Increased Activity of Prothrombinase Fgl-2 in Peripheral Blood Mononuclear Cells of Patients with B-Cell Lymphoma.
Authors: Wolach Ofir , Rabizadeh Esther , Lederfein Doron , Binkovski Natalia , Raanani Pia , Shpilberg Ofer , Inbal Aida 
Published: October 2019

Abstract
Abstract 2665

Background:
Hematologic and solid tumors are associated with hypercoagulability the reason for which has not been delineated. Prothrombinase named fibrinogen-like protein 2 (FGL-2) is a 70 kD transmembrane protein that was found to have a quality of a serine protease capable of directly cleaving prothrombin to thrombin. FGL-2 is synthesized by monocytes, T-lymphocytes and endothelial cells. FGL-2 protein and its mRNA have been previously found within different tumor cells.

Aim:
To study the role of FGL-2 in patients with lymphoproliferative disorders. Our hypothesis is that upregulation of FGL-2 activity in patients with B-cell malignancies may contribute to tumorigenesis via generation of thrombin leading to increased angiogenesis and spread/metastasis of malignant cells.

Methods:
Thrombin generation reflecting FGL-2 activity was measured in homogenized peripheral blood mononuclear cells (PBMC) from 29 patients with active lymphoproliferative disorders and 107 normal controls. Informed consent was obtained from every participant. PBMC extracts were incubated with an equal volume of human prothrombin (final concentration10 μM) for 30 min at 37 °C. Thrombin generation was measured at 405 nm using an automated plate reader after addition of chromogen S-2238. The thrombin activity of each sample was calculated by comparison with absorbance curve generated by known concentrations of human thrombin. FGL-2 was immunoprecipitated (IP) from PBMC with an anti-FGL-2 antibody and EZview Red Protein A Affinity Gel (sigma # P6486). The activity of IP FGL-2 was measured by thrombin generation assay. The expression of FGL-2 was analyzed in HUVEC and PBMC in the presence or absence of IF-γ at 20 ng/ml. Total RNA was isolated using RNAqueous™ (Ambion #AM1912) and RT-PCR, analysis was performed using Rotor-gene RG-3000 (Corbett). The difference in cycle time (ΔCT) was measured by comparing FGL-2 gene with ABL-1 gene (house keeping gene). The relative quantification was calculated by the formula RQ= 2−ΔCT. HUVEC were transfected with 0.5 μM SiRNA synthesized complementary to FGL-2 (Target SiRNA) using Dharmacon ON-TARGET plus SMART pool reagent (Thermo Fisher Scientific) according to manufacturer instructions. FGL-2 expression following addition of target SiRNA was compared to that obtained following addition of non-specific (non-target) SiRNA. Student's t-test was used for all comparisons.

Results:
As shown in the table, almost 3-fold increase in FGL-2 activity in PBMC was observed among patients with active B-cell lymphoma, either aggressive or indolent, as compared to that of healthy controls (p<0.0001 for all comparisons). No difference in FGL-2 activity in PBMC between patients with aggressive and indolent lymphoma was observed (p=0.68).
Three-fold increase in thrombin generation was obtained in PBMC from the patients following IP, similarly to that observed in non-IP PBMC. A significant increase in mRNA of FGL-2 was found in either PBMC of lymphoma patient or HUVEC treated with IF-γ. In HUVEC the increase in FGL-2 mRNA was inhibited by 80% following treatment with specific SiRNA.

Conclusions:

Disclosures:
No relevant conflicts of interest to declare.