The association of idiopathic venous thrombosis with occult cancer is generally recognized. However, it has not been fully appreciated that thrombin generated during thrombosis can augment the malignant phenotype. Thrombin activates tumor cell adhesion to platelets, endothelial cells, and subendothelial matrix proteins; enhances tumor cell growth; increases tumor cell seeding and spontaneous metastasis; and stimulates tumor cell angiogenesis. These mechanisms are reviewed. Evidence is also presented to support the hypothesis that thrombin serves to preserve dormant tumor cells in individuals, preventing host eradication. It is proposed that tumor malignancy may be regulated by a procoagulant/anticoagulant axis.

Platelet-adhesive protein-tumor cell interaction was studied in vitro and in vivo. Monoclonal antibody 1OE5, which inhibits binding of fibronectin and von Willebrand factor to the platelet membrane glycoprotein GPIIb-GPIIIa complex, inhibited the binding of mouse CT26 and human HCI8 colon carcinoma cells to platelets by 63-65%, whereas an irrelevant monoclonal antibody, 3B2, had no effect. Monoclonal antibody 6D1, which inhibits binding of von Willebrand factor to GPIb, also had no effect. RGDS, a tetrapeptide that represents the adhesive domain of fibronectin and von Willebrand factor inhibited binding of the tumors to platelets by 64-69%. Monospecific polyclonal antifibronectin antibody inhibited binding by 60-82%; anti-von Willebrand factor antibody inhibited binding by 75-81%.In vivo, polyclonal monospecific anti-mouse von Willebrand factor antibody inhibited pulmonary metastases induced by CT26 tumor cells by 53-64%, B16a amelanotic melanoma cells by 45% and T241 Lewis bladder cells by 46% without induction of thrombocytopenia. Pulmonary metastases with CT26 cells could be inhibited by induction of thrombocytopenia, and reconstituted by infusion of either murine or human platelets. Reconstitution of pulmonary metastases with human platelets could be inhibited 77% by preincubation of human platelets with monoclonal antibody 1OE5 before infusion of platelets into mice.Thus, platelets appear to contribute to metastases by their adhesive interaction with tumor cells via the adhesive proteins fibronectin and von Willebrand factor.

Recent studies have revealed a role for platelets and the platelet-adhesive proteins, fibronectin and von Willebrand factor (vWF) in platelet-tumor cell interaction in vitro and metastasis in vivo. The present report documents the effect of thrombin treatment of platelets on this interaction in vitro and in vivo. In vitro, thrombin at 100-1,000 mU/ml maximally stimulated the adhesion of six different tumor cell lines from three different species two-to fivefold. As little as 1-10 mU/ml was effective. The effect of thrombin was specific (inhibitable by hirudin, dansyl-arginine N43-ethyl-1,5 pentanediyl) amide and unreactive with the inactive thrombin analogue N-P-tosyl-L-phenylchloromethylketone-thrombin and D-phenylalanyl-L-propyl-L-arginine chloromethylketone-thrombin (PPACK-thrombin), and required high-affinity thrombin receptors (competition with PPACK-thrombin but not with N-P-tosyl-L-lysine-chloromethyl-ketone-thrombin). Functionally active thrombin was required on the platelet surface. Binding of tumor cells to thrombin-activated platelets was inhibitable by agents known to interfere with the platelet GPIIb-GPIIIa integrin: monoclonal antibody 10E5, tetrapeptide RGDS and 'y chain fibrinogen decapeptide LGGAKQAGDV, as well as polyclonal antibodies against the platelet adhesive ligands, fibronectin and vWF. In vivo, thrombin at 250-500 mU per animal increased murine pulmonary metastases fourfold with CT26 colon carcinoma cells and 68-413-fold with B16 amelanotic melanoma cells. Thus, thrombin amplifies tumor-platelet adhesion in vitro twoto fivefold via occupancy of high-affinity platelet thrombin receptors, and modulation of GPIIb-GPIIIa adhesion via an RGD-dependent mechanism. In vivo, thrombin enhances tumor metastases 4-413-fold with two different tumor cells lines. (J. Clin. Invest. 1991. 87:229-236.).

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...