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 (t1/2 (α) = 0.6 min, and t1/2 (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 t1/2 (α) to 48% and 21%, respectively. A corresponding increase in the β-elimination ot
t-PA was observed. The t1/2 (α) and t1/2 (β) were unchanged. Studies on the eleaiaucc of 125I-ovalbumin also showed a biphasic elimination with an initial rapid phase, t1/2 (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
