetin to VWF. “Potential” Type 2M VWD subjects with sequence variations in this region may not have VWF functional abnormalities. Therefore, such individuals may require additional studies – particularly if bleeding symptoms are not identified in the presence of significantly abnormal laboratory testing. Disclosures: No relevant conflicts of interest to declare.

Abstract 1635 There is evidence for increased levels of procoagulant proteins, thrombin generation and tissue factor activity in individuals with sickle cell disease. Both thrombin and tissue factor also have potent proinflammatory effects that likely further contribute to the vascular dysfunction and organ injury observed in sickle cell disease. In this study, we examine the effect of the LMW heparin, enoxaparin, on markers of vascular injury and inflammation in the Berkeley mouse model of severe sickle cell disease (HbSS mice). HbSS mice or control HbAA mice (that exclusively express normal human HbA) were treated with continuous subcutaneous infusion of enoxaparin (10 mg/kg/day) or saline for 14 days using a surgically implanted Alzet pump (n=12-14 mice per group). Blood was collected at baseline and following 14 days enoxaparin therapy. Anesthetized animals were perfused with PBS at constant physiologic pressure (80 mmg Hg) and lung and liver collected for pathologic analyses. Enoxaparin-treated mice had steady state LMW heparin levels of 0.45 ± 0.17 anti-Xa U/mL compared to Disclosures: Field: Novartis: Honoraria.

Abstract 2208 Diagnosis of von Willebrand disease (VWD) currently relies on two assays of von Willebrand factor (VWF), the VWF antigen ELISA (VWF:Ag) and the VWF ristocetin cofactor activity assay (VWF:RCo). The latter exploits the capacity of ristocetin to induce VWF – platelet interactions as a measure of VWF function. Ristocetin, however, is a non-physiologic agonist as shear stress is the physiologic stimulus inducing VWF to bind platelets in vivo. Recently we have reported that the VWF:RCo/VWF:Ag ratio is decreased in individuals with an A1 domain polymorphism, D1472H. The lack of bleeding in subjects with this polymorphism suggests D1472H does not create a physiologic defect in VWF – platelet interactions. D1472H is directly adjacent to a known ristocetin-binding area in the VWF A1 region (Leu 1457 – Pro 1471), supporting the hypothesis that D1472H affects the ability of ristocetin to bind VWF. Similarly, a heterozygous sequence change leading to P1467S (located in the same ristocetin binding domain) resulted in an undetectable VWF:RCo but no bleeding symptoms were noted in affected subjects. To further investigate the cause of this observation, we developed a method to study the binding of ristocetin to VWF directly. Maleic anhydride microtiter plates were used to capture ristocetin via its amine groups. A VWF source, either plasma or recombinant VWF (rVWF), was then added, wells washed, and VWF binding detected using anti-VWF antibodies. Both plasma and rVWF bound to the captured ristocetin similarly with ristocetin plating concentrations ranging from 0.01 to 1 mg/ml. Specificity of ristocetin dependent VWF binding was confirmed, as preincubation of ristocetin with rVWF decreased binding of rVWF to immobilized solid-phase ristocetin. No detectable binding was present for a full length rVWF construct with the entire A1 loop deleted (del 1242–1478) or a construct missing part of the A1 loop (del 1392–1402). VWF binding to ristocetin was inhibited by both monoclonal and polyclonal antibodies directed against the VWF A1 loop. VWF A1 loop constructs with the A1 domain polymorphisms D1472H and P1467S showed decreased binding to ristocetin when compared to a wild-type A1 loop construct. Wild-type A1 loop binding to ristocetin in our assay was 0.98 while 1472H A1 loop binding was reduced at 0.77 (p Disclosures: Montgomery: GTI Diagnostics, Inc: Consultancy.

