Download PDF
Thromb Haemost 1995; 74(06): 1501-1510
DOI: 10.1055/s-0038-1649973
Original Articles
Fibrinolysis
Schattauer GmbH Stuttgart

Uptake, Internalization and Degradation of the Novel Plasminogen Activator Reteplase (BM 06.022) in the Rat

J Kuiper
1   The Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Sylvius Laboratory, Leiden, The Netherlands
,
H van de Bilt
1   The Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Sylvius Laboratory, Leiden, The Netherlands
,
U Martin
2   The Dept. of Pharmacology, Boehringer Mannheim, Mannheim, Germany
,
Th J C van Berkel
1   The Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Sylvius Laboratory, Leiden, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 13 October 1994

Accepted after resubmission 08 September 1995

Publication Date:
27 July 2018 (online)

  PDF Download  Permissions and Reprints

Summary

The catabolism of the novel plasminogen activator reteplase (BM 06.022) was described. For this purpose BM 06.022 was radiolabelled with l25I or with the accumulating label l25I-tyramine cellobiose (l25I-TC).

BM 06.022 was injected at a pharmacological dose of 380 μg/kg b.w. and it was cleared from the plasma in a biphasic manner with a half-life of about 1 min in the α-phase and t1/2of 20-28 min in the β-phase. 28% and 72% of the injected dose was cleared in the α-phase and β-phase, respectively. Initially liver, kidneys, skin, bones, lungs, spleen, and muscles contributed mainly to the plasma clearance. Only liver and the kidneys, however, were responsible for the uptake and subsequent degradation of BM 06.022 and contributed for 75% to the catabolism of BM 06.022. BM 06.022 was degraded in the lysosomal compartment of both organs. Parenchymal liver cells were responsible for 70% of the liver uptake of BM 06.022. BM 06.022 associated rapidly to isolated rat parenchymal liver cells and was subsequently degraded in the lysosomal compartment of these cells. BM 06.022 bound with low-affinity to the parenchymal liver cells (550 nM) and the binding of BM 06.022 could be displaced by t-PA (IC50 5.6 nM), indicating that the low-density lipoprotein receptor-related protein (LRP) could be involved in the binding of BM 06.022. GST-RAP, which is an inhibitor of LRP, could in vivo significantly inhibit the uptake of BM 06.022 in the liver.

It is concluded that BM 06.022 is metabolized primarily in the liver and the kidneys. These organs take up and degrade BM 06.022 in the lysosomes. The uptake mechanism of BM 06.022 in the kidneys is unknown, while LRP is responsible for a low-affinity binding and uptake of BM 06.022 in parenchymal liver cells.

 

