Endothelial Protein C Receptor (EPCR), Oxide Nitric Synthase (NOS) and Tissue Factor (Factor III) Gene Expression: The Role Like Promising Biomarkers In Sickle Cell Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 987-987
Author(s):  
Bruno A. V. Cerqueira ◽  
Wendell Vilas Boas ◽  
Teresa Cristina Fonseca ◽  
Regiana Q. Souza ◽  
Milena Magalhães Aleluia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Tissue Factor ◽  
Sickle Cell ◽  
Protein C ◽  
Healthy Controls ◽  
Cell Disease ◽  
Endothelial Protein C Receptor ◽  
Factor Gene ◽  
Inflammatory Profile

Abstract Sickle cell anemia is a severe monogenic disorder characterized by the homozygous state of a single beta globin gene mutation, with heterogeneous clinic characteristics, associated with pro-inflammatory profile, oxidant state and hypercoagulable state. Vessels occlusion is likely initiated by intimal proliferation and amplified by inflammation, excessive adhesion of cells to activated endothelium and vascular tone dysregulation, normally modulated by NFkB pathway both endothelium cells as leukocytes. Herein, we investigated the gene expression of tissue factor (Factor III), oxide nitric synthase (NOS) and endothelial protein C receptor (EPCR) associating with biomarkers of prognosis like hemolysis markers, pro-inflammatory and anti-inflammatory profile. Forty two steady-state sickle cell disease (SCD) patients (16.5 ± 13.5 years, 20 female) from Northeast Brazil were enrolled in this study and were diagnosed in attendance of the outpatients’ clinic of the Sickle Cell Disease Center of Itabuna (CERDOFI). The control group was compound by 20 healthy Brazilian individuals with hemoglobin AA pattern matched by age, years and ethnic origin. The study was approved by the UESC ethical committee and informed consents were signed by patients or official responsible. Using real time quantitative PCR, we analyzed tissue factor, NOS and EPCR gene expression. We also measured hematological and hemoglobin parameters by electronic cell counter and HPLC respectively, biochemical profile was evaluated by colorimetric methodology and cytokine by flow cytometry. The statistical analysis was performed using the Kolmogorov–Smirnov test to access distribution of quantitative variables. Mean values of quantitative variables between groups were compared using an unpaired t-test for data distributed normally and a Mann–Whitney test for non-normal data. Oxide nitric synthase gene expression was increased in SCD patients 1.58-fold compared with healthy controls and higher tissue factor and EPCR gene expression were detected in patients than healthy controls, 4.82-fold and 5.46-fold respectively. The SCD cohort comprised pediatric and adult patients, and the medical history data was search from patient’s records where 95% of patients presented painful crisis at least once. Tissue factor gene expression was positive correlated with expression of NOS (p=0.005) and EPCR (p=0.0001). The increase tissue factor gene expression was detected in patients with high serum levels of bilirubin (p=0.026). Tissue factor gene expression above 75th percentile was associated high concentration of serum creatine kinase and serum calcium (p<0.005) in SCD patients. Our study reveals that genes associate to hemostasis and vascular integrity are upregulated in SCD patients, probably associated to chronic oxidative stress and pro-inflammatory state. Enhanced tissue factor, NOS and EPCR gene expression may influences in the pathophysiology of SCD. Studies tissue factor, NOS and EPCR should be carried out in order to explore mechanism, clarify participation and contributing to search of therapeutic strategies on prevention of vascular events among SCD patients. Disclosures: No relevant conflicts of interest to declare.

The Protein C Pathway in Human and Murine Sickle Cell Disease: Alterations in Protein C, Thrombomodulin (TM), and Endothelial Protein C Receptor (EPCR) at Baseline and during Acute Vaso-Occlusion

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 538-538 ◽  
Author(s):  
Yihe Guo ◽  
Teresa Uy ◽  
Nancy Wandersee ◽  
J. Paul Scott ◽  
Hartmut Weiler ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Mouse Liver ◽  
Protein C ◽  
Coagulation System ◽  
Activity Levels ◽  
Healthy Controls ◽  
Cell Disease ◽  
Endothelial Protein C Receptor ◽  
Protein C Activity

