The Role of the Low-Density Lipoprotein Receptor-Related Protein (LRP) in the Plasma Clearance and Liver Uptake of Recombinant Single-chain Urokinase-type Plasminogen Activator in Rats

 

PDF Download Buy Article Permissions and Reprints

Summary

Urokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of ¹²⁵I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91 % and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= deltal25- rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of ¹²⁵I-deltal25-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

• References

• 1 Kasai S, Arimura H, Nishida M, Suyama T. Proteolytic cleavage of singlechain pro-urokinase induces a conformational change which follows activation of the zymogen and reduction of its high affinity for fibrin. J Biol Chem 1985; 1260: 12377-12381
  PubMedGoogle Scholar
• 2 Wijngaards G, van Rijken DC, Wezel AL, van der Groeneveld E, Velden CAM. Characterization and fibrin-binding properties of different molecular forms of pro-urokinase from a monkey kidney cell culture. Thromb Res 1986; 42: 749-760
  CrossrefPubMedGoogle Scholar
• 3 DanØ K, Andreasen PA, Grondahl-Hansen J, Kristensen P, Nielsen LS, Skriver L. Plasminogen activators, tissue degradation and cancer. Adv CancerRes 1985; 44: 139-134
  PubMedGoogle Scholar
• 4 Eaton DL, Scott RW, Baker JB. 1984 Purification of human fibroblast urokinase proenzyme and analysis of its regulation by proteases and protease nexin. J Biol Chem 1984; 259: 6241-6247
  PubMedGoogle Scholar
• 5 Gurewich V, Pannel R, Louie S, Kelley P, Suddith RL, Greenlee R. Effective and fibrin-specific clot lysis by a zymogen precursor form of urokinase (pro-urokinase): a study in vitro and in two animal species. J Clin Invest 1984; 73: 1731-1739
  CrossrefPubMedGoogle Scholar
• 6 Rijken DC, Binnema DJ, Los P. Specific fibrinolytic properties of different molecular forms of pro-urokinase from a monkey kidney cell culture. Thromb Res 1986; 42: 761-768
  CrossrefPubMedGoogle Scholar
• 7 Collen D, Zamarron DC, Lijnen HR, Hoylaerts M. Activation of plasminogen by pro-urokinase. J Biol Chem 1986; 261: 1259-1266
  PubMedGoogle Scholar
• 8 van Lijnen HR, Hoef B, Collen D. Comparative kinetic analysis of the activation of human plasminogen by natural and recombinant single-chain urokinase-type plasminogen activator. Biochim Biophys Acta 1986; 884: 402-408
  CrossrefPubMedGoogle Scholar
• 9 Liu J, Pannell R, Gurewich V. A transitional state of pro-urokinase that has a higher catalytic efficiency against glu-plasminogen than urokinase. J Biol Chem 1992; 267: 15289-15292
  PubMedGoogle Scholar
• 10 Pannell R, Gurewich V. Pro-urokinase: a study of its stability in plasma and of a mechanism for its selective fibrinolytic effect. Blood 1986; 67: 1215-1223
  PubMedGoogle Scholar
• 11 Loscalzo J, Wharton TP, Kirschenbaum JM, Levine HJ, Flaherty JT, Topol EJ, Ramaswamy K, Kosowsky BD, Salem DN, Ganz P, Brinker JA, Gurewich V, Muller JE. and the Pro-urokinase for Mycardial infarction study group Clot-selective coronary thrombolysis with pro-urokinase. Circulation 1989; 79: 776-782
  CrossrefPubMedGoogle Scholar
• 12 PRIMI trial study group. A randomised double blind trial of recombinant prourokinase against streptokinase in acute myocardial infarction. Lancet. 1989: 863-868
  Google Scholar
• 13 Collen D, de Cock F, Lijnen HR. Biological and thrombolytic properties of proenzyme and active forms of human urokinase. Turnover of natural and recombinant urokinase in rabbits and squirrel monkeys. Thromb Haemost 1984; 52: 24-26
  PubMedGoogle Scholar
• 14 Spriggs DJ, Stassen JM, Hashimoto Y, Collen D. Thrombolytic properties of human tissue-type plasminogen activator single-chain urokinase-type plasminogen activator, and synergistic combinations in venous thrombosis models in dogs and rabbits. Blood 1989; 73: 1207-1212
  PubMedGoogle Scholar
• 15 Stump DC, Lijnen HR, Collen D. Purification and characterization of a novel low molecular weight form of single-chain urokinase-type plasminogen activator. J Biol Chem 1986; 261: 17120-17126
  PubMedGoogle Scholar
• 16 Ueno T, Kobayashi N, Maekawa T. Studies on metabolism of urokinase and mechanism of thrombolysis by urokinase. Thromb Haemost 1979; 42: 885-894
  Article in Thieme ConnectPubMedGoogle Scholar
• 17 Kuiper J, Rijken DC, de MunkGAW, van BerkelThJC. In vivo and in vitro interaction of high and low molecular weight single-chain urokinase-type plasminogen activator with rat liver cells. J Biol Chem 1992; 267: 1589-1595
  PubMedGoogle Scholar
• 18 Van der KaadenME, Kuiper J, Groeneveld E, van BerkelThJC, Rijken DC. Clearance of urinary urokinase-type plasminogen activator by the asialoglyco-protein receptor and proteoglycans on rat parenchymal liver cells (Submitted).
