In Vivo Clearance of Ternary Complexes of Vitronectin-Thrombin-Antithrombin Is Mediated by Hepatic Heparan Sulfate Proteoglycans

Michael J Wells,Morris A Blajchman

doi:10.1074/jbc.273.36.23440

Abstract

Thrombin is inhibited by its cognate plasma inhibitor antithrombin, through the formation of covalent thrombin-antithrombin (TAT) complexes that are found as ternary complexes with vitronectin (VN-TAT). To determine whether the metabolism of VN-TAT ternary complexes is different from that previously reported for binary TAT complexes, plasma clearance studies were done in rabbits using human VN-TAT. 125I-VN-TAT was shown to be cleared rapidly from the circulation (t1/2alpha = 3.8 min) in a biphasic manner mainly by the liver. 125I-TAT had a similar initial clearance (t1/2alpha = 5.3 min) but had a significantly faster beta-phase clearance (t1/2beta = 42.8 min versus 85.4 min for VN-TAT; p = 0.005). Protamine sulfate and heparin abolished the rapid initial alpha-phase of 125I-VN-TAT clearance and reduced its liver-specific association and in vivo degradation. Heparin also reduced the alpha-phase clearance of 125I-TAT and was associated with the appearance of high molecular weight complexes, suggesting enhanced complex formation between VN and TAT. 125I-VN-TAT binding to HepG2 cells was reduced by competition with VN-TAT or heparin but to a much lesser extent in the presence of TAT. The binding of VN-TAT to HepG2 cells was not inhibited by competition with the low density lipoprotein receptor-related protein ligand, methylamine-alpha2-macroglobulin. 125I-VN-TAT binding was also inhibited by treating HepG2 cells with heparinase or by growing the cells in the presence of beta-D-xyloside. Finally, both heparin and chloroquine, but not methylamine-alpha2-macroglobulin, reduced the internalization and degradation of VN-TAT by HepG2 cells. Taken together, these data indicate the importance of VN in TAT metabolism and demonstrate that VN-TAT binds to liver-associated heparan sulfate proteoglycans, which mediate its internalization and subsequent intracellular degradation.

Highlights

Vitronectin (VN), a 78-kDa glycoprotein synthesized by the liver, is found in the circulation at a plasma concentration of 200 – 400 ␮g/ml [12]
We have examined the metabolism of 125I-VN-TAT in vivo and have identified hepatocyte-associated heparan sulfate proteoglycans (HSPG) as major binding sites involved in the metabolism of VN-TAT both in vivo and in vitro
Purification and Radiolabeling of Human VN-TAT—In order to investigate the metabolism of VN-TAT in vitro and in vivo, VN-TAT was purified by heparin-Sepharose chromatography

Summary

Introduction

Vitronectin (VN), a 78-kDa glycoprotein synthesized by the liver, is found in the circulation at a plasma concentration of 200 – 400 ␮g/ml [12]. In the experiments with 125I-VN-TAT (ϳ12.6 ϫ 106 dpm) either alone or in competition with heparin (10,000 units/ml) (n ϭ 3 for each), the incubation period was reduced to only 30 min, and only the livers were removed and processed for determination of radioactive uptake.

Results

Conclusion