Abstract 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&lt;0.001). Assessment of protein C activity levels in plasma revealed that humans (n=8) with SCD have lower protein C activity levels than healthy controls (n=10) (78%±8.7% vs. 107%±5.3%, p=0.01). Additionally, we are the first to report that protein C activity levels decrease further during acute vaso-occlusive events (paired samples in 7 individuals, p=0.01). Another key protein of the PC pathway is TM, an endothelial-bound protein which activates protein C. 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&lt;0.05). Exposure to HR in SCD mice increased hepatic inflammation and ischemia and decreased hepatic TM levels compared to SCD mice at baseline (HR2 84%, HR4 60%, and HR21 85% of baseline SCD liver TM). In preliminary experiments, Western Blot analysis confirmed high TM expression in mouse SCD livers compared to HbA livers at baseline and after HR. Immunohistochemistry demonstrated widespread, increased TM staining in hepatic parenchymal vessels of SCD mice compared to HbA mice both at baseline and after HR, with decreased staining within mature infarcts. 4) Finally, we studied EPCR, a membrane-bound protein that binds circulating protein C and promotes both anti-thrombotic and anti-inflammatory functions. Similar to TM, immunohistochemical staining for EPCR was more prominent in hepatic parenchymal vessels of SCD mice compared to HbA mice at baseline and after HR (preliminary studies, n=3 per group). In summary, these data confirm that SCD is a prothrombotic state and suggest that the protein C pathway is altered in SCD. TAT levels are elevated in human and murine SCD, and increase further during vaso-occlusion, illustrating that SCD is a hypercoagulable state. Protein C activity levels are low in human SCD and decrease further during vaso-occlusive events, suggesting that protein C may be consumed both chronically and acutely. In SCD mice at baseline, elevated expression of TM and EPCR suggests that there may be a chronic, compensatory up-regulation of these proteins in SCD. Finally, an acute consumptive process could account for the transient, decreasing trend of these natural anticoagulant proteins in SCD mice after exposure to HR. Thus, targeted administration of activated protein C may provide a novel therapy to minimize tissue injury during acute vaso-occlusive events in SCD.

scholarly journals Tissue factor promotes activation of coagulation and inflammation in a mouse model of sickle cell disease

Blood ◽  
2012 ◽  
Vol 120 (3) ◽  
pp. 636-646 ◽  
Author(s):  
Pichika Chantrathammachart ◽  
Nigel Mackman ◽  
Erica Sparkenbaugh ◽  
Jian-Guo Wang ◽  
Leslie V. Parise ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Sickle Cell Disease ◽  
Tissue Factor ◽  
Sickle Cell ◽  
Plasma Levels ◽  
Cell Injury ◽  
Vascular Inflammation ◽  
Cell Disease ◽  
Endothelial Cell Injury ◽  
Cell Adhesion Molecule 1

Abstract Sickle cell disease (SCD) is associated with a complex vascular pathophysiology that includes activation of coagulation and inflammation. However, the crosstalk between these 2 systems in SCD has not been investigated. Here, we examined the role of tissue factor (TF) in the activation of coagulation and inflammation in 2 different mouse models of SCD (BERK and Townes). Leukocytes isolated from BERK mice expressed TF protein and had increased TF activity compared with control mice. We found that an inhibitory anti-TF antibody abrogated the activation of coagulation but had no effect on hemolysis or anemia. Importantly, inhibition of TF also attenuated inflammation and endothelial cell injury as demonstrated by reduced plasma levels of IL-6, serum amyloid P, and soluble vascular cell adhesion molecule-1. In addition, we found decreased levels of the chemokines MCP-1 and KC, as well as myeloperoxidase in the lungs of sickle cell mice treated with the anti-TF antibody. Finally, we found that endothelial cell-specific deletion of TF had no effect on coagulation but selectively attenuated plasma levels of IL-6. Our data indicate that different cellular sources of TF contribute to activation of coagulation, vascular inflammation, and endothelial cell injury. Furthermore, it appears that TF contributes to these processes without affecting intravascular hemolysis.

scholarly journals Identifying Potential Drug Targets for Sickle Cell Disease through Gene Expression and Pathway Analysis of GEO Data

