Regular Article The cell-membrane prothrombinase, fibrinogen-like protein 2, promotes angiogenesis and tumor development Esther Rabizadeh a,b , Izhack Cherny a , Doron Lederfein a , Shany Sherman a , Natalia Binkovsky a , Yevgenia Rosenblat c , Aida Inbal a,d, ⁎ a Hemato-Oncology Laboratory, Felsenstein Medical Research Center, Petach Tikva, Israel 1 b Hematology Laboratory, Rabin Medical Center, Beilinson Hospital, Petach Tikva, Israel c Pathology Institute, Rabin Medical Center, Beilinson Hospital, Petach Tikva, Israel d Thrombosis and Hemostasis Unit, Hematology Institute, Rabin Medical Center, Beilinson Hospital, Petach Tikva, Israel 1 abstract article info Article history: Received 7 July 2014 Received in revised form 9 November 2014 Accepted 30 November 2014 Available online xxxx Keywords: Angiogenesis Fibrinogen-like protein 2 Tumorigenesis The aim of the study was to further investigate the role of fibrinogen-like protein 2 (FGL-2), a transmembrane prothrombinase that directly cleaves prothrombin to thrombin, in angiogenesis and tumor development and the mechanism(s) underlying these processes. To study angiogenesis HUVEC clones with decreased fgl-2 mRNA were generated by specific siRNA. To study tumorigenesis SCID mice were implanted with intact (wild type) and fgl-2-silenced PC-3 clones. IFN-γ treated HUVEC expressing increased fgl-2 mRNA exhibited significant capillary sprouting that was not inhibited by hirudin, whereas fgl-2 silencing completely inhibited blood-vessel formation. Tumors (poorly differentiated carcinoma) developed in all 12 mice injected with wild type PC-3 com- pared with 8/12 mice injected with the fgl-2-silenced PC-3 clone. The tumors developed by fgl-2-silenced PC-3 clones were smaller and less aggressive and contained significantly fewer blood vessels (p b 0.05). All tumors’ sections were negative for thrombin staining, indicating that FGL-2-induced tumorigenesis was not mediated by thrombin. In fgl-2-silenced tumors there was a decrease in fgl-2 mRNA (p = 0.02) and ERK1/2 phosphoryla- tion (p b 0.05) by 80% and a 20%, respectively. The mechanism underlying these processes, studied in PC-3 clones, revealed that fgl-2 silencing was associated with a 65% decrease in FGF-2 mRNA (p b 0.01) and a 30% down reg- ulation of ERK1/2 phosphorylation (p b 0.05). Together, these results suggest that FGL-2 mediates angiogenesis and tumorigenesis not by thrombin-mediated mechanism but rather through FGF-2/ERK signaling pathway. FGL-2 may serve as a valuable therapeutic target in the future. © 2014 Elsevier Ltd. All rights reserved. Introduction The bidirectional relationship between cancer and thrombosis has been known for almost two centuries [1–3]. Thrombosis often precedes the diagnosis of cancer, and its presence is associated with a detrimental disease course [4,5]. These findings support the paradigm that coagula- tion and tumor growth form a vicious circle in which hypercoagulability facilitates the aggressive biology of cancer and vice versa. The mecha- nism underlying these events is still unclear. Malignant cells are known to directly activate blood coagulation in three ways: by produc- ing procoagulant, fibrinolytic, and proaggregating factors; by releasing proinflammatory and proangiogenic cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1ß; and by interacting directly with host endothelial cells, leukocytes, and platelets via adhesion mole- cules [5]. However, the precise procoagulant proteins that stimulate tu- morigenesis have not been identified. One potential candidate is the cell-membrane-associated protein, fibrinogen-like protein 2 (FGL-2)/fibroleukin, which has been shown to induce sprouting in vascular endothelial cells [6] and to be overexpressed in tumor cells [7]. FGL-2, also known as FGL-2- prothrombinase, is a member of the fibrinogen family of proteins [8]. It exerts serine protease activity and is capable of directly cleaving pro- thrombin to thrombin in the absence of factor VII or factor X. Like plas- matic prothrombinase, factor Xa, FGL-2 prothrombinase requires phospholipids, calcium, and factor Va for optimal catalytic activity [9]. However, unlike factor Xa, FGL-2 is a transmembrane protein which is not inhibited by antithrombin in the presence of heparin or by other protease inhibitors that inhibit factor Xa [9]. Thrombosis Research xxx (2014) xxx–xxx Abbreviations: ERK, extracellular-signal-regulated kinases; FGF-2, basic fibroblast growth factor; FGL-2, fibrinogen-like protein 2; HUVEC, human umbilical vein endothelial cells; IFN-γ, human interferon-gamma; MAPK, mitogen-activated protein kinases; SCID, Severe Combined Immunodeficiency ⁎ Corresponding author at: Thrombosis and Hemostasis Unit, Hematology Institute, Rabin Medical Center, Beilinson Hospital, Petach Tikva 49100, Israel. Tel.: +972 3 9377912; fax: +972 3 920 1568. E-mail addresses: erabi@clalit.org.il (E. Rabizadeh), izhackch@clalit.org.il (I. Cherny), doronle@clalit.org.il (D. Lederfein), shanyshnush@walla.com (S. Sherman), nataliabi@clalit.org.il (N. Binkovsky), blyrosenblat@clalit.org.il (Y. Rosenblat), aidai@clalit.org.il (A. Inbal). 1 Affiliated with Sackler Faculty of Medicine, Tel Aviv University, Israel. TR-05758; No of Pages 7 http://dx.doi.org/10.1016/j.thromres.2014.11.023 0049-3848/© 2014 Elsevier Ltd. All rights reserved. Contents lists available at ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromres Please cite this article as: Rabizadeh E, et al, The cell-membrane prothrombinase, fibrinogen-like protein 2, promotes angiogenesis and tumor development, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.11.023