S182 Abstracts / Thrombosis Research 140S1 (2016) S168–S200 PO-15 Antiangiogenic small molecule ligands of FGF2 derived from the endogenous inhibitor thrombospondin-1 D. Pinessi 1 , C. Foglieni 1 , A. Bugatti 2 , E. Moroni 3 , A. Resovi 1 , D. Ribatti 4 , M. Rusnati 2 , R. Giavazzi 1 , G. Colombo 3 , G. Taraboletti 1 1 Tumor Angiogenesis Unit, Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Bergamo, 2 Department of Molecular and Translational Medicine, University of Brescia, Brescia, 3 Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Milano, 4 Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, and National Cancer Institute “Giovanni Paolo II”, Bari; Italy Introduction: Platelet thrombospondin-1 (TSP-1) is a major endogenous regulator of growth factor activity in physiological and pathological processes, including tumor onset, progression and angiogenesis. We previously demonstrated that TSP-1 binds to FGF-2, sequestering the growth factor and inhibiting its angiogenic activity. We also identified a non-peptidic antiangiogenic compound (SM27) that retains the structural and functional properties of the FGF2-binding sequence of TSP-1. Aim: To identify new small molecule inhibitors of FGF2 that recapitulate the structure and functional properties of the FGF-2-binding site of TSP-1, by investigating the chemical space around SM27. Materials and Methods: A similarity-based screening of small molecule libraries has been used to identify candidates, followed by docking calculations, and evaluation of the activity of the resulting compounds in biochemical and biophysical assays, to assess interaction with FGF2, and in experimental models of angiogenesis, to assess biological activity. Results: The used integrated approach allowed selecting 7 bi-naphthalenic compounds that bound FGF2 inhibiting FGF2 binding to both heparan sulfate proteoglycans and FGFR1. The compounds inhibited FGF2-induced endothelial cell proliferation, vessel sprouting from aortic rings and angiogenesis in the chorioallantoic membrane assay, with improved potency over SM27. Conclusions: We have identified new compounds that are valuable as FGF inhibitors for potential therapeutic purposes. Moreover, these compounds are useful chemical tools to identify the minimal stereochemical require- ments for FGF2 binding and activity to improve the design of new agents for antineoplastic therapy. Acknowledgement: Supported by AIRC (Associazione Italiana per la Ricerca sul Cancro). PO-16 ASK1 regulates tumor lung metastasis and platelet functions M. Kamiyama, I. Naguro, H. Ichijo Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Introduction: Apoptosis signal-regulating kinase 1 (ASK1) is a MAP3K in the JNK and p38 MAPK pathways and responds to various stresses. Accumulating evidence indicates that ASK1 plays important roles in tumorigenesis by regulating apoptosis and inflammation. However, little is known about ASK1’s roles in tumor metastasis. Aim: To investigate ASK1’s roles in tumor metastasis. Materials and Methods: We performed experimental lung metastasis model by intravenous injection of Lewis lung carcinoma cells constitutively expressing luciferase (3LL-Luc2 cells). As for the analysis of platelet functions, tail bleeding assay and ferric chloride-induced thrombosis model were utilized. Results: We measured the transition of luciferase activity of the lung lysates up to 7 days as an indicator of lung metastasis. ASK1-/- mice showed markedly lower luciferase activity as early as 3 hours after injection compared to WT mice; hence ASK1 appears to be involved in the early stage of tumor lung metastasis, which is prior to the extravasation of tumor cells. Platelets aggregate and adhere to tumor cells in the early stage and are known to support hematogenous metastasis. ASK1-/- mice were normal in hematological parameters including platelet number, while analysis by western blot revealed that platelets of ASK1-/- mice exhibited markedly reduced phosphorylation of JNK and p38, both of which have been reported to regulate platelet functions such as platelet aggregation. We found that platelets of ASK1-/- mice were less responsive to specific aggregation agonists and that ASK1-/- mice showed bleeding tendency and defect in thrombosis. These phenotypes were also observed in megakaryocyte and platelet-specific ASK1 deficient mice. Conclusions: It is suggested that impaired platelet functions caused by ASK1 deficiency in platelets may attenuate tumor lung metastasis. PO-17 Identification of IgG bound to plasminogen in oncologic diseases V.N. Iakovlev 2 , E.I. Goufman 3 , N.B. Tikhonova 3 , R.B. Aisina 1 , L.I. Mukhametova 1 , K.B. Gershkovich 4 1 Lomonosov Moscow State University, Chemistry Faculty, 2 Angiogen Co. Ltd, 3 Institute of Human Morphology, Russian Academy of Medical Sciences, 4 Emanuel Institute of Biochemical Physics of Russian Academy of Sciences; Russia Introduction: The binding of plasminogen (Pg) to cell receptors and extracellular ligands facilitates its activation to plasmin, which stimulates the extracellular matrix degradation, neoangiogenesis and tumor invasion. Plasmin can also degrade IgG thereby exposing C-terminal lysine residues. Previously, we have found IgG specifically bounded to Pg in the plasma of patients with malignant tumors. Aim: To identify IgG degraded by plasmin in the plasma of cancer patients. Materials and Methods: Methods of ELISA were used for comparative research of levels of IgG bound to Pg in plasma of patients with the prostate cancer (PC, n=25) and lung cancer (LC, n=17). Plasma of healthy donors (n=29) was used as control. All patients signed informed consent for participation in this study. Affinity chromatography on Pg-sepharose was used for the quantification of IgG. Carboxypeptidase was used for remove of C-terminal lysine residues of the IgG. The program ATTESTAT was used for nonparametric analysis. Results: The frequency of occurence of elevated levels of IgG to Pg in plasma was detected in 68% of patients with PC, 59% of patients with LC and only 12% of healthy women and 10% of healthy men. The quantification of antibodies in plasma samples showed that the quantity of IgG to Pg in patients with PC was 27% from the total amount of IgG and in healthy men - 9%. Treatment of diluted plasma samples with carboxypeptidase B abolished the elevated levels of IgG to Pg, as well as the specific activity of the purified IgG to Pg- sepharose. Conclusions: C-terminal lysine residues which are formed as a result of degradation of native IgG with plasmin can bind to lysine binding sites on the kringle domains of Pg. Increased levels of these degraded IgG can be marker at cancer. PO-18 Fibronectin EDA/EDB is expressed in adherent SCLC NCI-H69 cells and in pleural effusions of lung cancer patients: possible implication for drug resistance U. Salge-Bartels 1 , M. Heiden 1 , R. Seitz 1 , F. Gieseler 2 1 Paul-Ehrlich-Institut, Division of Haematology and Transfusion Medicine, Langen, Germany, 2 University Hospital Schleswig-Holstein, Experimental Oncology, Lübeck, Germany Introduction: Small cell lung cancer (SCLC) is an extremely aggressive tumour which metastasizes early. Even if chemotherapy can achieve an initial regression, relapses due to chemo-resistance are almost inevitable. Sethi et al (Nat Med. 1999;5:662-668) reported that matrix proteins are essentially involved in the development of drug resistance. SCLC cells in suspension culture secrete negligible amounts of matrix proteins Aim: For a more detailed study of the SCLC ability to produce matrix proteins we applied a recently introduced cell culture model of adherence