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 15-16
Author(s):  
Sharjeel Syed ◽  
Jihad Aljabban ◽  
Jonathan Trujillo ◽  
Saad Syed ◽  
Robert Cameron ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Drug Targets ◽  
Meta Analysis ◽  
Cell Disease ◽  
Blood Samples ◽  
Disease Modifying Therapies ◽  
Disease Modifying

Background: The pathogenesis of sickle cell disease (SCD) and its complications have been well characterized down to the molecular level. However, there remains a relative dearth of disease modifying therapies that reduce the frequency and number of vas-occlusive crises, hospitalizations, and deaths. Recent advancements in utilizing hydroxyurea and L-glutamine, which both impact unique disease pathways, should pave way for the identification of other molecular pathways as ideal drug targets. In this regard, our meta-analysis serves to identify key genes and associated pathways that are differentially expressed in SC patients. Methods: We employed our STARGEO platform to tag samples from the NCBI Gene Expression Omnibus and performed meta-analysis to compare SC and healthy control transcriptomes. For the meta-analysis, we tagged 285 peripheral blood samples from SC patients and 86 samples from healthy subjects as a control. We then analyzed the signature in Ingenuity Pathway Analysis to elucidate top disease functions from our analysis. Results: From our meta-analysis, we identified iron homeostasis signaling, NRF2-mediated oxidative stress response, cell senescence, and pyrimidine interconversion/biosynthesis as top canonical pathways that were upregulated in the peripheral blood samples from SC patients. Top upstream regulators included membrane associated protein and transporter ABCB6, non-coding RNY3, and erythroid maturation transcription factors GATA1, KLF1, and HIPK2 (with predicted activation). The most upregulated genes included inflammatory modulators RNF182 and IFI27, the latter of which has been shown to inhibit vascular endothelial growth and repair. Several membrane-associated protein coding genes such as GYPA, RAP1GAP, and PAQR9 were also upregulated in the SC samples. RAP1GAP is known to modulate neutrophil cell adhesion and homing while PAQR9 has roles in regulating protein quality control: a role also seen in similarly upregulated YOD1, a deubiquitinating enzyme involved in trafficking of misfolded proteins. Expectedly, also upregulated were HBBP1 and SOX6, which regulate globin genes and have been shown to silence γ-globin expression. Lastly, SLC6A19, the neutral amino acid transporter mutated in Hartnup disease, was also upregulated. Of the downregulated genes, WASF3, a member of the Wiskott-Aldrich syndrome protein family, has been linked to poor survival in many malignancies, including AML and CMML, but has not previously been linked to SCD pathogenesis. ENKUR was also downregulated and has been annotated as a tethering protein to cation channels as well as linked to pathways involving vascular leakage. SIGLEC10, which binds to vascular adhesion proteins, is a key suppressor of inflammatory responses to damage; it's downregulation along with ELAPOR1, a transmembrane protein involved in cellular response to stress, was also observed. Finally, based off the focus genes in our analysis we identified several networks with most being involved in amino acid metabolism, cellular assembly, function, and maintenance, hematological disease, and organismal injury. The top pathway is illustrated in Figure 1. Conclusions: Our study illustrates differentially expressed gene activity in SCD consistent with known pathophysiology such as immune response, endothelial damage and adherence, heme metabolism, and globin regulation. We also showed evidence of genes not previously studied in SCD, which may have novel roles such as those part of the ubiquitin-proteasome system like YOD1 and RNF182. Additionally, while some genes in our analysis like EKLF and GAT1 have been shown to enhance δ-globin expression, paving way for possible drug therapies for B-hemoglobinopathies, others like IFI27, PAQR9, RAP1GAP, ENKUR, SIGLEC10, WASF3, and SOX9 have yet to be studied as mediators of disease pathogenesis in SCD. A target to SOX9, a known suppressor of γ-globin, or ABCB6, a known modulator of erythroid cell shape and hydration, have particularly promising potential as disease modifying therapies. Finally, HIPK2, HBBP1, and SLC6A19 have previously been shown to have intriguing effects on hydroxyurea dosing and responsivity in SC patients and may also be candidate target molecules to enhance existing therapies. These data identify potential candidate pathways for mechanistic studies seeking to confirm a causative role in the pathogenesis of sickle cell disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Paraoxonases (PON) 1, 2, and 3 Polymorphisms and PON-1 Activities in Patients with Sickle Cell Disease

