| Popis: |
The plasminogen activators, t-PA and u-PA, are glycoproteins known to be involved in homeostasis of the blood clotting system, and thus are of potential clinical use in the treatment of thrombosis. Several in vivo studies have shown that both t-PA and u-PA are quickly removed from the blood circulation, predominantly by the liver. The mechanism by which the liver removes these proteins is not understood. To delineate this, we conducted in vitro studies of binding of PAs or their derivatives to isolated mouse liver membranes utilizing a functional assay developed in our laboratory. The assay consisted of initial binding of t-PA to liver membranes followed by centrifugation to pellet the membranes and the assay of the activity of the membrane-bound t-PA by a fibrin-agar plate method. The bound t-PA, which retained complete enzymic activity, could be dissociated by SDS treatment in an undegraded form as shown by SDS-PAGE. The binding of t-PA as well as u-PA was very fast and did not compete with glycoproteins or sugars containing the terminal galactose, mannose and N-acetylglucosamine residues. Furthermore, the treatment of t-PA with neuraminidase and/or periodate oxidation did not affect its binding characteristics. These data suggest that the carbohydrate moieties of t-PA and u-PA, unlike many glycoproteins, do not mediate their binding to the liver. This raised the possibility of the liver binding sequence being located in the protein backbone, especially the non-protease domains which are known to determine the biological specificities of PAs. The relative binding of u-PA and its low molecular weight (LMW) derivative containing only the protease domain, to the liver membranes was studied. Unlike u-PA and t-PA, LMW-urokinase did not bind significantly. This suggests that the protein sequence containing the non-protease domains, rather than the carbohydrate moieties of PAs contain the information necessary for binding to the liver and possibly their clearance from the blood circulation. |
