Levels of Circulating Alternatively Spliced Tissue Factor (asTF) in the Plasma of Patients with Pancreatic Ductal Adenocarcinoma (PDAC) May Help Predict Aggressive Tumor Phenotype

Abstract Tissue Factor (TF) present in blood cells and plasma is referred to as blood-borne or circulating TF. TF has been implicated in the pathogenesis of several chronic disease states, most notably cardiovascular disease/thrombosis, diabetes, and cancer. Full-length TF is an integral membrane protein while alternatively spliced TF can be secreted in a free form and features a unique C-terminal domain enabling its selective detection in bio-specimens. Recently, asTF was shown to circulate in the blood of metastatic breast cancer patients at concentrations exceeding 1 ng/mL (Kocaturk et al, PNAS 2013), and it promoted tumor growth and spread in an orthotopic model of pancreatic ductal adenocarcinoma (PDAC, Unruh et al, Int J Cancer, 2014). asTF protein acts as a cell agonist driving angiogenesis, cancer cell proliferation, and monocyte recruitment via integrin binding. It is not known whether circulating asTF may contribute to or serve as a biomarker in patients suffering from cardiovascular disease, diabetes, and/or solid cancers including PDAC. We evaluated circulating asTF in healthy subjects and individuals with ongoing acute coronary syndrome (ACS); diabetes mellitus (DM); ongoing ACS+DM; and PDAC. Samples of platelet poor plasma from 204 subjects were obtained from University of Cincinnati Cancer Institute’s Tumor Bank and Diagnostica Stago collections, blood specimens drawn from emergency room visitors at four medical centers in the US, and George King Bio-Medical, Inc. ACS was defined by positive troponin levels; DM was self-identified. Blood was drawn into tubes containing heparin (ASC, DM, ACS+DM), acid citrate dextrose (PDAC), or sodium citrate (healthy subjects), centrifuged at 3000 rpm for 15 min at 4°C, and stored at -80°C until use. Blinded asTF ELISA was performed on plasma samples as per the prototype-tailored procedure (Diagnostica Stago). Samples with asTF concentrations ≥0.2 ng/mL were deemed positive. asTF concentrations are presented as mean±SD. Kruskal-Wallis one-way analysis of variance was used to compare differences in concentration levels between the cohorts; Chi-Square and/or Fisher’s exact test were used to compare proportions. asTF protein was detectable in the plasma of 3/19 (15.8%) subjects in the healthy cohort (CORE Set 50, George King Bio-Medical); 7/38 (18.4%) in the no ACS/no DM cohort (emergency room visitors’ control group); 2/40 (5%) in the DM cohort; 5/39 (12.8%) in the ACS cohort; 4/25 (16.0%) in the ACS/DM cohort; and 20/43 (46.5%) in the PDAC cohort; the proportion of PDAC patients positive for asTF was significantly higher compared to that in all other cohorts (p<0.01, Chi-Square test). The mean asTF concentrations in the cohorts were as follows: PDAC, 0.403±0.912 ng/mL; healthy subjects, 0.169±0.596 ng/mL; emergency room visitors’ control group, 0.159±0.357 ng/mL; ACS, 0.0925±0.258 ng/mL; DM, 0.0423±0.19 ng/mL; ACS+DM, 0.208±0.642 ng/mL; the differences between mean asTF levels in the cohorts did not reach significance. Next, we evaluated asTF’s potential as a biomarker to help detect a more aggressive PDAC phenotype. Among the 43 patients with PDAC, 36 were initially deemed resectable and 7 unresectable due to the presence of metastatic disease as determined by diagnostic screening; following exploratory laparoscopic surgery, 11 out of 36 patients initially deemed resectable were deemed unresectable due to the presence of metastatic disease. When the entire PDAC cohort was split into bona fide resectable (25) and unresectable (18) sub-cohorts, positivity for asTF was significantly more prevalent in the unresectable sub-cohort irrespective of the results of initial evaluation and/or pre-operative CA19-9 levels (asTF ≥0.2 ng/mL: 13 unresectable and 7 resectable patients; asTF<0.2 ng/mL: 5 unresectable and 18 resectable patients, p=0.0059, Fisher’s exact test). We here report that asTF at levels ≥0.2 ng/mL occurs more frequently in the plasma of patients with PDAC compared to healthy subjects and/or individuals with ACS, DM, and ACS/DM. Further, PDAC patients whose plasma asTF levels were equal to or exceeded 0.2 ng/mL had a significantly lower chance to qualify for tumor resection, irrespective of initial pre-surgical diagnostic evaluation. asTF may thus comprise a novel marker of aggressive PDAC phenotype with potential utility in patient stratification, warranting prospective evaluation of larger PDAC patient cohorts. Disclosures No relevant conflicts of interest to declare.