Antioxidants ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 252 ◽  
Author(s):  
Cadiele Oliana Reichert ◽  
Carolina Garcia de Macedo ◽  
Débora Levy ◽  
Bruno Carnevale Sini ◽  
Andréia Moreira Monteiro ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Protective Factor ◽  
Plasma Cholesterol ◽  
Transferrin Saturation ◽  
Density Lipoprotein ◽  
Ldl Oxidation ◽  
Healthy Controls ◽  
Cell Disease ◽  
Q192r Polymorphism

(1) Background: Oxidative stress, chronic inflammation, vasoocclusion, and free iron are all features present in sickle cell disease. Paraoxonases (PON) are a family (PON-1, PON-2, PON-3) of antioxidant enzymes with anti-inflammatory action. Here, for the first time, we described PON-1 activities and PON-1, PON-2, PON-3 polymorphisms in patients with sickle cell disease, homozygous for HbSS, compared with healthy controls. (2) Methods: The groups were matched for age and gender. PON-1 activities (arylesterase and paraoxonase) were determined by enzymatic hydrolysis of phenylcetate and paraoxon, respectively. Polymorphisms were determined by Restriction Fragment Length Polymorphism- Polymerase Chain Reaction (RFLP-PCR). (3) Results: Plasma cholesterol and fractions, ApoA1 and ApoB levels were all decreased in sickle cell disease patients, while anti-oxidized low-density lipoprotein (LDL) antibodies and C-reactive protein were increased. Serum arylesterase activity was lower in sickle cell disease patients when compared with healthy controls. In patients, paraoxonase activity was higher in those with PON-1 RR Q192R polymorphism. In these patients, the increase of serum iron and ferritin levels and transferrin saturation were less pronounced than those observed in patients with QQ or QR polymorphism. No differences were observed with PON-1 L55M, and PON-2 and PON-3 polymorphisms. Multivariate regression analysis showed that transferrin and ferritin concentrations correlated with arylesterase and paraoxonase activities. (4) Conclusions: Both transferrin and ferritin were the main predictors of decreased arylesterase and paraoxonase activities in patients with sickle cell disease. LDL oxidation increased, and RR PON-1 Q192R polymorphism is likely to be a protective factor against oxidative damage in these patients.

scholarly journals Serum Iron Levels and Copper-to-Zinc Ratio in Sickle Cell Disease

Medicina ◽  
2019 ◽  
Vol 55 (5) ◽  
pp. 180
Author(s):  
Charles Antwi-Boasiako ◽  
Gifty Dankwah ◽  
Robert Aryee ◽  
Charles Hayfron-Benjamin ◽  
Alfred Doku ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Serum Levels ◽  
Zinc Homeostasis ◽  
Healthy Controls ◽  
Cell Disease ◽  
Flame Atomic Absorption ◽  
Iron Copper ◽  
Copper And Zinc

Background and Objectives: Altered copper and zinc homeostasis may influence the antioxidant defense system and consequently lead to oxidative stress and associated complications in sickle cell disease (SCD) patients. Iron levels have been reported to increase in sickle cell patients due to frequent blood transfusion, chronic intravenous haemolysis and increased absorption of iron from the gastrointestinal tract. These elevated levels of iron may also lead to extensive oxidative damage. The current study evaluated serum levels of iron, copper and zinc in SCD patients and “healthy” controls. Materials and Methods: The study was a cross-sectional one, comprising 90 SCD patients with Haemoglobin SS and Haemoglobin SC genotypes and 50 HbAA “healthy” controls. Serum levels of iron, copper and zinc were measured using a Flame Atomic Absorption Spectrometer (Variant 240FS manufactured by VARIAN Australia Pty Ltd, VIC, Australia). Copper and zinc ratios were calculated and analyzed. Results: Serum levels of iron and copper were significantly elevated in the SCD patients, compared to their “healthy” counterparts (p < 0.001). These levels were further increased in patients with haemoglobin SS in vaso-occlusive crises (HbSS VOCs). Serum zinc levels were, however, significantly lower in the SCD patients, particularly during vaso-occlusion. The copper-to-zinc ratio was also found to be significantly higher in the SCD patients. Conclusion: Elevated copper-to-zinc ratio may be a biomarker of sickle cell oxidative stress and associated complications. The ratio may also be informative for the management of sickle cell oxidative burden. The significantly lower levels of zinc in the SCD patients may warrant zinc supplementation.

