Prolonged circulation of tissue-type plasminogen activator in vivo by blocking mannose receptor- and lrp-mediated liver uptake

A major complication of the use of of tissue-type plasminogen activator (t-PA) as fibrinolytic agent is its rapid clearance from the bloodstream. We have studied whether t-PA clearance can be reduced by preventing receptor-mediated endocytosis of t-PA in vivo via the mannose receptor and the low-density lipoprotein receptor related protein (LRP) in the liver. A series of synthetic cluster mannosides has been devised and validated by in vitro binding studies. The most promising mannoside, MeL5, displaying a nanomolar affinity for the mannose receptor (K,= 0.6 nM), was found to significantly inhibit 125l-tPA clearance (1 mg/kg) in rats (apparent serum half-life t,/2= 3.1±0.2 vs 1.1±0.1 min for the control; P<0.005). Although liver endothelial cells did not contribute any longer to the hepatic uptake of 1J5l-t-PA after preinjection of MjL; (2 mg/kg), an increased uptake by hepatocytes partly compensated for the reduced endothelial cell uptake of t-PA. As this parenchymal cell uptake may be mediated by LRP, it was studied whether an additional reduction in t-PA clearance could be achieved by simultaneous blocking of LRP via preinjection of the 39kDa protein (RAP; 40 mg/kg). RAP preinjection reduced the clearance of t-PA (t1/8= 5.9±0.5 min; P<0.001) and reduced liver uptake from 82.8±1.3% to 60.4 ±1.2% of the injected dose (P<0.01) The combined pretreatment with M and RAP almost fully abolished liver uptake (10.6 ±6% of the injected dose; P<0.0001 ) and t-PA clearance (tt/2=16 ± 2.5 min; P<0.0001). In conclusion, therapeutic levels of serum t-PA can be maintained for a prolonged time-span by circumventing both LRP and the mannose receptor-mediated liver uptake of t-PA in vivo.