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A First-In-Class, Humanized Antibody Targeting Alternatively Spliced Tissue Factor: Preclinical Evaluation in an Orthotopic Model of Pancreatic Ductal Adenocarcinoma

Frontiers in Oncology ◽

10.3389/fonc.2021.691685 ◽

2021 ◽

Vol 11 ◽

Author(s):

Clayton S. Lewis ◽

Aniruddha Karve ◽

Kateryna Matiash ◽

Timothy Stone ◽

Jingxing Li ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Tissue Factor ◽

Tumor Tissue ◽

Single Agent ◽

The United States ◽

Ductal Adenocarcinoma ◽

Boyden Chamber ◽

Orthotopic Model ◽

Alternatively Spliced

In 2021, pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer deaths in the United States. This is largely due to a lack of symptoms and limited treatment options, which extend survival by only a few weeks. There is thus an urgent need to develop new therapies effective against PDAC. Previously, we have shown that the growth of PDAC cells is suppressed when they are co-implanted with RabMab1, a rabbit monoclonal antibody specific for human alternatively spliced tissue factor (asTF). Here, we report on humanization of RabMab1, evaluation of its binding characteristics, and assessment of its in vivo properties. hRabMab1 binds asTF with a KD in the picomolar range; suppresses the migration of high-grade Pt45.P1 cells in Boyden chamber assays; has a long half-life in circulation (~ 5 weeks); and significantly slows the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice when administered intravenously. Immunohistochemical analysis of tumor tissue demonstrates the suppression of i) PDAC cell proliferation, ii) macrophage infiltration, and iii) neovascularization, whereas RNAseq analysis of tumor tissue reveals the suppression of pathways that promote cell division and focal adhesion. This is the first proof-of-concept study whereby a novel biologic targeting asTF has been investigated as a systemically administered single agent, with encouraging results. Given that hRabMab1 has a favorable PK profile and is able to suppress the growth of human PDAC cells in vivo, it comprises a promising candidate for further clinical development.

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Antibody-based targeting of alternatively spliced tissue factor: a new approach to impede the primary growth and spread of pancreatic ductal adenocarcinoma

Oncotarget ◽

10.18632/oncotarget.7955 ◽

2016 ◽

Vol 7 (18) ◽

pp. 25264-25275 ◽

Cited By ~ 9

Author(s):

Dusten Unruh ◽

Betül Ünlü ◽

Clayton S. Lewis ◽

Xiaoyang Qi ◽

Zhengtao Chu ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Tissue Factor ◽

Ductal Adenocarcinoma ◽

New Approach ◽

Primary Growth ◽

Alternatively Spliced

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Alternatively spliced tissue factor contributes to tumor spread and activation of coagulation in pancreatic ductal adenocarcinoma

International Journal of Cancer ◽

10.1002/ijc.28327 ◽

2013 ◽

Vol 134 (1) ◽

pp. 9-20 ◽

Cited By ~ 27

Author(s):