  PubMedGoogle Scholar
• 19 Van derKaaden M E, Rijken DC, van GroeneveldE, Berkel ThJC, Kuiper J. Native and non-glycosylated recombinant single-chain urokinase-type plasminogen activator are recognized by different receptor systems on rat parenchymal liver cells. Thromb Haemost 1995; 74: 722-729
  Article in Thieme ConnectPubMedGoogle Scholar
• 20 Nykjaer A, Petersen CM, Moller B, Jensen PH, Moestrup SK, Holtet TL, Etzerodt M, Thogersen HC, Munch M, Andreasen PA, Gliemann J. Purified α₂-macroglubulin receptor/LDL receptor-related protein binds urokinase plasminogen activator inhibitor type-1 complex: Evidence that the α₂-macroglubulin receptor mediated cellular degradation of urokinase receptor bound complexes. J Biol Chem 1992; 267: 14543-14546
  PubMedGoogle Scholar
• 21 Herz J, Kowal RC, Goldstein JL, Brown MS. Proteolytic processing of the 600 kd low density lipoprotein receptor-related protein (LRP) occurs in a trans-Golgi compartment. EMBO J 1990; 09: 1769-1776
  PubMedGoogle Scholar
• 22 Ashcom JD, Tiller SE, Dickerson K, Cravens JL, Argraves WS, Strickland DK. The human α₂-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of α₂-macro-globulin. J Cell Biol 1990; 110: 1041-1048
  CrossrefPubMedGoogle Scholar
• 23 Moestrup SK, Gliemann J. Purification of the rat hepatic α-macroglobulin receptor as an approximately 440-kDa single chain protein. J Biol Chem 1989; 264: 15574-15577
  PubMedGoogle Scholar
• 24 Herz J, Hamann U, Rogne SM, Myklebost O, Gausepohl H, Stanley KK. Surface location and high affinity for calcium of a 500 kD liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor. EMBO J 1988; 7: 4119-4127
  PubMedGoogle Scholar
• 25 Strickland DK, Ashcom JD, Williams S, Burgess WH, Migliorini M, Argraves WS. Sequence identity between the α-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor. J Biol Chem 1990; 265: 17401-17404
  PubMedGoogle Scholar
• 26 Bu G, Geuze HJ, Strous GJ, Schwartz AL. 39 kDa receptor-associated protein and molecular chaperone for LDL receptor-related protein. EMBOJ 1995; 14: 2269-2280
  PubMedGoogle Scholar
• 27 Kowal RC, Herz J, Weisgraber KH, Mahley RW, Brown MS, Goldstein JL. Opposing effects of apolipoproteins E and C on lipoprotein binding of low density lipoprotein receptor-related protein. J Biol Chem 1990; 265: 10771-10779
  PubMedGoogle Scholar
• 28 Beisiegel U, Weber W, Bengtsson-Olivecrona G. Lipoprotein lipase enhances the binding of chylomicron remnants to low density lipoprotein receptor-related protein. Proc Natl Acad Sci USA 1991; 88: 8342-8646
  CrossrefPubMedGoogle Scholar
• 29 Kounnas MZ, Morris RE, Thompson MR, Fitzgerald DJ, Strickland DK, Saelinge CB. The α₂-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas Pexotoxin A. J Biol Chem 1992; 267: 12420-12423
  PubMedGoogle Scholar
• 30 Bu G, Williams S, Strickland DK, Schwartz AL. Low density lipoprotein receptor-related protein/α₂MR is a hepatic receptor for tissue-type plasminogen activator. Proc Natl Acad Sci USA 1992; 89: 7427-7431
  CrossrefPubMedGoogle Scholar
• 31 Kounnas MZ, Henkin J, Argraves SW, Strickland DK. Low density lipoprotein receptor-related protein/α-macroglobulin receptor mediates cellular uptake of pro-urokinase. J Biol Chem 1993; 268: 21862-21867
  PubMedGoogle Scholar
