Thrombin-Thrombomodulin Interactions Are An Important Determinant of Metastatic Potential

Abstract 822

A substantial body of evidence indicates that tumor cell-associated (i.e., tissue factor) and circulating hemostatic system components (i.e., thrombin, fibrinogen, platelets) play a cooperative role in supporting metastasis. However, the role of endothelial regulators of hemostasis in metastasis remains largely unexplored. Thrombomodulin (TM) is the endothelial thrombin receptor central to thrombin-mediated activation of protein C. To test the hypothesis that thrombin-TM interactions are an important determinant of metastasis, we used mice carrying the Glu387Pro mutation in thrombomodulin (TM^Pro) known to decrease thrombin affinity ∼100 fold and APC generation ∼1000 fold. TM^Pro/Pro and control mice were intravenously injected with either a low dose (3 × 10⁴ cells/mouse) or high dose (3 × 10⁵ cells/mouse) of Lewis lung carcinoma cells (LLC) in separate experiments. At the low cell dose, the majority of wild-type mice developed no discernable pulmonary metastases, while TM^Pro/Pro mice each developed ∼100 grossly apparent pulmonary metastases. At the high cell dose the outcome was possibly even more striking, with few metastatic foci apparent in wild-type mice and fully confluent surface metastases too numerable to count in TM^Pro/Pro mice. Histological analyses confirmed that lung tissue from TM^Pro/Pro mice had been largely replaced with tumor. The dramatic augmentation in metastasis observed in TM^Pro/Pro mice did not appear to be due to genotype dependent differences in tumor growth potential as LLC cells transplanted into the dorsal subcutis of TM^Pro/Pro and control mice grew at similar rates and were histologically indistinguishable. Rather, tumor cell fate analyses using ¹²⁵I-radiolabeled LLC cells revealed that the imposition of the TM^Pro mutation dramatically improved the early survival of tumor cells in the lung. Twenty minutes after tumor cell injection >80% of the tumor cells were localized within the lungs regardless of animal genotypes, indicating that the TM^Pro mutation did not have a major impact on initial tumor cell adhesion/stabilization within the pulmonary vasculature. In contrast, 6 hours post-injection <20% of the initial inoculum remained in the lungs of control mice, while 75% remained in the lungs of TM^Pro/Pro mice. To determine whether the prometastatic phenotype conferred by the TM^Pro mutation is directly dependent on tumor cell-associated procoagulant function, TM^Pro/Pro and control mice were challenged with previously described fibrosarcoma cells genetically incapable of tissue factor (TF) expression, or fibrosarcoma cells in which TF expression had been genetically restored. The number of metastatic foci formed by TF-expressing cells was dramatically higher in TM^Pro/Pro mice relative to wild-type animals, whereas TF-deficient tumor cells were essentially incapable of forming metastases in mice of either genotype. Thus, the prometastatic effect of the TM^Pro mutation is contingent upon TF expression by the tumor cell. Depletion of circulating prothrombin levels to <5% of normal by an anti-sense oligonucleotide approach also profoundly limited the formation of metastatic foci in both TM^Pro/Pro and control mice, consistent with the conclusion that thrombin-TM interactions are a key determinant of metastasis. Taken together, these studies demonstrate for the first time that endothelial thrombin-thrombomodulin interactions strongly control metastatic potential and suggest that intervention at the level of thrombomodulin could represent a novel therapeutic strategy for preventing or treating metastatic disease.