Alternatively Spliced Tissue Factor (asTF) Is Elevated in the Plasma of Patients with Sickle Cell Disease: Pilot Studies Performed Using a Novel asTF-Specific ELISA

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2240-2240 ◽  
Author(s):  
Evgeny Ozhegov ◽  
Ramprasad Srinivasan ◽  
Vladimir Bogdanov
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Sickle Cell Disease ◽  
Healthy Subject ◽  
Tissue Factor ◽  
Sickle Cell ◽  
Healthy Subjects ◽  
Amino Acid Residues ◽  
Cell Disease ◽  
Alternatively Spliced

Abstract Abstract 2240 Background and Rationale: Vasoocclusive crises are a major hallmark of sickle cell disease (SCD) pathobiology; experimental evidence suggests that SCD vasoocclusion can be triggered by the increased adhesion of white blood cells, including monocytes, to the microvascular endothelium. Pro-coagulant activity of Tissue Factor, the trigger of blood coagulation, is heightened in the blood of patients with SCD. We recently reported that, compared to full length Tissue Factor (flTF), alternatively spliced Tissue Factor (asTF) acts as a very potent inducer of cell adhesion molecules E-selectin, VCAM-1, and ICAM-1 on microvascular endothelial cells, thereby raising the possibility that asTF may promote monocyte adhesion to the endothelium in vivo (Srinivasan et al, J Thromb Haemost 2011). Analogously to flTF, asTF is continuously present in circulation. Currently, no asTF-specific assay exists that can reliably detect asTF protein in plasma, and no data is available on the levels of asTF in the plasma of patients with SCD. We sought to develop monoclonal antibodies suitable for asTF-specific enzyme-linked immunosorbent assay (ELISA), to evaluate the levels of plasma asTF in SCD patients and age/gender matched healthy subjects. Methods: Two rabbit monoclonal antibodies were raised and characterized: i) antibody RabMab-95 recognizing amino acid residues 81–95 of mature asTF; ii) antibody RabMab-1 recognizing the last 11 amino acid residues of the asTF's unique C-terminus. By western blotting, both RabMab's recognized a) recombinant asTF produced in E. coli, b) eukaryotic asTF expressed in HEK293 cells using an inducible promoter system, and c) native asTF constitutively expressed in human pancreatic adenocarcinoma cell lines, with high specificity and sensitivity. In a sandwich ELISA of platelet poor plasma (PPP) samples, RabMab-95 was used as the capture antibody and horseradish peroxidase-conjugated RabMab-1 as the detection antibody; conventional blocking, sample incubation, and substrate development techniques were used. In addition, levels of flTF in PPP samples were assessed using ZYMUTEST Tissue Factor kit (RK035A, HYPHEN BioMed). Results: The SCD cohort comprised 16 pediatric and adult patients (10 females and 6 males, average age: 28.25±11.3 years); in the healthy subject cohort (n=17, 10 females and 7 males), the average age was 26.6±6.7 years. 14 out of 16 SCD patients had detectable levels of asTF, ranging from 25 pg/mL to 38,350 pg/mL (average: 5,323±9,934 pg/mL); in contrast, only 2 out of 17 healthy subjects had detectable levels of asTF: one PPP sample had 650 pg/ml and the other, 1,883 pg/mL (p=0.0397, SCD vs healthy subjects). The adult (>20 y.o., n= 10, average age: 35.2±7.8 years) and the pediatric (≤20 y.o., n=6, average age: 16.7±3.6 years) SCD sub-cohorts had average asTF values of 8,319±11,738 pg/mL and 329±446 pg/mL, respectively; while the difference between the adult SCD sub-cohort and the age-matched healthy subject sub-cohort was statistically significant (p=0.0337, adult SCD vs age-matched healthy subjects), there was a trend toward statistical significance in the pediatric asTF sub-cohort when compared to age-matched healthy subjects (p=0.1004, pediatric SCD vs age-matched healthy subjects). The levels of flTF in SCD plasma ranged from less than 1 pg/mL to 105 pg/mL (8 out of 16 patients), and did not correlate with asTF levels. Conclusions: We have developed a monoclonal ELISA for specific detection of asTF in human PPP. Our findings indicate that adult as well as pediatric SCD patients have heightened levels of asTF protein in circulation. Importantly, in ∼50% of SCD patients the levels of plasma asTF were in the range vastly exceeding the levels previously reported for any form of blood borne TF, likely sufficient to trigger a physiologically significant increase in leukocyte adhesion to the endothelium. Examination of circulating asTF levels in larger cohorts of pediatric and adult patients with SCD is thus highly warranted. Disclosures: No relevant conflicts of interest to declare.

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.

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 < .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.

