Low density lipoprotein receptor-related protein is necessary for the internalization of both tissue-type plasminogen activator-inhibitor complexes and free tissue-type plasminogen activator.

K Orth,T Willnow,J Herz,M.J Gething,J Sambrook

doi:10.1016/s0021-9258(17)31937-3

Abstract

Tissue-type plasminogen activator (t-PA) is used as a thrombolytic agent in treatment of myocardial infarction. However, large doses of this agent must be administered in treatment to maintain a thrombolytic state because t-PA is cleared rapidly from circulation. We designed specific ligands to distinguish between two major mechanisms by which t-PA is taken into cells and degraded. One of these mechanisms involves internalization of complexes between t-PA and its cognate inhibitor plasminogen activator inhibitor type-1 (PAI-1); the other mechanism is independent of PAI-1. Using specific inhibitors for low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP), we show that the degradation by hepatocytes of both free t-PA and t-PA.PAI-1 complexes involve the receptor LRP. We demonstrate that fibroblasts degrade both free t-PA (PAI-1-independent) and t-PA complexed with its specific inhibitor PAI-1 (PAI-1-dependent), whereas genetically altered fibroblasts that do not express LRP neither internalize nor degrade these ligands. We also show that a PAI-1-independent, t-PA ligand can inhibit the degradation of both free t-PA and t-PA.PAI-1 complexes. We propose LRP is the receptor for both PAI-1-independent and PAI-1-dependent t-PA ligands.

Highlights

Tissue-type plasminogen activator (t-PAi)s used as a to polymerized fibrinwhile the thrombolytic agent in treatment of myocardial infarc- kringle domains areresponsible for the consequent increase in tion
As with other proteins in threombolytidfibrinolytic pathways, t-PAis built of structural modules, each encoded by a separate exon or pair of exons, that fold into structurally conserved domains (Patthy, 1985): a finger-like domain, an epidermal growth factor (EGF)-like motif, two kringles, and a serine protease domain
The abbreviationsused are: t-PA,tissue-typeplasminogen activator; GST, glutathione S-transferase;LRP, low density lipoprotein receptorthese ligands is inhibited by specific inhibitors for LRP (RAP and polyclonal anti-LRP IgG) (Herzet al., 1991; Kowal et al, 1989)

Summary

THEJOURNALOF BIOIGCICCAHLEMISTRY

21117-21122, 1994 Printed in U.S.A. Low Density Lipoprotein Receptor-related ProteinIs Necessary for the Internalizationof Both Tissue-type Plasminogen Activator-Inhibitor Complexes and Free Tissue-type Plasminogen Activator*. The short clearance timeof t-PA from the cirdegraded Oneof these mechanisms involves internal- culation ( t , = 4-6 min in man; Nilsson et al, 1984) mandates ization of complexes between t-PAanitds cognate inhib- the use of large doses of t-PA to generate and sustain an aditor plasminogen activator inhibitor type-1 (PAT-1); the equate thrombolytic state The mannosereceptor, present primarily on endothelial independent) andt-PA complexed witihts specific inhib- cells, recognizes the mannose-richoligosaccharide onthe krinitoPrAI(-P1AI-1-dependenwt),heregaesnetically gle 1domain of t-PA (Rijken et al, 1990; Pohlet al., 1987; Otter altered fibroblasts that do not express LRP neither in- et al, 1991, 1992). Nal epitope thought tobe located onthe B-loop of the EGF-like domain of t-PA (Browne et al, 1990; Bassel-Duby et al, 1992)

The specific receptor has not beenidentified but studiesof both

Sites available for binding to receptors

RESULTS

GST Competitor