Dusten Unruh ◽

Kevin Turner ◽

Ramprasad Srinivasan ◽

Begüm Kocatürk ◽

Xiaoyang Qi ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Tissue Factor ◽

Ductal Adenocarcinoma ◽

Tumor Spread ◽

Alternatively Spliced

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Success Rate of Obturation of Root Canals by Different Techniques in Primary Molars: A Comparative Clinical Study

Current Dentistry ◽

10.2174/2542579x02999200503032916 ◽

2020 ◽

Vol 2 (1) ◽

pp. 36-44

Author(s):

Satyawan G. Damle ◽

Ritika Bansal ◽

Dhanashree D. Sakhare

Keyword(s):

Success Rate ◽

Study Groups ◽

Primary Molars ◽

Control Group ◽

Second Primary ◽

Chi Square ◽

Exact Test ◽

Root Canals ◽

Significant Difference ◽

Comparative Clinical Study

Objective: To compare the success rate of different obturation procedures in primary mandibular second molars clinically and also by digital radiovisiography. Methods: A total of 40 children aged between 4-8 years with deeply carious mandibular second primary molars indicated for single session pulpectomy were selected. Canals were obturated with Metapex. The 3 study groups (Endodontic plugger, Handheld lentulospiral, Navi Tip syringe) were compared with the control group (reamer) both clinically and radiovisiographically. The data collected were statistically analyzed using Pearson’s Chi-square and Fisher’s exact test. Results: The use of Navi tip syringe led to the least number of voids followed by Endodontic plugger and Reamer and the highest number of voids was reported with Lentulospiral. Navitip presented maximum number of optimally filled cases followed by Endodontic plugger and Lentulospiral and least number of optimally filled cases with reamer. However, there was no statistically significant difference (p>0.05) in any of the groups with clinical (pain and tenderness to percussion) and radiographic parameters (presence or absence of voids and length of obturation). Conclusion: Within the limitations of the present study, though the clinical outcome was statistically insignificant, Navitip syringe exhibited encouraging results and is a promising option for obturation in primary teeth.

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Abstract 16942: Alternatively Spliced Tissue Factor promotes plaque progression, inflammation and angiogenesis in experimental atherosclerosis

Circulation ◽

10.1161/circ.130.suppl_2.16942 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Daniel Alicea ◽

Aleksey Chudnovskiy ◽

David T Rodriguez ◽

Dong Kwon Yang ◽

Dongtak Jeong ◽

...

Keyword(s):

Tissue Factor ◽

Experimental Atherosclerosis ◽

Control Group ◽

Plaque Progression ◽

Neointimal Thickness ◽

Inflammatory Monocytes ◽

Atherosclerotic Lesions ◽

Plaque Stabilization ◽

Alternatively Spliced ◽

Oil Red O Staining

Introduction: Alternatively Spliced Tissue Factor (asTF) is novel isoform of tissue factor with angiogenic activity mediated via HIF-1α signaling (Giannarelli, AHA 13). asTF is highly expressed in human complicated atherosclerotic plaques (Giannarelli, ACC 13); however, it is unknown whether asTF has a functional role in atherogenesis. Hypothesis: asTF promotes atherosclerotic plaque progression, inflammation and angiogenesis. Methods: ApoE-/- mice (8-weeks old; n=15) were fed a Western-type diet from 2 weeks before surgery continuing through the experiment. Immediately after transluminal wire injury of the left common carotid artery (LCCA), LCCA was incubated with lentivirus encoding asTF-GFP (asTF+ group; n=10) or GFP (asTF- control group; n=5). Four weeks after, blood and spleen were collected for flow cytometry analysis of neutrophils and monocytes. LCCA was removed and processed for H&E, Oil-Red O staining and immunostaining for macrophages (MOMA-2 and MAC-3), vascular smooth muscle cells (VSMC, α-actin), endothelial cells (CD31) and HIF-1α. Results: Neointimal thickness and plaque lipid accumulation were significantly greater in asTF+ vs asTF- mice (Fig 1, A-C). An increase in plaque macrophages, neovessels and HIF-1α was observed in asTF+ vs asTF- (Fig 1, D-F). Medial thickness and VSMC density were similar between groups. Increased circulating neutrophils and Ly6C high (classical/inflammatory) monocytes were observed in asTF+ vs asTF- mice (Fig 1, G,H). In contrast, circulating Ly6C low (patrolling) monocytes were significantly reduced (Fig 1, I). Similar findings were observed in the spleen (Fig 1, J-L). Conclusions: Our results demonstrate that asTF expressed within atherosclerotic lesions promotes plaque progression towards a more advanced phenotype and is associated with systemic proinflammatory status. These data makes asTF an attractive marker of plaque vulnerability and a potential therapeutic target for plaque stabilization.

