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

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.

No relevant conflicts of interest to declare.