The coagulation system is activated in sickle cell disease (SCD) and acute vaso-occlusion may heighten hypercoagulability. Protein C, a natural anticoagulant, has been reported to be low in individuals with SCD. Therefore, the natural anticoagulation pathway may be disrupted in SCD. The objective of this study is to more fully evaluate the protein C pathway in murine and human SCD by examining levels of: coagulation activation; protein C activity; thrombomodulin (TM); and endothelial protein C receptor (EPCR). In order to assess the level of activation of the coagulation system, we measured plasma thrombin/antithrombin (TAT) complex levels in humans and mice. TAT levels were elevated in 22 humans with SCD versus 9 healthy controls at baseline, and levels increased further in 15 individuals with SCD during acute vaso-occlusive events (5.6±1.2 vs. 2.4±0.2 vs. 9.2±1.8ug/L respectively, p=0.02). In order to study acute vaso-occlusive events in mice, we developed a model of acute vaso-occlusion by exposing Berkeley SCD mice to 3 hours of hypoxia (FI02 8–10%) followed by 2, 4, or 21 hours of reoxygenation in room air (HR2, HR4, HR21). In support of our human findings, TAT was elevated in SCD mice compared to HbA mice at baseline, and increased further in SCD mice exposed to HR2 (n=5–14 per group, p TM is elevated in several chronic inflammatory diseases and acutely decreases in meningococcemia. We evaluated TM in mouse liver, an organ susceptible to vascular congestion, infarction, and inflammation in SCD mice. We first measured TM in mouse liver homogenates by ELISA. All SCD mice, at baseline and after HR, expressed elevated liver TM levels compared to HbA mice (n=6 per group, 1.7 to 2.9-fold increases in SCD livers, p

Stroke affects ∼10% of children with sickle cell anemia. There is evidence for a familial predisposition, and the strongest risk predictor is presence of Circle of Willis (CoW) disease. We hypothesize that inherited differences in endothelial biology influence whether or not the child with sickle cell anemia develops CoW disease. We have studied 20 subjects (age 200 cm/sec, or abnormal MRA); and 9 are not-at-risk, defined as having reached age 10 (at least) without a clinical stroke, plus having absence of CoW disease (TCD

Pulmonary hypertension affects approximately 30% of young adults with sickle cell disease (SCD) and is a risk factor for early death. There is increasing evidence that intravascular hemolysis contributes to the pathophysiology of pulmonary hypertension in SCD as well as other hemolytic disorders. In this study, we compare measures of vascular dysfunction in Berkeley sickle cell mice (SCD mice) with a murine model of hereditary spherocytosis (sph/sph mice) that exhibit severe hemolytic anemia due to alpha spectrin deficiency, but without HbS-induced RBC sickling. We assessed right ventricular systolic pressure in vivo as a measure of pulmonary arterial function and endothelial-dependent vasodilation of facialis arteries ex vivo as a measure of systemic arterial function. Right ventricular systolic pressures and right ventricle to body weight ratios were increased to similar levels in both SCD and sph/sph mice as compared to control mice (p

The brief report by Beullens et al[1][1] is misleading regarding the urine test that the World Anti-Doping Agency (WADA) uses to detect recombinant human erythropoietin (rhEpo). The WADA-recommended test is based on immunoelectorphoresis and double blotting (IEF/DB), and was developed by Lasne and

Children with sickle cell disease use different coping styles to control their pain. Those who use an affective coping style such as fear and anger self-statements or a passive coping style such as resting have more severe pain, higher utilization of health care services, and worse psychological adjustment. Those who use active coping styles such as diverting attention or praying and hoping engage in greater work and social activities. The association of coping style to the health-related quality of life (HRQL) in children with sickle cell disease is not known. Our study sought to determine the association of coping styles with HRQL in children with sickle cell disease. We hypothesized that children with sickle cell disease who used affective or passive coping styles would have worse HRQL. To determine the association between coping styles and HRQL, the Child Health Questionnaire-Child Form (CHQ-CF87) was completed by children with SCD age 10–18 years at the time of their annual comprehensive visit to 1 of 2 sickle cell disease clinics. Mean scores were calculated for each of the 12 concepts that make-up the CHQ-CF87. Scores range from 0, representing poor quality of life, to 100, representing a high quality of life. In addition, children also completed the Coping Strategies Questionnaire (CSQ) for sickle cell disease. This questionnaire assesses ways in which children cope with pain based on the child’s response on a 7-point Likert-type scale where 0 =never and 6=always. Mean scores were calculated for each of 3 types of coping styles: affective, passive adherence and active coping. Pearson correlation coefficients were calculated to determine the association of HRQL with coping style. Partial correlation coefficients, controlling for disease severity, were calculated for those relationships with significant associations. Forty-eight children completed both questionnaires for analysis. The mean age of the children was 13. 3 years (standard deviation 2.2) and 58% were female. Children who used an affective coping style had worse associated HRQL scores in the areas of mental health and self esteem (r of −0.38, p=0.010 and r of −0.35, p=0.015 respectively). Furthermore, children who used an affective coping style or passive coping style reported a greater frequency of disruption in their usual family activities or that their health more likely was a source of family tension (r of −0.39, p=0.006 and r of −0.31, p=0.037 respectively). These relationships persisted after controlling for disease severity. There was no significant association with other domains of HRQL and coping. In conclusion, children with sickle cell disease who report using affective and passive coping styles have worse mental health and self esteem and increased disruption of family activities. This maladaptive response to pain further impacts the well being of children with sickle cell disease and their families. In addition, these findings support the need to provide psychological intervention and therapy to improve children’s coping abilities to ultimately improve their HRQL.

