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 [S-35]Na2SO4. Cell extracts were then prepared and subjected to affinity chromatography on diisopropyl fluorophosphate (DFP)-inactivated TPA-Sepharose 4B. Approximately 6% of the incorporated S-35 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 S-35 radioactivity. Urea-containing polyacrylamide gel electrophoresis of the eluted material consistently revealed two main signals of S-35 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 S-35 signals (PGA and PGB), and chondroitinases AC and ABC abolished the S-35 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.