STUDIES ON THE EFFECT OF FUCOSYLATED AND NONFUCOSYLATED FINGER GROWTH-FACTOR CONSTRUCTS ON THE CLEARANCE OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR MEDIATED BY THE LOW-DENSITY-LIPOPROTEIN-RECEPTOR-RELATED PROTEIN/

Previously we observed that the finger/growth factor (FG) region of ti ssue-type plasminogen activator (t-PA) blocked the low-density-lipopro tein-receptor-related protein (LRP)-mediated clearance of t-PA by rat hepatocytes. However, the concentrations needed were much higher than those for intact t-PA. The FG region was expressed in yeast and lacked the fucose of Thr61, which was reported to be important for efficient clearance of t-PA by human hepatoma cells. At position 83 it had a se rine, whereas human t-PA has a free cysteine and rodent t-PA an argini ne at this position. To understand the reason for the low efficacy of the FG protein we produced in CHO cells chimeric molecules composed of two FG modules linked to the Fc portion of human IgG1 (FG(2)-Fc). Two variants were studied, one having Ser3, the other Arg83. The two fuc osylated FG(2)-Fc chimeras were compared with each other, with non-fuc osylated FG and with intact t-PA with regard to their effect on the cl earance of t-PA and t-PA . plasminogen-activator inhibitor type 1 (PAI -1). For this comparison, LRP-specific clearance models were used. In rat hepatoma cells and in mouse embryonic fibroblasts (MEF-1) the clea rance of t-PA and of t-PA . PAI-1 was inhibited more than 95% by recep tor-associated protein, an inhibitor of LRP-mediated clearance, wherea s no t-PA or t-PA . PAI-1 clearance was observed in LRP-deficient PEA- 13 mouse embryonic fibroblasts. The Ser83 and Arg83 FG(2)-Fc chimeras were equally efficient inhibitors in these models. Their efficacies in inhibiting t-PA and t-PA . PAI-1 degradation (IC50 750 mM and 890 nM, respectively) were similar to those of non-fucosylated FG (IC50 1950 nM and 1560 nM) and 75-fold lower than that of intact t-PA (IC50 9.9 n M and 21.1 nM). The results indicate that the presence of a serine of an arginine at position 83 and the presence of a fucose on Thr61 are n ot of major importance for the LRP-mediated clearance of t-PA.