Stroke affects ~10% of children with sickle cell anemia. There is evidence for a familial predisposition, and the strongest risk predictor is presence of Circle of Willis (CoW) disease. We hypothesize that inherited differences in endothelial biology influence whether or not the child with sickle cell anemia develops CoW disease. We have studied 20 subjects (ages 10–18) with HbSS or HbSβothal. Twelve are at-risk for stroke, defined as having had an actual stroke, or by having known CoW disease (TCD >200 cm/sec, or abnormal MRA); and 8 are not-at-risk, defined as having reached age 10 (at least) without a clinical stroke, plus having absence of CoW disease (TCD

The major cause of morbidity and mortality in sickle cell disease (SCD) is tissue ischemia and infarction due to vascular obstruction. One of the most devastating vascular complications of SCD is clinical stroke caused by occlusion of the large cerebral arteries in the Circle of Willis or its branches. Overt stroke occurs with obvious speech or motor deficits in approximately 10% of children with SCD. Additional complications due to stroke in SCD include an increased risk of secondary disabilities such as mental retardation and learning disabilities, with the most severe forms of neurocognitive impairments associated with large vessel disease (overt stroke). When compared to normal RBCs, sickle red blood cells (SS-RBCs) have markedly increased adhesion to the vascular endothelium and several subendothelial matrix proteins. The increased sickle RBC adhesion likely plays a significant role in the pathogenesis of vascular obstruction. We have previously shown that SS-RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In addition, we have observed that the level of adhesion of RBC adhesion varies from patient to patient (i.e., there are some patients whose RBCs tend to consistently be “stickier” or “less sticky” than other patients). In this study, we proposed that patients who develop large vessel (Circle of Willis and major branches) central nervous system (CNS) disease (e.g., clinical stroke) have higher baseline levels of RBC adhesion. Thus, we examined the level of sickle RBC adhesion to immobilized TSP under flow conditions from patients with SCD with and without stroke. We recruited 87 male and female patients over the age 3 yrs with HbSS disease from the Wisconsin Sickle Cell Disease Comprehensive Center. Of these, 8 patients later developed an overt stroke following their participation in this study. None of the patients were transfused prior to studying RBC adhesion. After obtaining informed consent, blood samples were collected into citrate from patients with HbSS disease. Washed RBCs were perfused through flow chambers previously coated with TSP (2 μg/cm2) at a wall shear stress of 1 dyne/cm2 that mimics the forces in post-capillary venules and optimizes sickle RBC adhesion. After rinsing, adherent RBCs per unit area were counted in four random fields in each of two duplicate wells by direct microscopic visualization. We found that patients with SCD who developed an overt stroke (n=8) had a higher level of RBC adhesion (1436 ±101 RBCs/mm2) compared to 79 unselected patients with SCD who had a mean RBC adhesion of 1004 ± 53 RBCs/mm2 (p=0.016, student t-Test). Thus, RBCs derived from the subpopulation of patients with SCD who developed an overt stroke associated with large vessel disease have an abnormally enhanced adhesive phenotype. These data suggest that enhanced levels of RBC adhesion may play a role in the development of large vessel disease in SCD.

