This study explores the potential of neural networks to detect the gender of a student given a student's score on the Carolina Automated Reading Evaluation (CARE), a collection of thirteen assessments of reading disabilities created by researchers at the University of South Carolina. Data from prior administrations of the CARE at two elementary schools in South Carolina was parsed and read into dictionaries that contained a randomly created ID number, the student's gender and grade level, and their scores on the individual assessments they were administered. A neural network was created in Python using prominent machine learning libraries, taking test scores as inputs and attempting to train gender as an output [16]. Since not every student was administered every assessment, only assessments that had been taken by more than 60% of the students were used in the neural network (see Fig. 1). These assessments were used to train the neural network to detect gender. After repeated attempts to train the network to this data set failed, a data set of similar size from the University of California, Irvine machine learning repository was used to test the network built to determine if there was a structural defect in its construction [15]. As shown in Figure 3, the constructed network was able to train to an appropriate degree on the data set from UC Irvine but despite repeated attempts was unable to train on the data with respect to gender from the CARE administrations. From this, it was interpreted that the CARE data follows similar patterns to previous studies of other tests of reading disabilities, which concluded that students in lower grades show insignificant differences in reading ability by gender.

Sickle cell disease (SCD) results from a point mutation in the β-globin gene forming hemoglobin S (HbS), which polymerizes in deoxygenated erythrocytes, triggering recurrent painful vaso-occlusive crises and chronic hemolytic anemia. Reactivation of fetal Hb (HbF) expression ameliorates these symptoms of SCD. Nuclear factor (erythroid derived-2)-like 2 (Nrf2) is a transcription factor that triggers cytoprotective and antioxidant pathways to limit oxidative damage and inflammation and increases HbF synthesis in CD34+ stem cell-derived erythroid progenitors. We investigated the ability of dimethyl fumarate (DMF), a small-molecule Nrf2 agonist, to activate γ-globin transcription and enhance HbF in tissue culture and in murine and primate models. DMF recruited Nrf2 to the γ-globin promoters and the locus control region of the β-globin locus in erythroleukemia cells, elevated HbF in SCD donor-derived erythroid progenitors, and reduced hypoxia-induced sickling. Chronic DMF administration in SCD mice induced HbF and increased Nrf2-dependent genes to detoxify heme and limit inflammation. This improved hematological parameters, reduced plasma-free Hb, and attenuated inflammatory markers. Chronic DMF administration to nonanemic primates increased γ-globin mRNA in BM and HbF protein in rbc. DMF represents a potential therapy for SCD to induce HbF and augment vasoprotection and heme detoxification.

Very Late Antigen-4 (VLA-4, α4β1-integrin, ITGA4) orchestrates cell-cell and cell-endothelium adhesion. Given the proposed role of VLA-4 in sickle cell disease (SCD) pathophysiology, we evaluated the ability of the VLA-4 blocking antibody natalizumab to inhibit SCD blood cell adhesion. Natalizumab recognized surface VLA-4 on leucocytes and reticulocytes in whole blood from SCD subjects. SCD reticulocytes were positive for VLA-4, while VLA-4 staining of non-SCD reticulocytes was undetectable. Titrations with natalizumab revealed the presence of saturable levels of VLA-4 on both SCD reticulocytes and leucocytes similar to healthy subject leucocytes. Under physiological flow conditions, the adhesion of SCD whole blood cells and isolated SCD leucocytes to immobilized vascular cell adhesion molecule 1 (VCAM-1) was blocked by natalizumab in a dose-dependent manner, which correlated with cell surface receptor binding. Natalizumab also inhibited >50% of whole blood cell binding to TNF-α activated human umbilical vein endothelial cell monolayers under physiological flow at clinically relevant concentrations (10 to 100 μg/ml). This indicates that VLA-4 is the dominant receptor that drives SCD reticulocyte and mononuclear cell adhesion to VCAM-1 and that the VLA-4 adhesion to VCAM-1 is a significant contributor to SCD blood cell adhesion to endothelium. Thus, VLA-4 blockade may be beneficial in sickle cell disease.