Whole Blood Tissue Factor Procoagulant Activity Is Elevated in Patients With Sickle Cell Disease

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4216-4223 ◽  
Author(s):  
Nigel S. Key ◽  
Arne Slungaard ◽  
Luke Dandelet ◽  
Stephen C. Nelson ◽  
Christopher Moertel ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Tissue Factor ◽  
Mononuclear Cell ◽  
Sickle Cell ◽  
Whole Blood ◽  
Procoagulant Activity ◽  
Cell Disease ◽  
Blood Samples ◽  
Significant Difference ◽  
Whole Blood Samples

Abstract We developed a simple assay for the measurement of tissue factor procoagulant activity (TF PCA) in whole blood samples that avoids the need for mononuclear cell isolation. This method combines convenience of sample collection and processing with a high degree of sensitivity and specificity for TF. Using this method, we have determined that TF PCA is detectable in whole blood samples from normal individuals, which is itself a novel observation. Essentially all PCA could be shown to be localized in the mononuclear cell fraction of blood. Compared with controls, whole blood TF levels were significantly (P &lt; .000001) elevated in patients with sickle cell disease (SCD), regardless of the subtype of hemoglobinopathy (SS or SC disease). No significant difference in TF PCA was observed between patients in pain crisis compared with those in steady-state disease. Because TF functions as cofactor in the proteolytic conversion of FVII to FVIIa in vitro, it was expected that an increase in circulating TF PCA would lead to an increased in vivo generation of FVIIa. On the contrary, FVIIa levels were actually decreased in the plasma of patients with SCD. Plasma TF pathway inhibitor (TFPI) antigen levels were normal in SCD patients, suggesting that accelerated clearance of FVIIa by the TFPI pathway was not responsible for the reduced FVIIa levels. We propose that elevated levels of circulating TF PCA may play an important role in triggering the activation of coagulation known to occur in patients with SCD. Because TF is the principal cellular ligand for FVIIa, it is possible that increased binding to TF accounts for the diminished plasma FVIIa levels.

Measurement of Urine Pyridinoline and Deoxypyridinoline as Markers of Increased Bone Degradation in Sickle Cell Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2572-2572
Author(s):  
Erfan Nur ◽  
Willem Mairuhu ◽  
Dees P. Brandjes ◽  
Ton van Zanten ◽  
Bart J. Biemond ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Bone Resorption ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Healthy Controls ◽  
Cell Disease ◽  
Skeletal Involvement ◽  
Asymptomatic Patients ◽  
Bone Degradation ◽  
Painful Crisis

Abstract Abstract 2572 Poster Board II-549 Introduction: Sickle cell disease (SCD) is commonly manifested through skeletal involvement. Besides the characteristic acute musculoskeletal pain, SCD is also associated with chronic skeletal complications such as osteopenia and osteoporosis. During bone resorption, the collagen cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) are released into circulation with subsequent urinary excretion. Measurements of urinary PYD and DPD could serve as valuable tools in detecting osteoporosis in the follow-up of SCD patients but perhaps also in determining the severity of bone infarction during painful crises. Therefore we compared urinary concentrations of PYD and DPD of SCD patients during asymptomatic state and painful crisis with those of race- and age-matched healthy controls. Methods: Urinary concentrations of PYD and DPD, adjusted for urine creatinine, were measured in SCD patients both during asymptomatic state (n=38) and painful crisis (n=27) and healthy controls with normal HbA hemoglobin (n=25) using high performance liquid chromatography (HPLC). Results: PYD and DPD concentrations were higher in asymptomatic SCD patients compared to controls ((54.8 (41.5–68.6) vs. 44.1 (37.7–49.9),P=0.005 and 11.6 (9.3–15.2) vs. 8.5 (6.8–10.4),P=0.004 respectively), with further increments during painful crisis (63.3 (51.8–76.0),P=0.041 and 15.3(13.0–21.5),P=0.003 respectively). In the asymptomatic patients levels of PYD and DPD were significantly correlated to the degree of hemolysis. Conclusion: In sickle cell patients bone resorption is increased and significantly correlated to the degree of hemolysis, compatible with their susceptibility to osteopenia and osteoporosis. Measurement of pyridinoline and deoxypyridinoline could have additional value as biomarkers of osteoporosis in SCD. During painful crises a further increment in bone degradation was observed. Disclosures: No relevant conflicts of interest to declare.

