Fig 2: Coagulome gene expression in human tumors. Dot plots showing the tumor type ranking according to the mRNA expression levels of six essential components of the tumor coagulome (F3, PLAU, PLAT, PLAUR, F2R and SERPINE1). Data from n = 33 tumor types, including OSCC, were retrieved from TCGA (total number of tumors analyzed was n = 10,071). Note that OSCC is the tumor type that presents the highest expression levels of F3, PLAU and SERPINE1 (OSCC in red).

Fig 3: Hirudin inhibits TGF-β-induced PAR1 upregulation in HK-2 cells and the renal tissues of UUO mice. Relative (A) mRNA levels and (B) protein expression levels of S1PR1-4 and PAR1 in HK-2 cells were detected by RT-qPCR and western blot analysis, respectively. (C) Expression levels of proteins, including S1PR1-4 and PAR1, in the renal tissues of UUO mice were determined by western blotting. (D) Relative mRNA levels of PAR1 in HK-2 cells were determined by RT-qPCR. PAR1 expression in (E) HK-2 cells and (F) renal tissues of UUO mice was quantified by western blotting. Error bars represent the mean ± SEM from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. the control. #P<0.05, ##P<0.01 and ###P<0.001 vs. TGF-β or UUO. UUO, unilateral ureteral ligation; PAR1, protease-activated receptor 1; S1PR, sphingosine-1-phosphate receptor; RT-q, reverse transcription-quantitative.

Fig 4: Hirudin suppresses TGF-β-induced epithelial-mesenchymal transition via S1P/S1PR2/S1PR3 signaling-mediated PAR1 inhibition. (A) Expression levels of N-cad, Slug and E-cad in HK-2 cells were determined using western blotting. (B and C) α-SMA expression in HK-2 cells was analyzed by immunofluorescence analysis. Scale bar, 100 µm. Error bars represent the mean ± SEM from three independent experiments. ***P<0.001 vs. the control. ###P<0.001 vs. TGF-β. ∆P<0.05 and ∆∆∆P<0.001 vs. TGF-β+hirudun (1 mg/ml). S1PR, sphingosine-1-phosphate receptor; PAR1, protease-activated receptor 1; N-cad, N-cadherin; E-cad, E-cadherin; α-SMA, α-smooth muscle actin; S1P, sphingosine-1-phosphate; TFL, TFLLR-NH2.

Fig 5: Hirudin suppresses TGF-β-induced fibrosis and MCP-1 expression via S1P/S1PR2/S1PR3 signaling-mediated PAR1 inhibition. (A) Expression levels of collagen IV, FN and MMP-9 in HK-2 cells were determined by western blotting. (B) Relative mRNA level of MCP-1 in HK-2 cells was analyzed by reverse transcription-quantitative PCR. (C) MCP-1 expression in HK-2 cells was quantified by western blotting. Error bars represent the mean ± SEM from three independent experiments. ***P<0.001 vs. the control. ###P<0.001 vs. TGF-β. ∆P<0.05, ∆∆P<0.01 and ∆∆∆P<0.001 vs. TGF-β+hirudun (1 mg/ml). MCP-1, monocyte chemoattractant protein 1; S1PR, sphingosine-1-phosphate receptor; PAR1, protease-activated receptor 1; FN, fibronectin; S1P, sphingosine-1-phosphate; TFL, TFLLR-NH2.