Clearance of Tissue Plasminogen Activator by Mannose and Galactose Receptors in the Liver

Summary

The mechanism of uptake of radio-iodinated tissue plasminogen activator (¹²⁵I-t-PA) was studied in rats. When trace amounts of ¹²⁵I-t-PA were injected alone, the clearance followed a biphasic pattern in which 65% and 35% were cleared with α- and β-kinetics (t_1/2 (α) = 0.6 min, and t_1/2 (β) = 6.4 min), respectively. Coinjection with excess unlabelled t-PA or inannan changed the uptake kinetics to the muiiupliasic β-elimination pattern. Mannosylated albumin and ovalbumin, both of which bind to the hepatic mannose receptor, reduced the proportion of t-PA cleared with t_1/2 (α) to 48% and 21%, respectively. A corresponding increase in the β-elimination ot t-PA was observed. The t_1/2 (α) and t_1/2 (β) were unchanged. Studies on the eleaiaucc of ¹²⁵I-ovalbumin also showed a biphasic elimination with an initial rapid phase, t_1/2 (α), accounting for only 39% of the clearance of ovalbumin, as compared to 65% in the case of t PA. Macromolecules with affinity for the galactose-receptor only, such as asialofetuin, or galactosylated albumin, did not significantly affect the clearance kinetics at the concentrations used. Asialoorosomucoid, which also carries galactosyl residues in the terminal position, reduced somewhat (from 65% to 48%) the proportion cleared with α-kinetics. Very high concentrations of galactose and N-acetyl-galactosamine, which are also known to compete for binding to the galactose receptor, lowered the proportion of t-PA cleared in the late β-phase (reduced from 35% to 26% with galactose and to 19% with N-acetyl-galactosamine).

To determine the hepatocellular site of uptake of t-PA, the protein was conjugated with ¹²⁵I-labelled tyramine cellobiose (¹²⁵I-TC) and injected intravenously (i.v.). This adduct is nonbiodegradable, and is trapped intralysosomally after endocytosis. I.v. injection of ¹²⁵I-TC-t-PA and subsequent isolation of the liver cells showed that the Kupffer cells (KC), liver endothelial cells (LEC) and parenchymal cells (PC) contained 11%, 44% and 45%, respectively, of the radioactive label recovered in liver (hepatic uptake 80% of injected dose). The in vivo uptake per cell was about three times higher in KC and LEC than in PC. Injection of ¹²⁵I-TC-t-PA together with mannose inhibited uptake in LEC and increased uptake in PC. Conversely, co-injection with galactose inhibited the uptake of ¹²⁵I-TC-t-PA in PC and increased the uptake in LEC. Co-injection with excess amounts of unlabelled t-PA shifted the site of uptake from LEC to PC and changed the clearance kinetics to a monophasic β-elimination. The inhibitors used had only marginal effects on the uptake of ¹²⁵I-TC-t-PA in KC. Although significant amounts of label were recovered in KC, the total size of the population of these cells is relatively small, so that the main hepatic uptake of ¹²⁵I-TC-t-PA was in LEC and PC.

In conclusion, the elimination of t-PA from the blood by the liver is strongly dependent on the structure of its carbohydrate side chains. The main cellular sites of clearance are LEC (via mannose receptors), and PC (via galactose receptors and an unsaturable noncarbohydrate uptake mechanism).

Keywords

Tissue plasminogen activator - Ovalbumin - ¹²⁵I-tyramine cellobiose - Clearance - Kinetics - Liver cells - Receptor - Rat

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