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Research protocol local ingredients-based supplementary food as an alternative to corn-soya blends plus for treating moderate acute malnutrition among children aged 6 to 59 months: a randomized controlled non-inferiority trial in Wolaita

BMC Public Health ◽

10.1186/s12889-019-8031-3 ◽

2019 ◽

Vol 19 (1) ◽

Author(s):

Debritu Nane ◽

Anne Hatløy ◽

Elazar Tadesse ◽

Bernt Lindtjørn

Keyword(s):

Intervention Group ◽

Trial Registration ◽

Acute Malnutrition ◽

Control Group ◽

Chi Square ◽

Clinical Trial Registration ◽

Exact Test ◽

Supplementary Food ◽

Randomized Controlled ◽

Moderate Acute Malnutrition

Abstract Background In Ethiopia, 12.5% of children below 5 years are wasted, and 9.7% are moderately wasted. The present strategy for the management of moderate acute malnutrition (MAM) is a supplementary feeding program; however, this is only provided to chronically food-insecure areas. This randomized controlled non-inferiority trial examines if Local ingredients-based supplement (LIBS) is as effective as corn-soya blends plus (CSB+) in treating moderate acute malnutrition among children aged 6–59 months. Methods A randomized controlled non-inferiority trial will be conducted with moderately wasted children aged 6 to 59 months in Wolaita, Ethiopia. The calculated sample size is 324 (i.e. with 162 children in each of two arms, to be assigned by randomization). The daily ration will be: 100 g of LIBS plus 25.2 g of sugar with 8 ml oil in the intervention group, and 150 g of CSB+ with 16 ml of oil in the control group. These interventions will be provided for a maximum period of 12 weeks, with follow-up performed on a weekly basis. Data analysis will be done using SPSS and STATA software. Both intention-to-treat and per protocol analyses will be done. Hazard ratio and Kaplan-Meier (log rank) curves of survival analysis will be done to predict the probability of recovery rate. Logistic regression will be used to test for interactions between independent and dependent variables. Analysis of variances, t-tests, fisher’s exact test and chi-square tests will be used to assess baseline characteristics. Conclusions This paper will introduce to the existing research locally available nutritious foods which have the potential to enhance recovery from moderate acute malnutrition and to reduce the burden of malnutrition. The perceptions of mothers on feeding children with local ingredient-based supplementary food to assist recovery from moderate acute malnutrition will be the focus of in a qualitative study to follow; this will provide a further contribution in an evolving area of research. Trial registration Pan-African Clinical Trial Registration number: PACTR201809662822990, retrospectively registered on 11/09/2018.

