The tissue factor (TF)/factor VIIa (FVIIa) complex not only controls hemostatic processes, but also contributes to thrombosis-related diseases and cancer. Genetic mouse models of human diseases (e.g. prostate- and colorectal cancer, sickle cell anemia) could serve to investigate further the role of TF/FVIIa in disease progression. However, such attempts are hampered by the lack of specific antimouse TF reagents. Here, we describe the generation of the monoclonal rat antimouse TF antibody 1H1. The antibody was produced by immunizing rats with recombinant soluble mouse TF(1–219) (muTF) expressed in a baculovirus expression system. Using standard hybridoma producing techniques, the 1H1 antibody (rat IgG2a/kappa) was identified by its ability to bind to muTF in an enzyme-linked immunosorbent assay (ELISA). Immunohistochemical staining of mouse frozen tissue sections with 1H1-IgG purified from ascites demonstrated specific staining in various tissues including lung and kidney. Figure 1A illustrates mouse TF expression in pulmonary alveolar epithelial cells [1,2]. Similar to human TF expression [1,2], strong mouse TF staining was also found in vascular adventitia, perineural tissue and autonomic ganglia, with weaker expression in medial smooth muscle of the renal artery (Fig. 1B).
In-vitro and in-vivo studies demonstrated that 1H1 inhibits mouse TF function. First, in clotting assays with the TF-expressing mouse melanoma cell line B16F10, we found 1H1 to prolong mouse plasma clotting in a concentration-dependent fashion (Fig. 1C). This was also observed in clotting assays with recombinant muTF and phospholipid vesicles. Similar to other function-blocking antibodies such as AP-1 [3,4], inhibitory activity was observed only when antibody and TF were allowed to preincubate before addition of plasma. Secondly, in a mouse lung metastasis model, B16F10 cells (0.1 × 106 cells) preincubated with 0.7 mg mL−1 of 1H1 or phosphate-buffered saline (PBS) (control) were injected via tail vein to isogenic C57Bl6J mice (8–12 weeks of age). After 2 weeks, control animals developed 47 ± 6 (n = 15, ± SEM) visible lung tumor foci. Treatment with 1H1 reduced the number of tumor foci by 68% (Fig. 1D) to 15 ± 2 (n = 15, P < 0.0001). These results demonstrated that 1H1 efficiently inhibits TF function in vivo and indicate that TF activity is essential for B16F10 melanoma cell metastasis, consistent with studies using murine tissue factor pathway inhibitor  or the thrombin inhibitor desulfatohirudin .
In conclusion, the monoclonal 1H1 antibody represents a useful reagent to investigate further the biology and pathophysiology of TF in mouse models.