Sickle Cell Disease (SCD) is caused by a point mutation in the beta- chain of hemoglobin, triggering a complex pathophysiology resulting in recurrent, painful vaso-occlusive events (VOCs) and chronic hemolytic anemia. Induction of fetal hemoglobin (HbF), is a well-established approach towards the treatment and potentially cure for SCD. This is exemplified by the condition known as Hereditary Persistence of Fetal Hemoglobin (HPFH), which when present in SCD patients results in asymptomatic disease. Reactivation of fetal hemoglobin can be achieved by different methodologies both genetic manipulation and pharmacological agents. The mechanism of action of hydroxyurea, the only FDA approved drug for the treatment of SCD, is by activating HbF. We have investigated the role of Nrf2 in activating fetal hemoglobin and it potential to ameliorate the symptoms of SCD. Nrf2 is a basic leucine zipper transcription factor that is bound to a cytosolic protein Keap1, a Kelch domain containing protein, which targets the factor to the proteasome. Previous studies have shown that chemical activation of Nrf2 leads to induction of HbF. Here we show that siRNA based knockdown of Keap1 led to ~70 % reduction in Keap1 mRNA levels in Human Umbilical cord Derived Eryrthroid Progenitor cells (HUDEP), and concomitantly mediated a 2-fold induction of g-globin mRNA levels. Peripheral blood derived mononuclear cells differentiated into erythroid progenitors treated with bardoxolone-Me, a potent Nrf2 activator, resulted in a significant 4-fold induction in γ-globin mRNA, 2.5 fold induction in F-cells and up to 2.5-fold up-regulation in both Aγ and Gγ globin protein. Bardoxolone-Me treated erythroid progenitors also demonstrated a significant reduction in hypoxia-induced shape changes of sickle cells - about 42 % decrease at 100 nM and 55 % decrease at 250 nM. Bardoxolone-Me was also evaluated in a Townes SCD mouse model carrying an inducible γ-globin gene. A single oral dose of bardoxolone-Me at 30mg/kg, showed 9-fold induction in γ-globin mRNA levels at 6 h compared to untreated mice. Based on these findings, Nrf2 activation, can potentially be utilized for HbF induction in the treatment of SCD. Disclosures Krishnamoorthy:Biogen: Employment, Equity Ownership, Other: share holder. Gupta:Biogen: Employment, Equity Ownership, Other: share holder. Hobbs:Biogen: Employment, Equity Ownership, Other: shareholder. Loh:Biogen: Employment, Equity Ownership, Other: share holder. Light:Biogen: Employment, Equity Ownership, Other: share holder. Peters:Biogen: Employment, Equity Ownership, Other: share holder. Sturtevant:Arietis: Employment. Pace:Augusta University: Employment; Biogen: Research Funding. Nakamura:Riken Institute: Employment. Lucas:Biogen: Employment, Equity Ownership, Other: share holder. Vieira:Biogen: Employment, Equity Ownership, Other: share holder.

Background: Vascular complications such as stroke and pulmonary hypertension are central features of sickle cell disease (SCD) pathophysiology and are associated with early mortality among patients with SCD. Better understanding of the abnormal blood flow patterns in sickle cell disease is critical to assessing the therapeutic benefit of emerging therapies. Previous studies have shown abnormal blood flow patterns in sickle cell patients using laser speckle contrast imaging (LSCI) (Ikeda et al. poster 1080 ASH annual meeting, December 8, 2012), however, no test re-test variability or changes in response to therapeutic intervention were assessed. As part of a larger study using multiple imaging technologies to concurrently evaluate blood flow and oxygenation in several organs of healthy subjects and subjects with SCD, the present study has analyzed skin blood flow by LSCI in these patients longitudinally using a multiple visit protocol. Furthermore, we analyzed the effect of Chronic Transfusion Protocol, the most effective intervention against vascular complications on skin blood flow.

