Adhesive interactions between cells and the subendothelial extracellular matrix take place at several stages during tumor progression and metastasis. We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which can be exposed in the presence of low concentrations of plasmin and cell-associated heparan sulfate proteoglycan. Thus, thrombin may act as a matrix-adhesive molecule via activation of the alpha v beta 3 integrin. We have now identified a 31 amino acid fragment as the minimal thrombin-generated cleavage product, which contains an active RGD site, following gel filtration analysis on FPLC Superdex 75 column. The role of membrane-associated heparan sulfate in thrombin conversion to an adhesive protein was demonstrated by using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Incubation of both thrombin and a low concentration of plasmin on the surface of wild type CHO cells resulted in a typical digestion cleavage profile upon gel filtration. No cleavage products were observed when thrombin and a suboptimal plasmin concentration were incubated on monolayers of CHO cell mutants lacking heparan sulfate. Next, we examined the possible role of the thrombin RGD site during the progression of tumor development and metastasis. Toward this, we tested murine melanoma cells expressing low (B16-F1 cells) and high (B16-BL6 cells) lung colonization potentials in cell adhesion assays in vitro. Differential adherence capability of the cells was observed: while high attachment levels of B16-BL6 cells were obtained, the low metastatic B16-F1 cells did not adhere to thrombin RGD. Antibodies raised against the RGD site in thrombin specifically recognized thrombin digested with plasmin, but were unable to interact with native thrombin or prothrombin and inhibited potently B16-BL6 melanoma cell adhesion. Furthermore, the antibodies failed to recognize RGD in other adhesive plasma proteins such as vitronectin, fibrinogen, or fibronectin. Provided that the RGD-containing fragments of thrombin are widely distributed throughout the vascular system, they may have a significant role during tumor progression and dislodgement of metastatic cells. The development of RGD mimetics and/or specific antibodies might thus be applied to inhibit a critical step in metastatic spread.