Fig 2: The coagulation–HGFA–HGF cleavage cascade supports the sustained supply of active-form HGFA and HGF in FF/PF. (A) Schematic of the extrinsic pathway linking coagulation to the activation of the HGFA–HGF axis. After initiation by overnight treatment with tissue factor (INNOVIN at 1:1 ratio), a fibrin clot formed in FF/PF, resulting in the coagulation factor–depleted supernatant (Dpl). (B) Anchorage-independent growth assay of FE25 cells treated with D0-FF/PF mix with or without a thrombin inhibitor (2 µM Dabigatran) or Dpl. **P < 0.01 compared with the vehicle (medium with 0.5% DMSO). ##P < 0.01 compared with no inhibitor/depletion treatment. (C-D) Western blot analysis with pro- and active-form HGFA and HGF in D0-, D7-, and D30-FF/PF (D) and D7-FF/PF with or without 2 µM Dabigatran or with Dpl (C).

Fig 3: The coagulation–HGF cascade and MET signaling supports the proliferation of cells of the corpus luteum, ovarian surface, and fallopian tube epithelium (A) IHC shows MET expression (brown) in the diestrous phase mouse ovary procured 6 h post hCG after PMSG induction. The enlarged picture (dotted box) shows the corpus luteum (CL), ovarian surface, and oviduct surface expression of MET (arrows) (scale bar, 25 µm). All images are representative of three independent mice. (B) Proliferation assay of corpus luteum cells (KGN) at 72 h with or without 0.6 IU PMSG-induced luteinization as well as ovarian surface cells (IOSE) and fallopian tube fimbrial epithelial cells (FE25), after treatment with 5% D0-FF/PF (D0) with or without 10 µM AMG337 or 100 nM Picropodophyllin. D30-FF/PF was also tested in luteinized KGN cells. (C–E) Expression of phosphory- and nonphosphory-forms of MET 1h after treatment with FF/PF with or without thrombin inhibition (Dab) or coagulation factor–depleted supernatant (Dpl). *P < 0.05, **P < 0.01 compared with the vehicle (medium with 0.5% DMSO) of each group. #P < 0.05, ##P < 0.01 compared with no inhibitor used for FF treatment in each group.

Fig 4: Decreasing concentration gradient of HGF from FF and PF to serum (A) ELISA analysis of HGF in 21 FF aspirates including 12 paired serum samples collected at the same time. (B) Linear regression analysis of the relationship between the level of FF–HGF and serum HGF in 12 paired samples. (C) ELISA analysis of HGF in 15 PF samples.

Fig 5: The coagulation–HGF cascade in FF/PF plays a major role in the transformation of fallopian tube epithelial cells, high-grade serous carcinoma and other epithelial ovarian cancer cells in vivo. (A) In the xenograft tumorigenesis of FE25 cells in NSG mice, 2 million FE25 cells were subcutaneously injected together with 200 µL of medium and 5% FF/PF with or without pretreatment with the thrombin inhibitor Dabigatran (Dab) or coagulation factor–depleted supernatant (Dpl) or with 20 ng/mL recombinant HGF (100-39H, PeproTech). Tumor growth was observed at 6 months. (B) Orthotopic xenograft tumorigenesis of OVSAHO cells in NSG mice. OVSAHO cells (2 million) were injected into the ovarian bursa (i.b.). Dabigatran was orally administered twice weekly for 1 month, and mice were sacrificed after 5 months. (C) Orthotoic allograft of mouse ID8 (non- high-grade serous carcinoma type epithelial ovarian cancer) cells to C57BL6 mice, with or without the same thrombin inhibitor. Ovarian tumor pictures and tumor weights are shown. **P < 0.01 vehicle (ddH2O with 5% DMSO) compared with dabigatran treatment.