Sickle cell disease (SCD) is caused by a point mutation in the β-chain of hemoglobin, which triggers a complex pathophysiology resulting in recurrent, painful vaso-occlusive crises (VOC) and chronic hemolytic anemia. Fetal hemoglobin (HbF) is the major species of hemoglobin during fetal and neonatal development, the expression of which is replaced by the adult beta globin perinatally. Reactivation of HbF in adult SCD patients is considered beneficial in ameliorating the symptoms of the disease. This is exemplified by a condition known as hereditary persistence of fetal hemoglobin (HPFH) in heterozygous sickle disease patients where symptoms of SCD are absent and the typical HbF level is over 30% (Steinberg, M.H. et al, 2014). Strategies to reactivate HbF have been used successfully in mouse models of SCD and led to amelioration of the disease phenotype, which is also partly the mechanism of action of hydroxyurea, the only FDA approved drug for SCD. Up-regulation of HbF can be achieved by several different approaches including pharmacologic or genetic manipulation of transcription activators or repressors of HbF. Nuclear factor (erythroid derived-2)-like 2 (Nrf2) is a basic leucine zipper transcription factor that has been shown to activate γ-globin transcription and increase HbF levels in cultured CD34+ erythroid cells. Nrf2 is well established for its role in cytoprotective and anti-oxidant actions by transcriptionally activating target genes that confer protection from oxidative damage triggered from injury and inflammation. Nrf2 is normally sequestered in the cytoplasm by Keap1, a Kelch-domain protein. Release of Nrf2 from Keap1 allows nuclear translocation of Nrf2 and activation of target genes via its binding to an anti-oxidant response element (ARE) in the promoter region of Nrf2 target genes. Previous studies (Macari and Lowrey, 2011) have demonstrated that the γ-globin promoter contains an ARE sequence supporting Nrf2 as an inducer of HbF. We investigated the role of dimethyl fumarate (DMF), a small molecule Nrf2 agonist, in activating γ-globin transcription and enhancing levels of HbF in tissue culture and murine SCD models. Delayed-release DMF, approved by the FDA as Tecfidera, is an oral therapeutic for the treatment of relapsing multiple sclerosis (MS). After oral administration of DMF, human exposure occurs to both DMF and the bioactive primary metabolite, monomethyl fumarate (MMF). We assessed the ability of increasing concentrations of DMF to induce γ-globin mRNA in human erythroleukemia cells, CD34+ cells isolated from bone marrow of non-SCD volunteer donors, and peripheral blood mononuclear cells from SCD volunteer donors. γ-globin mRNA increased by 3-4 fold compared to control as quantitated using real-time PCR. DMF incubation also resulted in 2-fold upregulation in HbF protein levels as demonstrated by UPLC and western blotting analysis and also a 2-fold induction in percentage of RBC containing HbF (F-cells) by flow cytometry. In addition, co-incubation of DMF with hydroxyurea produced an additive effect on γ-globin mRNA (8 - 10 fold in non-SCD; 2-4 fold in SCD) and F-cell induction in CD34+ stem cell derived erythroid progenitors (3-4 fold in both SCD and non-SCD). DMF treatment of KU812 erythroleukemia cells induced Nrf2 activation resulting in Nrf2 nuclear translocation and occupancy of the ARE in the γ-globin promoter by ChIP assay. Moreover, siRNA based knockdown of Keap1 resulted in up-regulation of HbF by flow cytometry, demonstrating that activation of the Nrf2 pathway in erythroid cells can result in the up-regulation of HbF. The effect of DMF on HbF induction was assessed in the Townes SCD mouse model. Townes mice were administered DMF at 100 mg/kg IP for 5 day per week for one month duration and compared to treatment with vehicle control and to treatment with hydroxyurea. This dose and route of administration of DMF resulted in clinically relevant plasma levels of MMF in Townes mice. After a month of dosing, there was a 3- to 4-fold increase in circulating F-cells in the DMF treated mice which was comparable to the hydroxyurea treated mice. Based on these findings, DMF may have potential as an HbF inducer in the therapy of SCD, in addition to its vascular protective and heme detoxifying properties (Belcher et al, ASH 2014). Further clinical studies are needed to evaluate the safety and efficacy of DMF in SCD. Disclosures Krishnamoorthy: Biogen: Employment, Equity Ownership. Gupta:Biogen: Employment, Equity Ownership. Sturtevant:Biogen: Employment. Li:Georgia Regents University: Employment. Makala:Georgia Regents University: Employment. Hobbs:Biogen: Employment, Equity Ownership. Light:Biogen: Employment, Equity Ownership.

