Heparin enhances active site-dependent binding of tissue-type plasminogen activator to endothelial cells.
Human umbilical vein endothelial cells (HUVEC) in culture express two classes of binding sites for tissue-type plasminogen activator (t-PA). The high-affinity binding site has been identified as PA inhibitor type 1 (PAI-1), which binds to the catalytic portion of the molecule, while the second site binds t-PA through an active-site independent domain. Because recombinant t-PA (rt-PA) is often administered concomitantly with heparin, we investigated the effects of heparin on rt-PA binding to HUVEC. Preincubation of HUVEC with heparin at 4 degrees C increased the binding of radiolabeled rt-PA in a time- and dose-dependent manner. One-half maximal increase in binding was observed within 10 minutes of heparin addition. When HUVEC were preincubated with optimal concentrations (5 U/mL) of heparin for 4 hours at 4 degrees C, a 2.5- +/- 0.2-fold increase in specific binding was observed (mean +/- SEM, n = 12, P less than .01). Other highly sulfated glycosaminoglycans and fucoidan (a sulfated polymer of fucose) stimulated rt-PA binding as well, whereas glycosaminoglycans with lower sulfate content than heparin did not. Several results suggested that heparin increased the binding of rt-PA to "cell-associated" PAI-1. First, only active-site-dependent binding was enhanced by heparin, whereas binding of active-site blocked rt-PA was not affected. Second, extracts from HUVEC preincubated with heparin contained increased amounts of rt-PA-PAI-1 complexes as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Third, antibodies to PAI-1 blocked the increased binding entirely. HUVEC preincubated with heparin also bound increased amounts of enzymatically active radiolabeled urokinase-type PAs. However, HUVEC preincubated with heparin did not express increased amounts of immunoreactive PAI-1. Therefore, heparin, at therapeutic concentrations, may enhance or stabilize the association of PAs with endothelial cell-associated PAI-1.
Rosenfeld
L
L
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia.
Kuo
A
A
Hirsh
J
J
Klugherz
B
B
Gardell
S J
SJ
Cines
D B
DB
Barnathan
E S
ES
D001665:Binding Sites; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D015849:Plasminogen Inactivators / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism
D006025:Glycosaminoglycans; D015849:Plasminogen Inactivators; D006493:Heparin; D010959:Tissue Plasminogen Activator