  • References

  • 1 The GUSTO Investigators. An international randomized trial comparing lour thrombolytic strategies for aeute myocardial infarction. N Engl J Med. 1993 329. 673-682
    PubMedGoogle Scholar
  • 2 Kuiper J, Otter M, Rijken DC, van Berkel Th JC. Characterization of the interaction in vivo of t-PA with liver cells. J Biol Chem 1988; 263: 18220-18224
    PubMedGoogle Scholar
  • 3 Einarsson M, Smedsrφd B, Pertoft H. Uptake and degradation of t-PA in rat liver. Thromb Haemost 1988; 59: 474-479
    Article in Thieme ConnectPubMedGoogle Scholar
  • 4 Smedsrφd B, Einarsson M, Pertoft H. t-PA is endocytosed by mannose and galactose receptors on rat liver cells. Thromb Haemost 1988; 59: 480-484
    Article in Thieme ConnectPubMedGoogle Scholar
  • 5 Bakhit C, Lewis D, Billings R, Malfroy B. Cellular catabolism of recombinant t-PA. J Biol Chem 1987; 262: 8716-8720
    PubMedGoogle Scholar
  • 6 Otter M, Kuiper J, Bos R, van Berkel Th JC, Rijken DC. Characterization of the interaction both in vivo and in vitro of tissue-type plasminogen activator with rat liver cells. Biochem J 1992; 284: 601-605
    PubMedGoogle Scholar
  • 7 Bu G, Williams S, Strickland DK, Schwartz AL. Low density lipoprotein receptor related protein/α2-macroglobulin receptor is a hepatic receptor for tissue-type plasminogen activator. Proc Natl Acad Sci USA 1992; 89: 7427-7431
    CrossrefPubMedGoogle Scholar
  • 8 Bu G, Warshawsky I, Schwartz AL. Cellular receptors for the plasminogen activators. Blood 1994; 83: 3427-3436
    PubMedGoogle Scholar
  • 9 Warshawsky I, Bu G, Schwartz AL. 39 kD protein inhibits tissue-type plasminogen activator clearance in vivo. J Clin Invest 1993; 92: 937-944
    CrossrefPubMedGoogle Scholar
  • 10 Hotchkiss A, Refino CJ, Leonard CK, O’Connor JV, Crowley C, McCabe J, Tate K, Nakamura G, Powers D, Levinson A, Mohler K, Spellman MW. The influence of carbohydrate structures on the clearance of recombinant t-PA. Thromb Haemost 1988; 60: 255-261
    Article in Thieme ConnectPubMedGoogle Scholar
  • 11 Krause J. Catabolism of t-PA, its variants, mutants and hybrids. Fibrinolysis 1988; 2: 133-142
    PubMedGoogle Scholar
  • 12 Ord JM, Owensby DA, Billadello JJ, Sobel BE. Determinants of clearance of t-PA and their pharmacological implications. Fibrinolysis 1990; 4: 203-209
    PubMedGoogle Scholar
  • 13 Hansen L, Blue Y, Barone K, Collen D, Larsen GR. Functional effects of asparigine-linked oligosaccharide on natural and variant human tissue-type plasminogen activator. J Biol Chem 1988; 263: 15713-15719
    PubMedGoogle Scholar
  • 14 Wilhelm J, Kalyan NK, Lee SG, Hum WT, Rappaport R, Hung PP. Deglycosylation increases the fibrinolytic activity of a deletion mutant of tissue-type plasminogen activator. Thromb Haemost 1990; 63: 464-471
    Article in Thieme ConnectPubMedGoogle Scholar
  • 15 Kohnert U, Rudolph R, Verheijen JH, Weening-Verhoeff EJ D, Stem A, Opitz U, Martin U, Lill H, Prinz H, Lechner M, Kresse GB, Buckel P, Fisher S. Biochemical properties of the kringle 2 and protease domain are maintained in the refolded t-PA deletion variant BM 06.022. Protein Engineering 1992; 5: 93-100
    CrossrefPubMedGoogle Scholar
  • 16 Martin U, von Mollendorf E, Akpan W, Kientsch-Engel R, Kaufmann B, Neugebauer G. Dose-ranging study of the novel recombinant plasminogen activator BM 06.022 in healthy volunteers. Clin Pharmacol Ther 1991; 50: 429-436
    CrossrefPubMedGoogle Scholar
  • 17 Martin U, von Mollendorf E, Akpan W, Kientsch-Engel R, Kaufmann B, Neugebauer G. Pharmacokinetic and hemostatic properties of the recombinant plasminogen activator BM 06.022 in healthy volunteers. Thromb Haemost 1991; 66: 569-574
    Article in Thieme ConnectPubMedGoogle Scholar
  • 18 Neuhaus KL, von Essen R, Vogt A, Tebbe U, Rustige J, Wagner HJ, Appel KF, Stienen U, Konig R, Meyer-Sabellek W. Dose finding with a novel recombinant plasminogen activator (BM 06.022) in patients withacute myocardial infarction: results of the German recombinant plasminogen activator study. JACC 1994; 24: 55-60
    CrossrefPubMedGoogle Scholar
  • 19 Seifried E, Haerer W, Ellbrϋck D, Mϼller M, Martin U, Kӧnig R. Bolus application of a novel recombinant plasminogen activator in AMI patients: pharmacokinetics and effects on the hemostatic system. Arteriosclerosis Thromb 1991; 11: 1590
    PubMedGoogle Scholar
  • 20 Martin U, Fisher S, Kohnert U, Rudolph R, Sponer G, Stern A, Strein K. Coronary thrombolytic properties of a novel recombinant plasminogen activator (BM 06.022) in a canine model. J Cardiovasc Pharmacol 1991; 18: 111-119
    CrossrefPubMedGoogle Scholar
  • 21 Martin U, Sponer G, Strein K. Evaluation of thrombolytic and systemic effects of the novel recombinant plasminogen activator BM 06.022 compared with alteplase, anistreplase, streptokinase, and urokinase in a canine model of artery thrombosis. JACC 1992; 19: 433-440
    CrossrefPubMedGoogle Scholar
  • 22 Pittman RC, Carew TE, Glass CK, Green SR, Taylor CA, Attie AD. A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo. Biochem J 1983; 212: 791-800