Central venous access devices (CVADs), such as tunneled central venous catheters (tCVADs) and peripherally inserted central catheters (PICCs), help provide essential care for some patients with sickle cell disease (SCD). CVADs facilitate administration of multiple intravenous (IV) medications and blood products, as well as blood draws for laboratory analysis. Understanding CVAD use and complications is particularly relevant for SCD patients because of their high risk of having insufficient peripheral IV access. Prior studies describing CVAD use and complications in SCD patients were limited by small sample sizes, typically including 15-20 SCD patients in a single treatment center. The resulting estimates for CVAD use and complications in SCD patients vary widely, are insufficient for development of evidence-based guidelines, and may not be representative of the treatment burden in the broader SCD population. The purpose of this study was to describe the frequency of CVAD use and CVAD-associated complications among SCD patients in a large US population sample.

Beta(β)-thalassemia is a rare disease, with limited data available on the typical clinical course and outcomes of patients in the US. There are currently no nationwide surveillance efforts to monitor the prevalence or changes in outcomes among those affected, or to inform the development of standards of care, policy, or advocacy efforts. Most information on β-thalassemia treatment in the US comes from studies involved with the Thalassemia Clinical Research Network, which ended data collection in 2012. Large administrative claims databases have been used to study numerous diseases, but insufficient information on β-thalassemia diagnosis codes has precluded their use for β-thalassemia research until recently. In 2011, new International Classification of Disease-9 (ICD9) codes for thalassemia subtypes were introduced, allowing analyses of various thalassemia populations in diverse settings across the US. The purpose of this study was to describe the prevalence of β-thalassemia diagnoses among pediatric and adult patients in two large administrative databases covering broad US geographic areas, and to examine treatment patterns and health outcomes in these samples of β-thalassemia patients.

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

Objective—Cocaine use is associated with arterial thrombosis, including myocardial infarction and stroke. Cocaine use results in increased plasma von Willebrand factor (VWF), accelerated atherosclerosis, and platelet-rich arterial thrombi, suggesting that cocaine activates the endothelium, promoting platelet–VWF interactions. Approach and Results—Human umbilical vein endothelial cells, brain microvasculature endothelial cells, or coronary artery endothelial cells were treated with cocaine or metabolites benzoylecgonine, cocaethylene, norcocaine, or ecgonine methylester. Supernatant VWF concentration and multimer structure were measured, and platelet–VWF strings formed on the endothelial surface under flow were quantified. Cocaine, benzoylecgonine, and cocaethylene induced endothelial VWF release, with the 2 metabolites being more potent than the parent molecule. Brain microvasculature endothelial cells were more sensitive to cocaine and metabolites than were human umbilical vein endothelial cells or coronary artery endothelial cells. Coronary artery endothelial cells released VWF into the supernatant but did not form VWF–platelet strings. Intracellular cAMP concentration was not increased after treatment with cocaine or its metabolites. Conclusions—Both cocaine and metabolites benzoylecgonine and cocaethylene induced endothelial VWF secretion, possibly explaining thrombotic risk after cocaine ingestion. VWF secretion is likely to vary between vascular beds, with brain endothelial cells being particularly sensitive. These results suggest that clinical management of cocaine-induced ischemia may benefit from therapies aimed at disrupting the VWF–platelet interaction.

