Determinants of the distribution of radiolabeled congeners of tissue-type plasminogen activator and its modification for improved clot imaging

Background: This study was performed to elucidate determinants of the distribution in diverse tissues of radiolabeled congeners of tissue-type plasminogen activator (t-PA) and to modify the distribution to enhance clot imaging. Methods: Uptake and degradation studies in rabbit alveolar macrophages in culture were performed with native t-PA rendered enzymatically inactive with PPACK (D-Phe-L-Pro-L-Arginyl-Chloromethyl Ketone) and conjugated to a residualizing label, radioiodinated dilactitol tyramine, (¹²⁵I-DLT). Pharmacokinetics and biodistribution of radiolabeled t-PA congeners were evaluated in rabbits. Results: Concentration-dependent and saturable uptake and degradation in macrophages of ¹²⁵I-DLT-t-PA and ¹²⁵I-DLT-PPACK-t-PA were demonstrated. Uptake and degradation of ¹²⁵I- DLT-t-PA were inhibited by mannosylated bovine serum albumin (BSA) or mannan, specific ligands having high affinity for mannose fucose receptors. In vivo, clearance in rabbits was rapid after intravenous injection of both t-PA and its congeners. A high mannose- deficient t-PA congener was cleared only slightly less rapidly. Results of autoradiographic, histologic, and immunocytochemical analyses showed that clearance was associated with uptake of PPACK-t-PA in macrophages in bone marrow, spleen, and liver. Accordingly, we hypothesized that interference with high mannose-mediated macrophage uptake would reduce uptake by tissues that can interfere with clot imaging. Compared with PPACK-t-PA, accumulation of the high mannose- deficient PPACK- t-PA congener in bone-marrow and in spleen was reduced by an average of 61% and 59% respectively. Results of experiments with ligands competing for mannose fucose receptors were consistent with these observations. Uptake by liver macrophages was decreased substantially with high mannose- deficient PPACK-t-PA. Accumulation was prominent, however, along edges of hepatic sinusoids adjacent to endothelial cells. Conclusions: Thus, inhibition of the interaction of PPACK-t-PA with high mannose receptors in macrophages reduces tissue uptake that can interfere with clot imaging without compromising rapid clearance from the blood pool mediated by mannose fucose receptor-independent mechanisms in the liver.