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3D-analysis of unwanted tooth movements despite bonded orthodontic retainers: a pilot study

BMC Oral Health ◽

10.1186/s12903-020-01304-2 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Katharina Klaus ◽

Faidra Xirouchaki ◽

Sabine Ruf

Keyword(s):

Pilot Study ◽

Control Group ◽

3D Analysis ◽

Chi Square ◽

Exact Test ◽

Interindividual Variations ◽

Mandibular Plane ◽

Chi Square Test ◽

Multibracket Appliance ◽

Orthodontic Retainers

Abstract Background Recently, reports of unwanted tooth movements despite intact orthodontic bonded retainers have increased. These movements are not subject to relapse but are classified as a new developed malocclusion. The aims of the present pilot study were to analyze the prevalence of unwanted tooth movements despite intact bonded cuspid-to-cuspid retainers and to identify possible predisposing factors. Materials and methods Plaster casts of all patients finishing orthodontic treatment during three consecutive years were assessed before treatment (T0), after multibracket appliance debonding (T1) and after two years of retention (T2). After multibracket appliance treatment, all patients received a cuspid-to-cuspid flexible spiral wire retainer bonded to each tooth of the retained segment in the upper and lower jaw. The study group (SG) consisted of 44 patients (16 male, 28 female) with tooth movements (T1–T2) of the retained segment despite intact bonded cuspid-to-cuspid retainer and the control group (CG) of 43 patients (19 male, 24 female) without unwanted tooth movements. The casts of the SG were digitized, superimposed and measured. Using the Chi-square test, Fisher´s exact test and Mann–Whitney-U-test (p < 0.05), mandibular plane angle, incisor proclination, oral dysfunctions or habits (T0) and intercanine distance, overjet and interincisal relationship (T0, T1, T2) were compared between SG and CG. Results The prevalence of patients with unwanted tooth movements in one or both jaws was 27.0%. Maxillary retainers were affected more often (20.9%) than mandibular retainers (14.1%). The median amount of tooth movements was 0 to 0.66 mm with large interindividual variations. Oral dysfunctions or habits at T0, such as a lack of interincisal contact at all time points, were associated with unwanted tooth movements. Conclusion Unwanted tooth movements occurred more often with maxillary than mandibular retainers. Patients with oral dysfunctions/habits and without interincisal contact had a higher prevalence of unwanted tooth movements.

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The Effects of Recombinant Human Granulocyte–Macrophage Colony-Stimulating Factor Gel on Third-Degree Frostbite Wounds in Northeastern China: A Randomized Controlled Trial

Journal of Burn Care & Research ◽

10.1093/jbcr/iraa019 ◽

2020 ◽

Author(s):

Xiu-Hang Zhang ◽

Chang-Lei Cui ◽

Hao-Yue Zhu ◽

Jian Wang ◽

Yan Xue ◽

...

Keyword(s):

Bacterial Culture ◽

Controlled Trial ◽

Healing Time ◽

Control Group ◽

Fisher Exact Test ◽

Chi Square ◽

Exact Test ◽

Positive Effects ◽

Positive Bacterial Culture ◽

Better Than

Abstract The aim of the study was to investigate the effects of the rhGM-CSF gel on third-degree frostbite wounds. Sixty-two patients who had suffered third-degree frostbite on their hand or foot (91 wounds in total) were selected using a convenience sampling method and randomly allocated to two groups: the rhGM-CSF group(31patients,45 frostbite wounds) received the rhGM-CSF gel when wound dressing change daily; however, the control group (31patients, 46 frostbite wounds) received aloe glue. The wound healing time, the score of inflammation about the wound and the positive bacterial culture of wound secretions were used to measure outcomes, respectively. Data were analyzed using SPSS (25.0), Student’s t test or Mann–Whitney U test and chi-square test or Fisher exact test were selected, as appropriate. The healing time of the rhGM-CSF group was (12.2 ± 5.0) days, which was significantly shorter than that of the control group (15.5 ± 4.7) days (P < .0001). The rhGM-CSF group’s wound inflammation scores on the 7th and 14th day of treatment were (0.96 ± 0.21) and (1.88 ± 0.29), respectively, which were better than those of the control group (1.12 ± 0.24) and (1.38 ± 0.15) (both P < .0001). The positive bacterial culture of wound secretions in the rhGM-CSF group was also better than that in the control group on the 3rd, 7th, and 14th day after treatment (P = .027, .004, .030, respectively). According to the results, using rhGM-CSF gel considerably increases the speed of frostbite wounds healing, and have an effect on protecting third-degree frostbite wounds regarding the positive effects. Trial Registration: This trial was registered in the Chinese Clinical Trial Register, ChiCTR1900021299.

