Asialoglycoprotein Receptor (ASGP-R) is Liver Parenchymal Cells is Involved in Elimination of Recombinant Human TFPI

Abstract

We here report on a study carried out to determine the early clearance kinetics, and organ, cell(s) and receptor(s) responsible for the clearance of full length TFPI purified from BHK cells (TFPI^BHK). Following intravenous administration, ¹²⁵I-TFPI^BHK was cleared with a biphasic elimination curve, and with a significantly slower t_1/2α compared to recombinant human TFPI from <i>E.Coli</i> (TFPI^E.Coli) (1.95±0.10 versus 1.42±0.07 min, respectively, p<0.001). Studies on organ and cell distribution revealed that liver parenchymal cells (PCs) were responsible for 96% of the uptake of TFPI^BHK and 81% of TFPI^E.Coli, whereas the nonparenchymal cells (NPCs) were responsible for 4% and 19%, respectively. Pre-administration of excessive amounts of unlabeled TFPI^BHK prolonged blood clearance of ¹²⁵I-TFPI^BHK. Unlabelled TFPI^BHK inhibited endocytosis of ¹²⁵I-TFPI^BHK in PCs <i>in vitro</i>, whereas blocking of LDL-receptor related protein-1 (LRP-1) by receptor-associated protein (RAP) affected neither blood clearance nor endocytosis of ¹²⁵I-TFPI^BHK in PCs. In addition, TFPI^BHK was also found in the kidneys and this could be reduced in the presence of RAP. Asialoorosomucoid (ASOR), a potent inhibitor of the asialoglycoprotein receptor (ASGP-R), prolonged the circulatory survival of ¹²⁵I-m-TFPI by 1.5-fold (p<0.001). <i>In vitro</i>, ASOR and other ASGP-R antagonists significantly inhibited endocytosis of ¹²⁵I-TFPI^BHK in PCs. Moreover, unlabelled TFPI^BHK markedly decreased endocytosis of ¹²⁵I-asialofetuin. In conclusion, our findings suggest that ASGP-R mediated endocytosis in the liver is involved in the clearance of TFPI^BHK.