ISTH

20

17 Poster presented at

ISTH2017 on:

Elevated activity of fibrinogen-like protein 2 (Fgl2/fibroleukin) in platelets from cancer patients

Rabizadeh Esther, PhD1,2, Gurion Ronit, MD3, Prof. Peled Nir, MD4, Shepshelovich Daniel, MD5 Shezar Judi2, Zimra Yael PhD1,2, Cherny Izhack, PhD1,2

1Hemato-oncology Laboratory, Felsenstein Medical Research Center, Beilinson Campus, Tel Aviv University. 2Hematology Laboratory, Beilinson Hospital. 3HematoOncology

Unit and 4Oncology Unit, Davidiff Center , 5Internal Medicine, Beilinson Hospital. Rabin Medical Center, Petah Tikva, Israel

Summary of findings

• FGL2 expression is correlated with tumor development and aggressiveness both in vivo and in vitro

• We show that FGL2 directly induce coagulation in platelets independent of the classic coagulation cascade via FGL2.

• We show that FGL2 coagulation activity from peripheral blood is increased in aggressive lymphomas but decreased during remission

Introduction• The bidirectional relationship between cancer and thrombosis has been known for

almost two centuries. The mechanism underlying these events is still unclear.

• Fibrinogen-like protein 2 (FGL2) is a 60-70KD transmembrane serine protease expressed in by a an assortment of cells and tissues. In the blood, FGL2 is expressed by monocytes, endothelial cells and peripheral blood T cells (CD4+, CD8+).

• FGL2 exerts a prothrombinase activity, capable of directly cleaving prothrombin into thrombin (similar to Factor Xa), independent of the classical coagulation cascade

• FGL2 expression is up-regulated in tumor tissues cells but not in the normal surrounding tissue, thus believed to play a central role in cancer development.

• For the reasons above, FGL2 is a potential biomarker and a therapeutic target of cancer.

• In our research we studied the relationships between FGL2, coagulation and cancer by pursuing the following specific aims:

Catalytic serine

FGL2

Aim 3To devise FGL2 toward a liquid

biopsy biomarker

FGL2 activity in lymphoma patient vs. healthy control (A) Activity was increased by 3±0.3-fold in patients (n=53) as compared to control (n=145). p<0.001 (B) FGL2 activity over a cutoff value of 150% exhibited a sensitivity of 73.6% and specificity of 80.7%

Follow up of FGL2 activity in aggressive lymphoma patients. Patients with aggressive lymphomas (i.e., Burkitt, DLBCL, ALCL, TCL) were tested for FGL2 activity before the start of the treatment (‘active’), at the conclusion of the treatment (‘remission’) and recurrence of the disease (‘relapse’). In one case FGL2 activity was tested 17 months prior the development of the disease (‘healthy’). Best correlation between disease and FGL2 activity is observed in Aggressive diseases.

1. Increased activity of FGL2 in the peripheral blood cells of B-cell lymphoma patients

2. In aggressive lymphoma, FGL2 activity increases during disease and decreases in remission

ReferencesRabizadeh et al, (2014), Increased activity of cell membrane-associated prothrombinase, fibrinogen-like protein 2, in peripheral blood mononuclear cells of B-cell lymphoma patients. PLoS One 10;9(10):e109648Rabizadeh et al, (2015), The cell-membrane prothrombinase, fibrinogen-like protein 2, promotes angiogenesis and tumor development. Thromb Res 136(1):118-24Sherman et al, (2017), Fibrinogen-like Protein 2 Activity as a Potential Biomarker for Diagnosis of Early Mycosis Fungoides. Acta Derm Venereol 97(3):370-372

ContactIzhack Cherny izhackch@clalit.org.ilEsther Rabizadeh erabi@clalit.org.il

FundingNofar - Israel Ministry of Trade and Industry

FGL2 is a potential blood biomarker of lymphomas

Aim 1To uncover the role of FGL2 in

tumorigenesis

SCID mice model of Prostate carcinoma cell line (PC-3) induced tumors. Macroscopic picture of the tumor in mice injected with (A) WT PC-3 or (B) fgl-2-knock down PC-3. (C) Mean±SEM volume of the tumors weekly followup. (D) Mean±SEM of number of blood vessels in developed tumors (according to anti-VWF staining). *p<0.05.

1. Fgl2 knockdown of inhibits PC-3 subcutaneous xenografts tumor development and angio-genesis in SCID mice in vivo

2. Fgl2 knockdown inhibits tumorigenesis in vitro

Sprouting & tube formation (PC-3 cells, EC matrix angiogenesis assay in vitro)

15 hours incubation

6 hours incubation

Fgl2, knockdown

fgl2 over-expression

PC-3, WT

PC-3, WT

Cell cycleFACS analysis of PC3 cells (propidium iodide DNA staining)

Fgl2 knockdownG0/G1 G2/M

SG0/G1

G2/M

S

ERK1/2By MAPK phosphor-rylation profile assay (ELISA test)

20%

WT knockdown D

FGL2 plays a key role intumorigenesis

Felsenstein Research Medical Centeraffiliated with Sackler School of Medicine,Tel aviv Universityand Clalit Health Services

Aim 2To study the mechanism of FGL2

procoagulant activity in peripheral blood

Evidence of FGL2 in platelets (A) Purified platelet sample was lysed and immuno-precipitated using antibodies indicated below. Presence of FGL2 and absence of factor X (FX ) were confirmed. (B) FACS analysis of whole blood sample for presence of FGL2 on platelet. Antibodies: AF488 conjugated monoclonal anti human FGL2 (IgG2a); AF488 conjugated mouse IgG2a (control); PE conjugated mouse anti CD41 (platelet antigen).

1. FGL2 pro-coagulant activity in peripheral blood cells is correlated with platelet count

FGL2 activity vs. platelets and WBC counts. FGL2 induced coagulation time (CT) was determined for peripheral blood cell samples of (A) normal controls (n=166) with 1.5x106 mononuclear cells but different platelets count, (B) single normal donor with 58x106 platelet count and increasing amounts of white blood cells. ‘r’ represents non-linear correlation value.

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2. FGL2 activity is located in platelets

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FGL2 is located Platelets, via FGL2, induce coagulation - independent

of classic coagulation cascade

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2216--PBEsther Rabizadeh Wednesday, July 12DOI: 10.3252/pso.eu.ISTH2017.2017

Platelet Function and Interactions