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71 EFFECT OF CELL QUANTITY IN MICE CHIMERA PRODUCTION BY DEMI-BLASTOCYST AGGREGATION

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab71 ◽

2012 ◽

Vol 24 (1) ◽

pp. 148

Author(s):

C. Pontes Godoi ◽

P. D. Moço ◽

B. Cazari ◽

P. T. Mihara ◽

P. V. Silva ◽

...

Keyword(s):

Cell Mass ◽

Farm Animals ◽

Control Group ◽

Chi Square ◽

Cell Stage ◽

Exact Test ◽

Chi Square Test ◽

Aggregation Technique

Eight-cell-stage to pre-compaction morula are the most used embryonic stages to aggregation, because the embryos, in these early stages, synthesise cell adhesion molecules that increase the aggregation chances among them (Vestweber et al. 1987 Develop. Biol. 124, 451–456). Although post-compaction embryos produce reduced aggregation rates, they are not refractory to this process (Nogueira et al. 2010 Transgenic Res. 19, 344–345). Based on the evidence of less permissive aggregation in post-compaction-stage embryos and the need to expose the inner surface of those embryos to improve aggregation rate, the aim of this study was to evaluate, in mice, the influence of cell quantity (i.e. the quantity of half-embryos put together to aggregate themselves) in the chimerism rate of split blastocysts. Embryos, with preferentially different phenotypes, were obtained from C57BL/6/EGFP and Swiss Webster strains. Females ranging from 21 to 45 days old were superstimulated and mated according to Mancini et al. (2008 Transgenic Res. 17, 1015). Eight-cell-stage embryos (8C) and pre-compaction morula (PCM) were recovered (2 to 2.5 days post coitum) and had their zona pellucida removed using pronase treatment (2 mg mL–1 for 15 min), whereas blastocysts (recovered 3.5 dpc) were split with a microblade controlled by micromanipulator in an inverted microscope (NK2; Eppendorf, Hamburg, Germany and Eclipse Ti; Nikon, Tokyo, Japan, respectively). The aggregation groups were a control (C) with 2 pre-compaction whole embryos (8C or PCM, or both) and 2 experimental with post-compaction embryos [i.e. 2 (2DB) or 4 (4DB) demi-blastocysts]. The structures (2 or 4) of the groups were stuck to each other with the use of phytohemagglutinin (1 mg mL–1) and cultured in vitro by 24 h (37°C, 5% CO2 and saturated humidity). After culture, the presence of chimeric embryos was verified by detection of a single, cohesive cell mass or a structure in an 8 shape with more than one-half of its total diameter aggregated. For the 4DB group, a successful aggregation was considered when, at least 2 of 4 DB had aggregated. The results were analysed using chi-square test, Fisher's exact test and Kruskal-Wallis (to compare among groups, between groups and among medians of group replicates, respectively) and significance was considered when P < 0.05. The aggregation rates for the groups C, 2DB and 4DB were, respectively, 77.3a; 8.3b and 36.4%c (P < 0.001). The increasing of the aggregation technique efficacy, in post-compaction stages, would be particularly interesting in farm animals (e.g. bovine species), where it is not feasible to obtain, in vivo, pre-compaction stages embryos (as 8 cells) and when only trophectoderm aggregation is wanted. It was concluded that cell increasing (from 2 to 4 DB) improved the chimerism rate, but not enough to be similar to the control group. Supported by FAPESP of Brazil.

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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 ◽

10.1182/blood.v118.21.2240.2240 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2240-2240 ◽

Cited By ~ 1

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.

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