This Article Full Text Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Böhm, T. Articles by Binder, B. R. Search for Related Content PubMed PubMed Citation Articles by Böhm, T. Articles by Binder, B. R.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:665-672.)
© 1996 American Heart Association, Inc.

Articles

Isolation and Characterization of Tissue-Type Plasminogen Activator–Binding Proteoglycans From Human Umbilical Vein Endothelial Cells

Thomas Böhm; Margarethe Geiger; Bernd R. Binder

From the Department for Vascular Biology and Thrombosis Research, University of Vienna (Austria).

Correspondence to Prof Dr Bernd Binder, Department for Vascular Biology and Thrombosis Research, Schwarzspanierstrasse 17, A-1090 Vienna, Austria.

Abstract We analyzed the tissue-type plasminogen activator (TPA)–binding proteoglycans (PGs) on human umbilical vein endothelial cells (HUVECs), which were metabolically labeled with [³⁵S]Na₂SO₄. Cell extracts were then prepared and subjected to affinity chromatography on diisopropyl fluorophosphate (DFP)–inactivated TPA–Sepharose 4B. Approximately 6% of the incorporated ³⁵S radioactivity bound to DFP-treated TPA–Sepharose 4B and was eluted with 2 mol/L NaCl. In addition to NaCl, heparin, arginine, and lysine but not glycine, -amino-n-caproic acid, or aspartic acid inhibited this binding and eluted the bound ³⁵S radioactivity. Urea-containing polyacrylamide gel electrophoresis of the eluted material consistently revealed two main signals of ³⁵S radioactivity (one with an M_r between 600 000 and 750 000 [PGA] and the other with an M_r between 120 000 and 180 000 [PGC]). Occasionally a less intense signal with an M_r between 340 000 and 440 000 (PGB) was seen. Heparitinase treatment markedly decreased the intensities of both ³⁵S signals (PGA and PGB), and chondroitinases AC and ABC abolished the ³⁵S signal of PGC, indicating that most of the HUVEC-incorporated radioactivity with an affinity for TPA could be attributed to heparan sulfate– and chondroitin sulfate–like structures. Reductive elimination, which was performed to separate the possible glycosaminoglycan moieties from the core proteins, confirmed the PG-like nature of this material and again revealed heparan sulfate and chondroitin sulfate as the major glycosaminoglycan components. We therefore conclude that HUVECs synthesize TPA-binding, heparan sulfate– and chondroitin sulfate–containing PGs. In vivo, similar PGs may play a role in TPA binding to endothelial cells and thereby possibly influence TPA activity and/or provide an intravascular storage pool of TPA.

Key Words: glycosaminoglycans • tissue-type plasminogen activator • endothelial cells • proteoglycans

This article has been cited by other articles:

				B. P. Schick, J. F. Gradowski, and J. D. S. Antonio Synthesis, secretion, and subcellular localization of serglycin proteoglycan in human endothelial cells Blood, January 15, 2001; 97(2): 449 - 458. [Abstract] [Full Text] [PDF]