Abstract Background Red blood cell (RBC) exchange transfusion is a commonly used therapy for acute and chronic complications of sickle cell disease (SCD). However, the therapeutic benefit of RBC exchange in many clinical situations is not well established. RBC exchange can be performed in two ways: 1) manually, involving repeated cycles of whole blood phlebotomy followed by simple transfusion of donor RBC, or 2) automated exchange utilizing apheresis to rapidly remove patient RBC while returning patient plasma, platelets, and leukocytes along with donor RBC to the patient. Unlike manual exchange transfusion, automated RBC exchange exposes patient blood to high shear stress as blood transits the apheresis machine, reaching wall shear stresses of 30-50 dyne/cm2 (within the range of arterial shear) with regional peak wall shear stresses of over 500 dyne/cm2 (supraphysiologic shear). Increased wall shear stress increases the reactivity of plasma von Willebrand Factor (VWF), in part by enhancing exposure of the VWF A1 domain which is directs binding to the platelet glycoprotein (GP) Ib-IX-V. Increased VWF reactivity has been implicated in SCD pathogenesis, likely by promotion of adhesive interactions between VWF and sickle erythrocytes, platelets, leukocytes, and the vessel wall. Hypothesis We hypothesized that automated RBC exchange would increase the reactivity of VWF as compared to manual RBC exchange in SCD patients. Methods We assessed VWF parameters in the plasma of SCD patients before and after manual exchange transfusion and/or automated RBC exchange transfusion performed for non-acute clinical indications. The VWF parameters assessed were VWF antigen level (VWF:Ag), VWF activity (VWF:Act) as assessed by ELISA (DiaPharma) detecting the spontaneous Gp Ib-IX-V binding conformation in the VWF A1 domain, total active VWF (VWF:TA) calculated by VWF:TA = (VWF:Ag) x (VWF:Act), VWF ristocetin cofactor activity (VWF:RCo), and VWF multimer composition. Results Six SCD patients on chronic, monthly exchange transfusion support were enrolled in the study. Blood samples were obtained prior to and immediately following multiple separate exchange transfusion episodes for each patient. Three patients underwent only automated RBC exchange transfusion, one underwent only manual RBC exchange transfusion, and two patients underwent both manual and automated RBC exchange transfusion. Compared to normal pooled plasma (NPP), SCD patients prior to RBC exchange transfusion had increased VWF:Ag (1.3-2.1 fold), VWF:Act (mean 124% of normal), and VWF:TA (0.6-2.2, mean 1.52; VWF:TA of NPP= 1.02). There was no statistically significant change in VWF:Ag following manual or automated exchange. There was a statistically significant increase in VWF:Act in patients following automated RBC exchange (mean 25% increase, p<0.05 with range 12%- 53%) but not manual exchange (mean 3.4% increase, range 1.4%-6.3%). In addition, we found an increased VWF:TA following automated (mean 31% increase, p<0.05) but not manual exchange transfusion. Patients exhibiting the highest VWF:TA post-exchange transfusion also had increased VWF:Rco in response to low dose and high dose ristocetin. The patient plasma with the highest VWF:TA post-RBC exchange (3.59) was noted to initiate spontaneous platelet agglutination in the absence of ristocetin. The observed increases in VWF:TA or VWF:RCo did not correlate with differences in VWF multimer composition on non- reducing agarose gels. Conclusions VWF activity is increased after automated RBC exchange but not manual RBC exchange in adult sickle cell patients on chronic exchange transfusion. These increases are likely a result of shear induced activation of VWF in the apharesis machine circuit. We speculate that the increased VWF reactivity observed following automated RBC exchange transfusion could contribute to VWF-mediated adhesive interactions and aggravate SCD vascular complications, potentially conferring a microangiopathic effect, in certain clinical situations. Maneuvers to decrease automated RBC exchange transfusion-induced VWF activation may lead to improved outcomes for SCD patients requiring exchange transfusion. Disclosures: No relevant conflicts of interest to declare.

Erythropoietic protoporphyria (EPP) is a rare and usually autosomal dominant disorder characterized by ferrochelatase deficiency and accumulation of protoporphyrin in red blood cells (RBCs), skin, and liver. A small minority of patients develop severe liver dysfunction for which optimum treatment is lacking. Therapeutic plasma exchange (TPE) and RBC exchange (RCE) have been anecdotally reported to benefit patients with EPP and liver failure. A 50‐year‐old female with EPP developed severe liver dysfunction after knee replacement surgery and high‐dose acetaminophen use. Liver biopsy showed cholestatic liver injury without fibrosis. A total of 20 TPE procedures, six RCE procedures, and then 14 more TPE procedures were performed as adjunctive therapy with the purpose of preventing progression to end‐stage liver failure. After initial TPE, the plasma and RBC protoporphyrin levels decreased from 834.9 to 180.4 μg/dL (normal, ≤1 μg/dL), and from 3,905 to 2,879 μg/dL (normal, ≤80 μg/dL), respectively, without liver function improvement. RCE decreased RBC protoporphyrin levels from 2,879 to 1,225 μg/dL but plasma protoporphyrin increased from 180.4 to 1,044.1 μg/dL, and liver function failed to improve. Additional TPE again stabilized plasma protoporphyrin and improved RBC protoporphyrin levels but the patient ultimately died owing to end‐stage liver disease complications. This case illustrates that TPE and RCE may improve the plasma and RBC biochemical markers of EPP activity but liver function abnormalities may persist and patients may still progress to liver failure either because of irreversible liver injury or independent pathobiological factors unrelated to EPP‐induced hepatotoxicity. J. Clin. Apheresis, 2012. © 2012 Wiley Periodicals, Inc.