• 32 Herz J, Clouthier DE, Hammer RE. LDL receptor-related protein internalizes and degrades uPA-PAI-1 complexes and is essential for embryo implantation. Cell 1992; 71: 411-421
  CrossrefPubMedGoogle Scholar
• 33 Orth K, Madison EL, Gething MJ, Sambrook JF, Herz J. Complexes of tissuetype plasminogen activator and its serpin inhibitor plasminogen-activator inhibitor type 1 are internalized by means of the low density lipoprotein receptor-related protein/α-macroglobulin receptor. Proc Natl Acad Sci USA 1992; 89: 7422-7426
  CrossrefPubMedGoogle Scholar
• 34 Willnow TE, Armstrong SA, Hammer RE, Herz J. Functional expression of low density lipoprotein receptor-related protein is controlled by receptor associated protein in vivo. Proc Natl Acad Sci USA 1995; 92: 4537-4541
  CrossrefPubMedGoogle Scholar
• 35 Orsini G, Brandazza A, Sarmientos P, Molinari A, Lansen J, Cauet G. Efficient renaturation and fibrinolytic properties of pro-urokinase and a deletion mutant expressed in Escherichia coli as inclusion bodies. Eur J Biochem 1991; 195: 691-697
  CrossrefPubMedGoogle Scholar
• 36 Van DijkM CM, Ziere GJ, Boers W, Linthorst C, Bijsterbosch MK, van BerkelThJC. Recognition of chylomicron remnants and β-migrating very-low-density lipoproteins by the remnant receptor of parenchymal liver cells is distinct from the liver α2-macroglobulin-receptor site. Biochem J 1991; 279: 863-870
  CrossrefPubMedGoogle Scholar
• 37 Davidsen o, Christensen El, Gliemann J. The plasma clearance of human α2-macroglobulin-trypsin complex in the rat is mainly accounted for by uptake into hepatocytes. Biochim Biophys Acta 1985; 846: 85-92
  CrossrefPubMedGoogle Scholar
• 38 Herz J, Goldstein JL, Strickland DK, Ho YK, Brown MS. 39 kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/;-macroglobulin receptor. 1991; J Biol Chem 1991; 266: 21232-21238
  CrossrefPubMedGoogle Scholar
• 39 Bijsterbosch MK, Ziere GJ, van BerkelThJC. Lactosylated low density lipoprotein: a potential carrier for the site-specific delivery of drugs to Kupffer cells. Mol Pharmacol 1989; 36: 484-489
  PubMedGoogle Scholar
• 40 Caster WO, Simon AB, Armstrong WD. Evans blue space in tissues of the rat. Am J Physiol 1955; 183: 317-321
  PubMedGoogle Scholar
• 41 Kuiper J, Otter M, Rijken DC, van BerkelThJC. Characterization of the interaction in vivo of tissue-type plasminogen activator with liver cells. J Biol Chem 1988; 263: 18220-18224
  PubMedGoogle Scholar
• 42 Van Berkel ThJC, de Rijke YB, Kruyt JK. Differential fate in vivo of oxidatively modified LDL and acetylated LDL in rats. Recognition by various scavenger receptors on Kupffer and endothelial cells. J Biol Chem 1991; 266: 2282-2289
  PubMedGoogle Scholar
• 43 Nagelkerke JF, Barto KP, van BerkelThJC. In vivoandin vitrouptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer and parenchymal cells. J Biol Chem 1983; 258: 12221-12227
  PubMedGoogle Scholar
• 44 Van BerkelThJC, Dekker CJ, Kruyt JK, van Eijk HG. The interaction of transferrin and asialotransferrin with liver cells. Biochem J 1987; 243: 715-722
  CrossrefPubMedGoogle Scholar
• 45 Seglen PO. Isolation of liver cells. Meth Cell Biol 1976; 13: 29-83
  PubMedGoogle Scholar
• 46 van CasteleynE, Rooij HJC, van BerkelThJC, Koster JF. Mechanism of glucagon stimulation of fructose-1,6-biphosphatase in rat hepatocytes. Involvement of a low-Mr activator. FEBS Lett 1986; 201: 193-197