A Novel Connection Between Stress Erythropoiesis and Coagulation Activation in Sickle Cell Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 905-905
Author(s):  
Julia E. Brittain ◽  
David Manly ◽  
Leslie V. Parise ◽  
Nigel Mackman ◽  
Kenneth I. Ataga
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell Disease ◽  
Tissue Factor ◽  
Sickle Cell ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Cell Disease ◽  
Coagulation Activation ◽  
Monocytic Cells ◽  
Stress Erythropoiesis

Abstract Abstract 905 Introduction: Sickle cell disease (SCD) is associated with a hypercoagulable state. Multiple studies show that plasma from these patients exhibit: 1) increased thrombin generation; 2) decreased levels of natural anticoagulant proteins; and 3) a defect in the activation of fibrinolysis. The mechanism of coagulation activation in SCD is presumed to be multi-factorial, with contributions from abnormal erythrocyte phospholipid asymmetry and induction of tissue factor (TF) following hemolysis. In addition, hemolysis in SCD leads to elevated levels of erythropoietin (EPO) in patients, increased reticulocyte counts and the presence of stress (or shift) reticulocytes in circulating blood. These stress reticulocytes retain expression of the α4b1 integrin and are demonstrably adhesive to vascular factors in SCD. We have previously reported that these stress reticulocytes bind to blood monocytes in SCD patients via the α4b1 integrin, but the effect of this interaction on either cell remained unknown in SCD. Objective: With the increasing evidence that hemolysis and subsequent stress erythropoiesis associates with coagulation activation, we sought to evaluate the role of erythropoietin and the effect of stress reticulocyte adhesion to monocytes on coagulation activation in SCD patients. Methods: Coagulation activation in plasma samples was examined by evaluating TF activity on microparticles derived from patients with SCD. Stress reticulocytes were visualized and enumerated from these same patients using Wright Giemsa stained blood smears counter stained with new methylene blue to detect reticulocytes. Reticulocytes were scored as a stress reticulocytes based on the amount of punctuate reticular material, cell size, and presence of nuclear material. Stress reticulocyte induction of monocyte tissue factor expression was measured by flow cytometry after incubation of THP-1 monocytic cells with purified SS RBCs or control RBCs. To determine if induced THP-1 TF expression was due stress reticulocyte binding, THP-1 TF expression was examined in the presence or absence of known inhibitors of the monocyte/stress reticulocyte interaction. TF expression on CD14+ monocytes was examined in whole blood from SCD patients using flow cytometry. Plasma erythropoietin levels were quantified by ELISA. Results: We found that direct binding of the stress reticulocyte increased THP-1 TF expression 2.5 fold. This increase in TF expression was completely ablated by function blocking antibodies against the α4 integrin, but not by an isotype-matched control IgG. In whole blood samples, we also found increased TF expression on CD14+ monocytes with stress reticulocytes directly bound, compared to those monocytes in the same patient without stress reticulocytes bound (p = 0.002, n =3).We noted a strong correlation between stress reticulocyte count and TF activity on plasma microparticles in SCD (rspearman = 0.8656, CI = 0.5382 – 0.9660, p = 0.0006, n=11). Furthermore, we found that EPO induced α4b1 activation on the stress reticulocyte. This activation may promote both adhesion to the monocyte and an increase in TF expression. Consequently, we noted a strong trend towards an association of EPO with microparticle TF activity in SCD (rspearman = 0.5740, CI=-0.06 – 0.8780, p=0.068, n= 11) suggesting that EPO, by promoting the interaction between the stress reticulocyte and the monocyte, may contribute to TF activity in SCD. Conclusion: Taken together, we find that stress reticulocyte adhesion to monocytes and monocytic cells induces TF expression and may promote TF activity in patients. These data suggest a novel connection between stress erythropoiesis and coagulation activation in SCD. Disclosures: No relevant conflicts of interest to declare.

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