Cellular Internalization and Degradation of Antithrombin III-Thrombin, Heparin Cofactor II-Thrombin, and -Antitrypsin-Trypsin Complexes Is Mediated by the Low Density Lipoprotein Receptor-related Protein

doi:10.1074/jbc.271.11.6523

Cellular Internalization and Degradation of Antithrombin III-Thrombin, Heparin Cofactor II-Thrombin, and -Antitrypsin-Trypsin Complexes Is Mediated by the Low Density Lipoprotein Receptor-related Protein (*)

From the (1)Holland Laboratory, Department of Biochemistry, American Red Cross, Rockville, Maryland 20855 and the
(2)Department of Pathology and Medicine and Center for Thrombosis and Hemostasis, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

^¶ To whom correspondence should be addressed:
15601 Crabbs Branch Way, Rockville, MD 20855
. Tel.: 301-738-0726; Fax: 301-738-0794.

↵^§ Current address: SIBIA, 505 Coast Blvd., La Jolla, CA 92037.

Abstract

The inhibition of proteinase activity by members of the serine proteinase inhibitor (serpin) family is a critical regulatory mechanism for a variety of biological processes. Once formed, the serpin enzyme complexes (SECs) are removed from the circulation by a hepatic receptor. The present study suggests that this receptor is very likely the low density lipoprotein receptor-related protein (LRP), a prominent liver receptor. In vitro binding studies revealed that antithrombin III (ATIII)•thrombin, heparin cofactor II (HCII)•thrombin, and α₁-antitrypsin (α₁AT)•trypsin bound to purified LRP, and their binding was inhibited by the 39-kDa receptor-associated protein (RAP), an antagonist of LRP-ligand binding activity. In contrast, native or modified forms of the inhibitors were unable to bind to LRP. Mouse embryonic fibroblasts, which express LRP, mediate the cellular internalization leading to degradation of these SECs, while mouse fibroblasts genetically deficient in LRP showed no capacity to internalize and degrade these complexes. SECs were also degraded by HepG2 cells, and this process was inhibited by LRP antibodies, RAP, and chloroquine. The cellular-mediated uptake and degradation was specific for SECs; native or modified forms of the inhibitors were not internalized and degraded. Finally, in vivo clearance studies in rats demonstrated that RAP inhibited the clearance of ATIII•I-thrombin complexes from the circulation. Together, these results indicate that LRP functions as a liver receptor responsible for the plasma clearance of SECs.

Footnotes

↵* This work was supported by National Institutes of Health Grants HL50787 (to D. K. S.), GM42581 (to D. K. S.), DK45598 (to W. S. A.), and HL32656 (to F. C. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SEC

serpin-enzyme complex

LDLR

low density lipoprotein receptor

LRP

LDLR-related protein

gp330

glycoprotein 330

RAP

receptor-associated protein

ATIII

antithrombin III

HCII

heparin cofactor II

α₁AT

α₁-antitrypsin

PAI-1

plasminogen activator inhibitor type-1

uPA

urokinase-type plasminogen activator

α₂M

α₂-macroglobulin

α₂M*

methylamine-activated α₂M

PR3

proteinase 3

ELISA

enzyme-linked immunosorbent assay

BSA

bovine serum albumin

TBS

Tris-buffered saline

MEF

mouse embryonal fibroblast.
- Received September 6, 1995.
- Revision received December 12, 1995.