Abstract Abstract 1148 Cocaine ingestion is associated with increased risk of myocardial infarction, stroke, and deep vein thrombosis. Elevated risk for these thrombotic events persists for 7–10 days after cocaine ingestion, despite the fact that cocaine is rapidly cleared from the blood (with a half-life in circulation of less than 1 hour) being rapidly converted into numerous metabolites that circulate for 1–2 weeks. Thus, it is likely that these longer-lived cocaine metabolites account for the long-term thrombotic risk with cocaine use. Cocaine ingestion has been associated with increased plasma VWF concentration, early atherosclerosis, and platelet-rich arterial thrombi. VWF is the major adhesive ligand that attaches platelets to the vessel wall, either to the subendothelium at sites of injury where the endothelium has been denuded or, during inflammation, to intact endothelium, from which it is secreted. VWF is secreted by endothelial cells from intracellular storage granules (Weibel-Palade bodies) in a large, hyperadhesive multimeric form (ultra-large VWF, or ULVWF) that either remains tethered to the endothelial surface or is released into bulk flow. Platelet-VWF adhesion, and subsequent thrombus formation, may be augmented further by cocaine-induced vaso-constriction, increasing shear stress. Thus, we hypothesized that cocaine and/or its metabolites would stimulate endothelial VWF secretion as a mechanism of thrombotic risk. To test this possibility, we exposed cultured human endothelial cells from umbilical vein (HUVEC), brain microvasculature (BMVEC), or coronary artery (CAEC) to cocaine or one of its four major metabolites at concentration ranges reported to occur in plasma following cocaine use. The cocaine metabolites we tested were benzoylecgonine (BE), cocaethylene (CE), norcocaine (NC), and ecgonine methyl ester (EME). We assayed VWF release by platelet-VWF string formation in a parallel-plate flow chamber (2.5 dyne/cm2) and by measuring the concentration of VWF released into the supernatant. Cocaine concentrations as low as 0.1 μg/ml induced VWF release from HUVEC; 1–2 μg/ml cocaine was as effective in releasing VWF as 25 μM histamine or 4 mg/ml dDAVP. Of the cocaine metabolites, only BE and CE induced VWF release from endothelial cells. BMVEC were 10-fold more sensitive to cocaine and metabolites BE and CE than HUVEC in both platelet-string formation and VWF antigen assays. In CAEC, VWF release was slightly reduced compared to HUVEC in response to cocaine, BE, or CE. Consistent with this pattern, staining for intracellular VWF revealed the following hierarchy of intracellular VWF: BMVEC > HUVEC >> CAEC. In BMVEC and HUVEC, VWF staining was restricted to Weibel-Palade bodies, with CAEC also demonstrating a diffuse cytoplasmic pattern. We tested whether intracellular cAMP levels increased after cocaine or cocaine metabolite exposure to explore whether VWF release was dependent on Protein Kinase A, as is the case with dDAVP. Intracellular cAMP did not increase following exposure to cocaine or its metabolites in any of the endothelial cell lines. These results suggest that cocaine contributes to thrombosis by activating endothelial cells to secrete ULVWF via a mechanism that does not involve increased intracellular cAMP. The prolonged thrombotic risk after cocaine ingestion likely relates to the continued action of cocaine metabolites BE and/or CE on endothelial ULVWF secretion. VWF secretion is likely to vary between vascular beds, with brain endothelial cells being particularly sensitive. Furthermore, these results suggest that clinical management of cocaine-induced ischemia or vaso-occlusion may benefit from therapies aimed at disrupting the ULVWF–platelet interaction. Disclosures: No relevant conflicts of interest to declare.

