In vivo clearance of ternary complexes of vitronectin-thrombin- antithrombin is mediated by hepatic heparan sulfate proteoglycans

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 (t( 1/4 α) = 3.8 min) in a biphasic manner mainly by the liver. 125ITAT had a similar initial clearance (t( 1/4 α) = 5.3 min) but had a significantly faster β-phase clearance (t( 1/4 β) = 42.8 min versus 85.4 min for VN-TAT; p = 0.005). Protamine sulfate and heparin abolished the rapid initial α-phase of 125I-VN-TAT clearance and reduced its liver- specific association and in vivo degradation. Heparin also reduced the α- 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- α2-macroglobulin. 125I-VN-TAT binding was also inhibited by treating HepG2 cells with heparinase or by growing the cells in the presence of β-D- xyloside. Finally, both heparin and chloroquine, but not methylamine-α2- 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.