The advent of safer treatment for patients with mild congenital bleeding disorders has led to increasing numbers of surgical procedures such as T & A, one of the most common surgical procedures in the pediatric age group. Recommendations for prophylaxis of surgical hemorrhagic complications vary significantly even among clinicians from the same institution. We examined the records of 700 patients with mild type 1 VWD and mild PFD seen in the Comprehensive Center for Bleeding Disorders from January 1995-December 2003. Seventy-three underwent adenotonsillectomy; 8 were excluded from the analysis for lack of follow-up and one was treated with Humate-P because of failure to respond to desmopressin. Of the 64 included in the analysis, 28 had mild type 1 VWD. Thirty-six had a mild PFD characterized by failure of platelets to release ATP (all 36 patients) in response to ADP and disaggregation following initial aggregation in 19. All 64 patients were given 0.3 mcg/kg desmopressin IV over 30 min. about 1 hr prior surgery; post-operatively, patients received additional desmopressin for 0–3 days and oral EACA for 0–14 days. Thirteen of the 64 (20%) patients had excessive bleeding in the peri-operative period (1 intra-operative and 12 post-operative); whereas published rates of bleeding in normal patients range from 1–4% of cases. Bleeding time and historical bleeding scores were not different between the bleeder and non-bleeder groups. Among VWD patients, there was no difference in baseline VWF:RCo levels or rise in VWF:RCo levels following a challenge dose of desmopressin in those who did not bleed compared to those who did. However, bleeding rates were significantly higher among patients with PFD; nine of 36 (25%) of patients with a PFD had a hemorrhagic complication compared to 4 of 28 (14%) of those with VWD (p

The sickle red blood cell (SS-RBC) membrane manifests many abnormal properties, including oxidative damage of membrane proteins and lipids, disruption of the lipid bilayer, abnormal clustering of surface proteins, dehydration, and increased adhesive properties. Conditions that alter the RBC membrane may modify or abnormally expose its native components and contribute to the adhesive properties of the RBC. We have previously reported that sickle RBCs have enhanced adhesion to the plasma and subendothelial matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. Since a significant proportion of sickle RBCs are inherently dehydrated, in part due to intracellular K+ loss via the Gardos and K-Cl co-transport channels, we test the hypothesis that dehydration-induced alterations in membrane organization or composition contribute to sickle cell adhesion in sickle cell disease (SCD). To examine the role of RBC hydration in adhesion to the subendothelial matrix protein thrombospondin-1 (TSP), normal and sickle RBCs were incubated in buffers of varying tonicity and tested for adhesion to immobilized TSP under flow conditions. Expanding on our previous preliminary results, we found that sickle RBCs exhibited a decrease in TSP binding with increasing cell hydration (p

A 9-yr-old female presented with a Wilm's tumor and a coagulopathy consistent with von Willebrand's disease. Factor VIII procoagulant activity (VIII C), factor VIII related antigen (VIIIR:Ag), and von Willebrand factor activity (VIII:vWf) were decreased. There was no evidence for a circulating inhibitor of the factor VIII molecular complex. von Willebrand's antigen II (vW AgII), which is deficient in hereditary von Willebrand's disease, was decreased below detectable levels in this patient. The coagulation studies, VIIIR:Ag, and vW AgII levels returned to normal following therapy of the Wilm's tumor. Wilm's tumor must be included as one of the malignancies associated with acquired von Willebrand's disease. Immunofluorescent studies of the tumor specimen showed normal endothelial staining of VIIIR:Ag by semiquantitative techniques and a lack of specific tumor adsorption of VIIIR:Ag The presence of normal amounts of tissue VIIIR:Ag has not previously been demonstrated in acquired von Willebrand's disease. Since we failed to demonstrate an inhibitor in the plasma in this patient, the etiology of the acquired von Willebrand's disease in this patient appears to differ from other cases of acquired von Willebrand's disease. The finding that vW AgII is decreased in this patient, similar to that reported in hereditary von Willebrand's disease, supports the close association of vW AgII to VIIIR:Ag, even though they are immunologically and biochemically distinct.

von Willebrand's antigen II (vW AgII), a plasma and platelet antigen immunologically and biochemically distinct from factor-VIII-related antigen (VIIIR:Ag), is decreased in von Willebrand's disease. vW AgII is released from platelets during aggregation and clotting. The release of platelet vW AgII was studied in washed platelets following aggregation by thrombin, collagen, and adenosine diphosphate (ADP). Total platelet vW AgII was 3.39 U/10(9) platelets. Thrombin and collagen yielded release of 85% and 86% of platelet vW AgII, respectively, at the highest concentrations. Release of platelet vW AgII was correlated with the release of 5-hydroxytryptamine (5HT). Release of vW AgII by collagen and thrombin was inhibited by metabolic inhibitors. In addition, collagen release of vW AgII was inhibited by aspirin. In clinical syndromes associated with intravascular platelet destruction, marked elevations of plasma vW AgII were noted. Thus, vW AgII is released by a metabolically active process from platelets during aggregation. In addition, vW AgII appears to be a marker of intravascular platelet destruction.