Binding of tissue-type plasminogen activator with human endothelial cell monolayers. Characterization of the high affinity interaction with plasminogen activator inhibitor-1.
The formation and release of covalent complexes between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) limits the application of equilibrium radioligand binding analysis to characterize the interaction between t-PA and human umbilical vein endothelial cell (HUVEC) monolayers. To avoid this difficulty, we used a recombinant mutant of t-PA, S478A rt-PA, in which alanine has been substituted for the active-site serine. Although the mutant is incapable of covalently reacting with PAI-1, 125I-labeled S478A rt-PA binding to HUVEC monolayers is specific and reversible and is characterized by a high affinity (Kd of 1.5 nM) and a large number of sites (1.5 x 10(6)/cell). This binding was shown to occur through noncovalent interaction with PAI-1 in the HUVEC monolayer by the fact that a monoclonal anti-PAI-1 antibody (MA-7D4) completely blocked S478A rt-PA binding. Two solution-phase assays with recombinant PAI-1 (rPAI-1) confirmed this noncovalent interaction: complexes between 125I-S478A rt-PA and rPAI-1 could be isolated by immunoprecipitation with anti-PAI-1 antibodies, and S478A rt-PA competed with rt-PA for inactivation by rPAI-1. In contrast diisopropylphosphate rt-PA (in which the active site serine is chemically modified) showed minimal binding to HUVEC monolayers, as a result of impaired interaction with PAI-1, in the two assays. Thus, both wild-type rt-PA and S478A rt-PA interact with the HUVEC monolayer through PAI-1. With rt-PA this results in the formation of covalent rt-PA.PAI-1 complexes that are released from the monolayer into the supernatant. With S478A rt-PA this results in the formation of noncovalent complexes that remain associated with the HUVEC monolayer, thereby identifying a large pool of reactive PAI-1 molecules in the monolayer.
Russell
M E
ME
Cardiac Unit, Medical Service, Massachusetts General Hospital, Boston 02114.
Quertermous
T
T
Declerck
P J
PJ
Collen
D
D
Haber
E
E
Homcy
C J
CJ
D000409:Alanine; D001665:Binding Sites; D001667:Binding, Competitive; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009154:Mutation; D015849:Plasminogen Inactivators / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D014471:Umbilical Veins
D015849:Plasminogen Inactivators; D011994:Recombinant Proteins; D012694:Serine; D010959:Tissue Plasminogen Activator; D000409:Alanine