Vaso-occlusion, hemolysis, and oxidative stress are hallmarks of sickle cell disease (SCD). This pathology is accompanied by systemic endothelial activation, rendering the endothelium more adhesive for blood cells, including sickle erythrocytes. Activated endothelial cells display or secrete several adhesive molecules, including von Willebrand factor (VWF). We assessed several VWF parameters in SCD patients at baseline: multimer pattern, antigen concentration (VWF:Ag), activation factor (VWF:AF), and total active VWF (VWF:TA). VWF:AF was determined using a llama nanobody (AU/VWFa-11) that detects a platelet-binding conformation of the A1 domain; VWF:TA was calculated by multiplying VWF:Ag by VWF:AF. SCD plasma contained elevated VWF:Ag and ultralarge VWF multimers. VWF:TA, a measure of total VWF reactivity, correlated closely with hemolysis, as determined by serum lactate dehydrogenase. ADAMTS13 activity and antigen were normal in all patients. These findings suggest an important role for hyperreactive VWF in SCD pathology and connect SCD to other microangiopathies, particularly thrombotic thrombocytopenic purpura.

Abstract Abstract 3204 A high pressure circulatory system has two diametrically opposed requirements for its function: it must be able to rapidly gel to prevent blood loss when the integrity of the vasculature is compromised while simultaneously maintaining fluidity when the vasculature is intact. The endothelium is primarily responsible for maintaining blood fluidity, producing rapidly acting labile substances that inhibit both the clotting of blood and the adhesion and aggregation of platelets. Among these substances are the prostaglandins (PGE1, PGI2, PGD2), which bind platelet membrane receptors, raise concentrations of intracellular cyclic adenosine monophosphate (cAMP), and inhibit platelet functions. The major effector of increased cAMP is the serine/threonine kinase protein kinase A (PKA). Of the numerous targets for PKA, one of the most highly phosphorylated upon cAMP increase is glycoprotein (GP) Ibβ, a component of the GPIb-IX-V complex, the platelet receptor for VWF that mediates the initial adhesion of platelet to the vessel wall at sites of injury. The GPIb-IX-V complex consists of 4 type I transmembrane polypeptides, GPIbα, GPIbβ, GPV and GPIX. GPIbα and GPIbβ are disulfide linked in a 1:2 ratio, and the resulting GPIb is non-covalently associated with GPIX and GPV in a 2:2:1 ratio. The VWF-binding site resides within the N-terminal 300 amino acids of GPIbα 500 Å above the platelet surface. Although current data indicate that PKA phosphorylation of the GPIbβ cytoplasmic domain (at Ser166) inhibits the ability of GPIbα to bind VWF, the molecular mechanism(s) have yet to be elucidated. The cytoplasmic domain of GPIbβ associates with calmodulin (in the juxtamembrane 20 amino acids) in resting platelets; calmodulin dissociates upon platelet activation. With elevated cytosolic cAMP, GPIbβ Ser166 becomes phosphorylated and associates with 14-3-3ζ. An interesting feature of the cytoplasmic sequence N-terminal to Ser166 is its extreme cationic nature, containing 8 Arg residues in a stretch of 17 amino acids. Other cytosolic proteins with similar polybasic sequence (MARCKS, GAP43) function as organizers of the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), and promote the formation of lipid rafts; we reasoned that the polybasic region of GPIbβ might function similarly, organizing rafts when unbound by protein, but not when occupied by calmodulin or 14-3-3ζ. Platelet activation increases raft-associated GPIb-IX-V two fold, with concomitant dissociation of calmodulin from GPIbβ. Here we present evidence that the cytoplasmic domain of GPIbβ plays a role in the localization of the GPIb-IX-V complex to lipid rafts. Treatment of platelets with agents that increase cAMP (PGI2 or forskolin) inhibited GPIb-IX-V-dependent platelet functions, including ristocetin-induced aggregation, shear-induced aggregation and adhesion to VWF under flow. This effect was prevented by the cell-permeable PKA-specific inhibitor H-89. Consistent with the functional importance of GPIb-IX-V localization to lipid rafts, PGI2 and forskolin reduced the raft content of GPIb-IX-V by 35%, and this effect was reversed by H-89. We have thus uncovered a mechanism for long-observed inhibition of platelet adhesion by agents that elevate cytosolic cAMP concentrations, which depends on modulating the quantity of GPIb-IX-V complexes associated with lipid rafts. “Resting” platelets ex vivo are relatively quiescent because calmodulin occupies the GPIbβ polybasic region. The situation changes rapidly when platelets are activated, with more of the complex assuming a ligand-competent state as calmodulin dissociates and the complex organizes rafts. Elevations of cAMP promote phosphorylation of GPIbβ, enabling 14-3-3ζ association, which also displaces the GPIbβ tail from the membrane, disrupting raft association and adhesive function. Disclosures: No relevant conflicts of interest to declare.