    CrossrefPubMedGoogle Scholar
  • 23 Herz J, Goldstein JL, Strickland DK, Ho YK, Brown MS. 39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/α2-macroglobuiin receptor. J Biol Chem 1991; 266: 21232-21238
    PubMedGoogle Scholar
  • 24 Kuiper J, Rijken DC, de Munk GA W, van Berkel Th JC. In vivo and in vitro interaction of highand low molecular weight single chain urokinase type plasminogen activator with rat liver cells. J Biol Chem 1992; 267: 1589-1595
    PubMedGoogle Scholar
  • 25 De Duve C, Pressman BC, Gianetto R, Wattiaux R, Appelmans F. Tissue fractionation studies 6. Intracellular distribution patterns of enzymes in rat liver tissue Biochem J 1955; 60: 604-617
    CrossrefPubMedGoogle Scholar
  • 26 Van Berkel Th JC, de Rijke YB, Kruyt JK. Different fate in vivo of oxidatively modified-LDL and acetylated-LDL in rats. Recognition by various scavenger receptors on Kupffer and endothelial liver cells J Biol Chem 1991; 266: 2282-2289
    PubMedGoogle Scholar
  • 27 Nagelkerke JF, Barto K, Berkel Th JC. In vivo and in vitro uptake and degradation of acetylated low-density lipoprotein by ratliver endothelial, Kupffer, and parenchymal cells. J Biol Chem 1983; 258: 12221-12227
    PubMedGoogle Scholar
  • 28 Martin U, Fisher S, Kohnert U, Lill H, Rudolph R, Sponer G, Stem A, Strein K. Properties of a novel plasminogen activator (BM 06.022) produced in Escherichia coli. Z Kardiol 1990; 79: 167-170
    PubMedGoogle Scholar
  • 29 Martin U, Fisher S, Kohnert U, Rudolph R, Sponer G, Stern A, Strein K. Pharmacokinetic properties of an Escherichiacoli-produced recombinant plasminogen activator (BM 06.022) in rabbits. Thromb Res 1991; 62: 137-146
    CrossrefPubMedGoogle Scholar
  • 30 Martin U, Kohler J, Sponer G, Strein K. Pharmacokinetics of the novel recombinant plasminogen activator BM 06.022 in rats, dogs, and nonhuman primates. Fibrinolysis 1992; 6: 39-43
    PubMedGoogle Scholar
  • 31 Lucore CL, Fry ET A, Nachowiak DA, Sobel BE. Biochemical determinants of clearance of tissue-type plasminogen activator from the circulation. Circulation 1988; 77: 906-914
    CrossrefPubMedGoogle Scholar
  • 32 Collen D, Stassen J, Larsen G. Pharmacokinetics and thrombolytic properties of deletion mutants of human tissue-type plasminogen activator in rabbits. Blood 1988; 71: 216-219
    PubMedGoogle Scholar
  • 33 Tans well P, Schliiter M, Krause J. Pharmacokinetics and isolated liver perfusion of carbohydrate modified recombinant tissue-type plasminogen activator. Fibrinolysis 1989; 3: 79-84
    PubMedGoogle Scholar
  • 34 Stang E, Kindberg GM, Berg T, Roos N. Endocytosis mediated by the mannose receptorin liver endothelial cells. An immunocytochemical study. Eur. J. Cell Biol 1990; 52: 67-76
    PubMedGoogle Scholar
  • 35 Emeis JJ, Verheijen JH. Thrombolytic properties in a rabbit jugular vein thrombosis model of a tissue-type plasminogen activator mutant lacking the growth- and kringle one-domains. Drug Res 1992; 42: 358-362
    PubMedGoogle Scholar
  • 36 Kalyan NK, Lee SG, Wilhelm J, Fu KP, Hum WT, Rappaport R, Hartzell RW, Urbano C, Hung PP. Structure-function analysis with tissue-type plasminogen activator. J Biol Chem 1988; 263: 3971-3978
    PubMedGoogle Scholar
  • 37 Browne MJ, Chapman CG, Dodd I, Reavy B, Esmail AF, Robinson JH. The role of tissue-type plasminogen activator A-chain domains in plasma clearance. Fibrinolysis 1989; 3: 207-214
    PubMedGoogle Scholar
  • 38 Bassel-Duby R, Jiang NY, Bittick T, Madison E, McGookey D, Orth K, Shohet R, Sambrook J, Gething M. Tyrosine 67 in the epidermal growth factor-like domain of tissue-type plasmninogen activator is important for clearance by a specific hepatic receptor. J Biol Chem 1992; 267: 9668-9677
    PubMedGoogle Scholar
  • 39 Martin U, Sponer G, Strein K. Influence of hepatic and renal failure on pharmacokinetic properties of the novel recombinant plasminogen activator BM 06.022 in rats. Drug Met Disp 1993; 21: 236-241
    PubMedGoogle Scholar
  • 40 Otter M, Zockova P, Kuiper J, Van Berkel Th JC, Barrett-Bergshoeff MM, Rijken DC. Isolation and characterization of the mannose receptor from human liver involved in the plasma clearance of tissue-type plasminogen activator. Hepatology 1992; 16: 54-59
    CrossrefPubMedGoogle Scholar
  • 41 Bu G, Maksymovitch EA, Schwartz AL. Receptor-mediated endocytosis of tissue-type plasminogen activator by low-density lipoprotein receptor-related proetin on human hepatoma cells. J Biol Chem 1993; 268: 13002-13009
    PubMedGoogle Scholar
  • 42 Van Dijk MC M, Ziere GJ, Boers W, Linthorst C, Bijsterbosch MK, Van Berkel Th JC. Recognition of chylomicron remnants and 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
  • 43 Moestrup SK, Nielsen S, Andreasen P, Jprgensen KE, Nykjoer A, Rpigaard H, Gliemann J. Christensen EL Epithelial glycoprotein-330 mediates endocytosis of plasminogen activator inhibitor type-I complexes. J Biol Chem 1993; 268: 16564-16570
    PubMedGoogle Scholar