Comparison of recombinant and plasma-derived antithrombin biodistribution in a rabbit model

Leslie R. Berry; Bruce Thong; Anthony K. C. Chan

doi:10.1160/TH09-01-0062

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2009; 102(02): 302-308
DOI: 10.1160/TH09-01-0062

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Comparison of recombinant and plasma-derived antithrombin biodistribution in a rabbit model

Authors

Leslie R. Berry

¹Henderson Research Centre, Hamilton, Ontario, Canada

²Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada
Bruce Thong

¹Henderson Research Centre, Hamilton, Ontario, Canada

²Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada
Anthony K. C. Chan

¹Henderson Research Centre, Hamilton, Ontario, Canada

²Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada

Abstract

Summary

Antithrombin (AT) is a native plasma protein that acts as the main inhibitor of enzymes generated by the coagulation cascade. In extreme thrombotic conditions, consumption of plasma AT can make treatment with AT-associated heparin therapies less effective. Supplementation with recombinant human AT (rhAT) has shown promise but altered pharmacokinetics were observed when comparing stable heparin complexes of the plasma-derived AT (pAT) and rhAT proteins. To understand the differential clearance mechanisms,biodistribution of rhAT and pAT was determined. ¹²⁵I-labelled ATryn (rhAT) or Kybernin P (pAT) was intravenously injected into rabbits. At various time points, animals were sacrificed and organs analysed for bound radioactivity. ¹³¹I-albumin, injected shortly before termination, was used as a marker for trapped blood. Levels of circulating protein + metabolites were significantly less for rhAT than pAT (p < 0.001) and removal of acid soluble fragments confirmed that differences were due to more rapid rhAT clearance. More rhAT (28% dose) than pAT (3% dose) was liver-associated by the earliest time points, corresponding to elevated rhAT degradation products in urine/feces. However, at intermediate times (4 hours), rhAT showed significantly increased arterial and venous uptake over pAT (p ≤0.001).These vessel wall interactions accounted for the primary differences between clearance of rhAT and pAT during these time periods. Overall, circulating rhAT is more rapidly lost to the liver and vessels than pAT. Increased vessel wall binding may facilitate rhAT treatment of vascular thrombosis.

Keywords

Antithrombin - biodistribution - pharmacokinetics - protein glycosylation - vessel wall

