Recombinant human prothrombin kringles have potent anti-angiogenic activities and inhibit Lewis Lung Carcinoma tumor growth and metastases.

TH Kim, EK Kim, DW Yoon, Jaebeum Kim, TaiYoun Rhim, SS Kim.

Department of Biochemistry, College of Science, Yonsei University, 120-749, Seoul, Korea

Prothrombin, a protein involved in blood coagulation, is a plasma glycoprotein which is composed of the Gla domain, two adjacent kringle domains, and a serine protease domain. Kringles are three-disulfide triple-loop folding domains which are found in several other blood proteins. Previously, we reported that the rabbit prothrombin kringle-2 and human prothrombin fragments had the anti-endothelial cell proliferative activity (Lee et al., J. Biol. Chem.,1998 ,Rhim et al., Biochem. Biophys. Res. Commun.,1998). In this study, we showed that recombinant human prothrombin kringle-1,-2,and1-2 also have potent anti-angiogenic activities and recombinant human prothrombin kringle-1,-2,and-1-2 all inhibit Lewis lung carcinoma tumor growth and metastases. Recombinant human prothrombin kringles were expressed in E.coli expression system and purified to apparent homogeneity from E.coli crude extracts. Purified recombinant human prothrombin kringle-1,-2,and-1-2 migrated with a molecular mass of 14 kDa, 19 kDa, and 31 kDa on SDS-PAGE under reducing condition. The recombinant human prothrombin kringle-1,-2,and-1-2 exhibited potent inhibitory effects on bFGF-stimulated BCE cell growth with half-maximal concentrations(ED50) of approximately 41 nano;M, 55 nano;M, and 156 nano;M, respectively. All of the recombinant human prothrombin kringles also inhibited angiogenesis in the chorioallantoic membrane (CAM) of chick embryos at a dose of 20 µg. Systemic administration of recombinant human prothrombin kringle-1,-2,and-1-2 at a dose of 0.5 mg/kg/day suppressed the growth of primary Lewis Lung Carcinoma and at dose of 0.5 mg/kg/day and 1.0 mg/kg/day inhibited the Lewis Lung Carcinoma metastases to lung in C57BL6/J mice.

For more information, contact

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Angiogenesis.