  CrossrefPubMedGoogle Scholar
• 47 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 255: 6111-6120
  PubMedGoogle Scholar
• 48 Van Berkel ThJC, Kruijt JK, Spanjer HH, Nagelkerke JF, Harkes L, Kempen HJM. The efficacy of a water-soluble tris-galactoside-terminated cholesterol derivative on the fate of low density lipoproteins and liposomes. J Biol Chem 1983; 260: 2694-2699
  PubMedGoogle Scholar
• 49 Van BerkelThJC, Kruijt JK, Kempen HJM. Specific targeting of high density lipoprotein to liver hepatocytes by incorporation of tris-galactoside-terminated cholesterol derivative. J Biol Chem 1985; 260: 12203-12207
  PubMedGoogle Scholar
• 50 van Dijk, MC M, Boers W, Linthorst C, Van BerkelThJC. Role of the scavenger receptor in the uptake of methylamine-activated α-macroglobu-lin by rat liver. Biochem J 1992; 287: 447-455
  CrossrefPubMedGoogle Scholar
• 51 Zheng G, Bachinsky DR, Stamenkovic I, Strickland DK, Brown D, Andres G, McCluskey RT. Organ distribution in rats of two members of the low-density lipoprotein receptor gene family, Gp330 and LRP/α2MR, and the receptor associated protein (RAP). J of Histochem and Cytochem 1994; 42: 531-542
  PubMedGoogle Scholar
• 52 Nykjaer A, Kjoller L, Cohen RL, Lawrence DA, Gami-Wagner BA, Todd III RF, van ZonneveldA-J, Gliemann J, Andreassen PA. Regions involved in binding of urokinase-type-1 inhibitor complex and pro-urokinase to the endocytic α-macroglobulin receptor/low density lipoprotein receptor-related protein. J Biol Chem 1994; 269: 25668-25676
  PubMedGoogle Scholar
• 53 Breton J, Pezzi N, Molinari A, Bonomini L, Lansen J, Gonzalez de, Buitargo G, Prieto I. Prolonged half-life in the circulation of a chemical conjugate between a pro-urokinase derivative and human serum albumin. Eur J Biochem 1995; 231: 563-569
  CrossrefPubMedGoogle Scholar
• 54 Appella E, Robinson EA, Ullrich SJ, Stoppelli MP, Corti A, Cassani G, Blasi F. The receptor-binding sequence of urokinase. A biological function for the growth factor module of proteases. J Biol Chem 1987; 262: 4437-4440
  PubMedGoogle Scholar
• 55 Estreicher A, Wohlwend A, Belin D, Schleuning WD, Vassalli JD. Characterization of the cellular binding site for the urokinase-type plasminogen activator. J Biol Chem 1989; 264: 1180-1189
  PubMedGoogle Scholar
• 56 Rabbani SA, Rajwans N, Achbarou A, Murthy KK, Goltzman D. Isolation and characterization of multiple isoforms of the rat urokinase receptor in osteoblasts. FEBS Lett 1994; 338: 69-74
  CrossrefPubMedGoogle Scholar
• 57 Pannell R, Gurewich V. Pro-urokinase: a study of its stability in plasma and of a mechanism for its selective fibrinolytic effect. Blood 1986; 5: 1215-1223
  PubMedGoogle Scholar
• 58 Kuiper J, Kamps JAAM, van Berkel ThJC. Identification of the inhibitor of the plasminogen activator as the major secreted protein by endothelial rat liver cells. FEBS Lett 1989; 245: 229-234
  CrossrefPubMedGoogle Scholar
• 59 Quax PHA, van denHoogen C M, Verheijen JH, Padro T, Zeheb R, Gelehrter TD, Van BerkelThJC, Kuiper J, Emeis JJ. Endotoxin induction of plasminogen activator and plasminogen activator inhibitor type-1 mRNA in rat tissues in vivo. J Biol Chem 1990; 265: 15560-15563
  CrossrefPubMedGoogle Scholar