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.

scholarly journals Thrombin-independent contribution of tissue factor to inflammation and cardiac hypertrophy in a mouse model of sickle cell disease

Blood ◽  
2016 ◽  
Vol 127 (10) ◽  
pp. 1371-1373 ◽  
Author(s):  
Erica M. Sparkenbaugh ◽  
Pichika Chantrathammachart ◽  
Kasemsiri Chandarajoti ◽  
Nigel Mackman ◽  
Nigel S. Key ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Mouse Model ◽  
Cardiac Hypertrophy ◽  
Tissue Factor ◽  
Sickle Cell ◽  
Cell Disease ◽  
Independent Contribution

scholarly journals Evaluation of Baseline Hematologic Parameters of Steady State Sickle Cell Disease Patients at The Chantal Biya’s Foundation, Yaounde- Cameroon

2019 ◽  
Vol 7 (07) ◽  
Author(s):  
Bih Adelaide ◽  
Ngala Solange Mudih ◽  
Alima Yanda ◽  
Akaba Fergus Ambe ◽  
Jutcha Florent ◽  
...  
Keyword(s):  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Reference Values ◽  
Blood Cells ◽  
Healthy Subjects ◽  
White Blood Cells ◽  
Great Decrease ◽  
Cell Disease ◽  
Mean Values

Introduction Sickle cell disease is a genetic abnormality involving the hemoglobin. Although it is primarily a red blood cell disorder, the white blood cells and platelets are also affected by the mutation. The consequence hemoglobin S causing polymerization of hemoglobin, results in hemolysis and anemia. This study aims to provide baseline hematologic parameters in steady state sickle cell disease patients compared with the reference values of normal healthy subjects used at the Chantal Biya Foundation (CBF), in order to monitor other sicklers in Cameroon. Methodology A comparative analysis of sickle cell hematologic parameters with control hematologic parameters of normal healthy subjects (reference values of healthy subjects used at the Chantal Biya Foundation) was carried out. Results A total of 62 sickle cell disease patients in steady state who complied with the selection criteria were recruited. Of the 62, 29 were females and 33 were males. The age range was from 1 year to 19 years and an average age of 6 ± 4.19 SD. Results from sickle cell patients showed an increase in white blood cells (WBCs), neutrophils and lymphocytes and a great decrease in the mean values of hematocrit Hct. as well as RBC indices, but no great or slight difference in the values of basophils compared with the reference values of normal healthy subjects in the CBF Yaoundé, 2015. Conclusion Sickle cell disease patients in steady state have lower values of red cells parameters, but higher values of white cells and platelets count when compared with the reference values of normal healthy subjects used at the Chantal Biya Foundation.

Tissue factor-dependent activation of coagulation contributes to the inflammation in a mouse model of sickle cell disease

2012 ◽  
Vol 56 (5-6) ◽  
pp. 376
Author(s):  
Pichika Chantrathammachart ◽  
Nigel S. Key ◽  
Nigel Mackman ◽  
Rafal Pawlinski
Keyword(s):  
Sickle Cell Disease ◽  
Mouse Model ◽  
Tissue Factor ◽  
Sickle Cell ◽  
Cell Disease

Sickle blood contains tissue factor–positive microparticles derived from endothelial cells and monocytes

Blood ◽  
2003 ◽  
Vol 102 (7) ◽  
pp. 2678-2683 ◽  
Author(s):  
Arun S. Shet ◽  
Omer Aras ◽  
Kalpna Gupta ◽  
Mathew J. Hass ◽  
Douglas J. Rausch ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Steady State ◽  
Sickle Cell Disease ◽  
Tissue Factor ◽  
Sickle Cell ◽  
Endothelial Activation ◽  
Cell Disease ◽  
Control Subjects ◽  
Inflammatory State ◽  
And Control

Abstract Blood microparticles (MPs) in sickle cell disease (SCD) are reportedly derived only from erythrocytes and platelets. Yet in SCD, endothelial cells and monocytes are activated and abnormally express tissue factor (TF). Thus, sickle blood might contain TF-positive MPs derived from these cells. With the use of flow cytometry to enumerate and characterize MPs, we found total MPs to be elevated in crisis (P = .0001) and steady state (P = .02) in subjects with sickle cell disease versus control subjects. These MPs were derived from erythrocytes, platelets, monocytes, and endothelial cells. Erythrocyte-derived MPs were elevated in sickle crisis (P = .0001) and steady state (P = .02) versus control subjects, as were monocyte-derived MPs (P = .0004 and P = .009, respectively). Endothelial and platelet-derived MPs were elevated in sickle crisis versus control subjects. Total TF-positive MPs were elevated in sickle crisis versus steady state (P = .004) and control subjects (P < .0001) and were derived from both monocytes and endothelial cells. Sickle MPs shortened plasma-clotting time compared with control MPs, and a TF antibody partially inhibited this procoagulant activity. Markers of coagulation were elevated in patients with sickle cell disease versus control subjects and correlated with total MPs and TF-positive MPs (P < .01 for both). These data support the concept that SCD is an inflammatory state with monocyte and endothelial activation and abnormal TF activity. (Blood. 2003;102:2678-2683)

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