Full Text

References

References
1 Olson ST, Bjork I, Shore JD. Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin. Methods Enzymol 1993; 222: 525-559.
2 Roldan V, Ordonez A, Marin F. et al. Antithrombin Cambridge II (A384S) supports a role for antithrombin deficiency in arterial thrombosis. Thromb Haemost 2009; 101: 483-486.
3 Maclean PS, Tait RC. Hereditary and acquired antithrombin deficiency: epidemiology, pathogenesis and treatment options. Drugs 2007; 67: 1429-1440.
4 Mitchell L, Andrew M, Hanna K. et al. Trend to efficacy and safety using antithrombin concentrate in prevention of thrombosis in children receiving L-asparaginase for acute lymphoblastic leukemia. Results of the PAARKA study. Thromb Haemost 2003; 90: 235-244.
5 Sorg H, Hoffmann JN, Menger MD. et al. Antithrombin is as effective as heparin and hirudin to prevent formation of microvascular thrombosis in a murine model. Thromb Haemost 2006; 96: 371-377.
6 Hoffmann JN, Wiedermann CJ, Juers M. et al. Benefit/risk profile of high-dose antithrombin in patients with severe sepsis treated with or without concomitant heparin. Thromb Haemost 2006; 95: 850-856.
7 GTC Biotherapeutics. Recombinant human antithrombin III: rhATIII. Drugs RD 2004; 05: 110-112.
8 Konkle BA, Bauer KA, Weinstein R. et al. Use of recombinant human antithrombin in patients with congenital antithrombin deficiency undergoing surgical procedures. Transfusion 2003; 43: 390-394.
9 Avidan MS, Levy JH, van Aken H. et al. Recombinant human antithrombin III restores heparin responsiveness and decreases activation of coagulation in heparin-resistant patients during cardiopulmonary bypass. J Thorac Cardiovasc Surg 2005; 130: 107-113.
10 Edmunds T, Van Patten SM, Pollock J. et al. Transgenically produced human antithrombin: structural and functional comparison to human plasma-derived antithrombin. Blood 1998; 91: 4561-4571.
11 Chindemi PA, Klement P, Konecny F. et al. Biodistribution of covalent antithrombin-heparin complexes. Thromb Haemost 2006; 95: 629-636.
12 Manson HE, Austin RC, Fernandez-Rachubinski F. et al. The molecular pathology of inherited human antithrombin III deficiency. Transfus Med Rev 1989; 03: 264-281.
13 Zhou Q, Kyazike J, Echelard Y. et al. Effect of genetic background on glycosylation heterogeneity in human antithrombin produced in the mammary gland of transgenic goats. J Biotechnol 2005; 117: 57-72.
14 Witmer MR, Hatton MW. Antithrombin III-beta associates more readily than antithrombin III-alpha with uninjured and de-endothelialized aortic wall in vitro and in vivo. Arterioscler Thromb 1991; 11: 530-539.
15 Carlson TH, Kolman MR, Piepkorn M. Activation of antithrombin III isoforms by heparan sulphate glycosaminoglycans and other sulphated polysaccharides. Blood Coagul Fibrinolysis 1995; 06: 474-480.
16 Linehan SA. The mannose receptor is expressed by subsets of APC in non-lymphoid organs. BMC Immunol 2005; 06: 4.
17 Higuchi Y, Nishikawa M, Kawakami S. et al. Uptake characteristics of mannosylated and fucosylated bovine serum albumin in primary cultured rat sinusoidal endothelial cells and Kupffer cells. Int J Pharm 2004; 287: 147-154.
18 Weigel PH, Yik JH. Glycans as endocytosis signals: the cases of the asialoglycoprotein and hyaluronan/ chondroitin sulfate receptors. Biochim Biophys Acta 2002; 1572: 341-363.
19 Mertens G, Cassiman JJ, Van Den Berghe H. et al. Cell surface heparan sulfate proteoglycans from human vascular endothelial cells. Core protein characterization and antithrombin III binding properties. J Biol Chem 1992; 267: 20435-20443.
20 Camani C, Kruithof EK. Clearance receptors for tissue-type plasminogen activator. Int J Hematol 1994; 60: 97-109.
21 Rijken DC, Otter M, Kuiper J. et al. Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA) by liver cells cells. Thromb Res 1990; 10: 63-71.
22 Ezekowitz RA, Stahl PD. The structure and function of vertebrate mannose lectin-like proteins. J Cell Sci Suppl 1988; 09: 121-133.
23 Stockert RJ. The asialoglycoprotein receptor: relationships between structure, function, and expression. Physiol Rev 1995; 75: 591-609.
24 Pries AR, Secomb TW, Gaehtgens P. The endothelial surface layer. Pflugers Arch 2000; 440: 653-666.
25 Lightfoot EN. Transport Phenomena and Living Systems. Biomedical aspects of momentum and mass transport. Toronto: John Wiley & Sons; 1974
26 Carrell RW, Christey PB, Boswell DR. Serpins: Antithrombin and other inhibitors of coagulation and fibrinolysis: Evidence from amino acid sequences. In: Thrombosis and Haemostasis. Leuven, Belgium: Leuven University Press; 1987: 1-15.
27 Weigel PH. Galactosyl and N-acetylgalactosaminyl homeostasis: a function for mammalian asialoglycoprotein receptors. Bioessays 1994; 16: 519-524.
28 Nishikawa M. Development of cell-specific targeting systems for drugs and genes. Biol Pharm Bull 2005; 28: 195-200.
29 Justus AC, Roussev R, Norcross JL. et al. Antithrombin binding by human umbilical vein endothelial cells: effects of exogenous heparin. Thromb Res 1995; 79: 175-186.
30 Paredes N, Wang A, Berry LR. et al. Mechanisms responsible for catalysis of the inhibition of factor Xa or thrombin by antithrombin using a covalent antithrombin-heparin complex. J Biol Chem 2003; 278: 23398-23409.