Tumor/host-generated thrombin (endogenous thrombin) was investigated with tumor growth and metastasis experiments in mice by the use of hirudin, a highly potent specific inhibitor of thrombin. Pretreatment with hirudin inhibited tumor implantation in nude or syngeneic mice, following subcutaneous injection of 2 human and 2 murine tumors. Hirudin induced a considerable lag period in the appearance of tumor growth, compared with phosphate-buffered saline (PBS) treatment, but had no effect on established tumor nodule growth in vivo or on tumor growth in vitro. Hirudin treatment induced central necrosis of the tumor nodule compared with no effect with PBS treatment. Greater protection was noted with longer duration of treatment. Tumor seeding into blood was examined with green fluorescent protein (
IntroductionThe association of thrombosis and cancer (platelet and fibrin deposition) is well established, 1-5 but the role that activation of the coagulation pathway plays in promoting neoplastic progression is not well defined. We and others have shown that exogenous thrombin (1 U/mL) acting through its PAR-1 receptor, is capable of enhancing tumor adhesion to platelets, 6-8 endothelial cells, 9 fibronectin, and von Willebrand factor 7 in vitro. Studies have also revealed that exogenous thrombin promotes tumor growth in vitro 10 and in vivo 6,7,11 as well as experimental (tail-vein injection) metastasis. [6][7][8]12 Exogenous thrombin is also capable of inducing angiogenesis in a chorioallantoic membrane model. 13 However, the pathophysiologic relevance of thrombin in the host, that is, the end result of endogenous generation of host thrombin during tumor growth and spontaneous metastasis, has not been examined. Indeed, the concentration of thrombin generated by a developing tumor in the plasma at the interface is unknown and uncertain. In addition, the effect of thrombin on tumor growth in vitro is biplasic (activation at Ͻ 0.5 U/mL and inhibition at higher concentration). 10 Most tumor cells have constitutively active tissue factor on their surface capable of generating thrombin in vitro. However, the host has antithrombin mechanisms capable of neutralizing this activity.An approach was therefore designed to explore the mechanism of endogenously generated thrombin with its possible effect on tumor growth, seeding, and spontaneous metastasis by employing the highly potent and specific inhibitor of thrombin, hirudin. Hirudin is twice as potent as heparin in animal thrombosis models. [14][15][16] Unlike heparin, it is capable of neutralizing thrombin bound to the tumor thrombus as well as in the plasma, 17,18 with a Ki (inhibitory constant) of 0.05 pM. 19 It has other antithrombotic properties. It is able to dissociate thrombin-platelet receptor complexes (greater affinity of hirudin for thrombin than for platelets). 20 It can displace reversibly bound Factor Xa from vascular endothelium, 20 which then becomes inactivated by plasma protease inhibitors, leading to reduced prothrombin activation. It has also been reported...
