Isolation and characterization of tissue-type plasminogen activator- binding proteoglycans from human umbilical vein endothelial cells.
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, epsilon-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.
Böhm
T
T
Department for Vascular Biology and Thrombosis Research, University of Vienna, Austria.
Geiger
M
M
Binder
B R
BR
D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000302:isolation & purification / Q000378:metabolism; D006497:Heparan Sulfate / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D011509:Proteoglycans / Q000302:isolation & purification* / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism*
D002809:Chondroitin Sulfates; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D010959:Tissue Plasminogen Activator