A new study by Shida et al provides an elegant explanation for how vessel wall injuries are repaired without occluding the vessel. The hemostatic repair of damaged endovasculature resembles an intricate symphony orchestrated by platelets and a variety of plasma proteins, all tightly regulated to

Cocaine use is associated with sudden cardiac death, cardiac ischemia, and stroke in patients with no additional risk factors and is a frequent cause of these syndromes in patients with sickle cell disease. Pathologic findings include platelet-rich microthrombi and an increased plasma concentration of von Willebrand Factor (VWF). These findings suggest that, in addition to the well-known cocaine effects of vasoconstriction and increased tissue oxygen demand, activation of platelets and/or endothelial cells contributes to cocaine-induced ischemia. However, studies investigating the effect of cocaine on platelet functions have been inconclusive, finding both platelet activation and inhibition depending on the assay used. Further, the ability of cocaine to activate the vascular endothelium has not been examined, in particular, the endothelial secretion of the most adhesive forms of VWF, the ultralarge forms (ULVWF). ULVWF are long VWF multimers that remain tethered to the endothelial surface upon secretion, extend into the blood vessel lumen under laminar flow in long strings measuring up to 0.5 cm in length, and have multiple exposed binding sites for receptors on platelets, erythrocytes, and leukocytes. Elevated levels of ULVWF, due either to enhanced secretion or defective processing, have been implicated in diseases such as thrombotic thrombocytopenic purpura (TTP) and sickle cell anemia. We hypothesized that a major consequence of cocaine exposure is activation of the vascular endothelium to secrete ULVWF, which would provide a platform for blood cell adhesion and subsequent thrombosis or vaso-occlusion. We evaluated the ability of cocaine to stimulate ULVWF from cultured endothelial cells in a parallel-plate flow chamber assay and found that 1 μg/ml cocaine, a level comparable to peak blood levels detectable in cocaine abusers, efficiently induced secretion of ULVWF capable of binding platelets under flow conditions similar to that induced by histamine 6 μg/ml (3.92 ULVWF strings/field with cocaine vs. 4.62 strings/field with histamine). We also assessed the activation of platelets exposed in vitro to cocaine by flow cytometry, using two markers of platelet activation: P-selectin expression (which signals a-granule release) and conformational activation of the platelet integrin aIIbb3, detected with the antibody PAC-1. We found that when platelet-rich plasma was incubated with cocaine at concentrations from 0.1 μg/ml to 10 μg/ml, there was no increase in P- selectin exposure or PAC-1 binding. Furthermore, pretreatment of platelets with cocaine inhibited the ability of platelets to subsequently be activated by ADP in a dose-dependent manner. We did not observe any increase in mean fluorescence above background in ADP stimulated platelets pre-incubated with 1 μg/ml cocaine for P-selectin or PAC-1 binding. However, exposure of platelets in whole blood to 1 μg/ml cocaine resulted in a 3.2-fold increase in P- selectin exposure and a 5.4-fold increase in PAC-1 binding. These results indicate that cocaine directly activates the vascular endothelium to secrete ULVWF, and activates platelets indirectly, involving as yet unknown factors in whole blood, resulting in the formation of microthrombi. These effects of cocaine are likely to have pathogenic roles in cardiovascular syndromes associated with cocaine use, including the triggering of vaso-occlusive crises in sickle cell anemia and may explain the observed association of cocaine use with TTP.