between thrombin and NSCLC prognosis, and VM formation were different in patients with various PAR-1 expression levels. Thrombin expression was closely associated with VM formation and survival in NSCLC patients with higher PAR-1 levels; however, it was not significantly related with VM formation and survival in NSCLC patients with low PAR-1 expression. NSCLC patients with thrombin-positive/PAR-1-high expression had the poorest prognosis and were the most likely to form VM. These findings suggest that thrombin, together with PAR-1, substantially contributes to VM formation and NSCLC malignancy.

To further investigate the effect of thrombin on PAR-1-mediated VM formation, PAR-1-deficient NSCLC cells were treated with thrombin in vitro. We found that the ability of PAR-1-deficient NSCLC cells to form VM tubes was lost; thrombin had no effect on their VM formation capacity.

The cleavage of PAR-1 by thrombin leads to the activation of NF-κB signaling cascades.^[@CR33]^ Studies have shown that NF-κB plays an important role in the EMT process. In this study, we found that thrombin could activate NF-κB signaling and induce EMT, and that r-hirudin and DTIP could inhibit thrombin-induced NF-κB activation and EMT. PDTC, an inhibitor of NF-κB, could inhibit thrombin-induced EMT. Thrombin is the main activator of PAR-1. In PAR-1-deficient NSCLC cells, thrombin had no effect on NF-κB activation, EMT, and VM formation, whereas LPS could rescue NF-κB activation and EMT. These results suggested that thrombin promoted the activation of NF-κB through the activation of PAR-1, thereby inducing EMT and VM formation. r-hirudin and DTIP could bind to thrombin specifically and block thrombin binding to PAR-1, thereby inhibiting NF-κB activation and reducing the expression of N-cadherin and snail, while increasing the expression of E-cadherin in NSCLC cells, and this mechanism is very beneficial for suppressing VM formation. This study is the first to illustrate the effect of thrombin inhibitors on the formation of VM.

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