Single-chain and two-chain tissue-type plasminogen activator (t-PA) bind differently to cultured human endothelial cells.
It has recently been shown that the fibrinolytic components plasminogen and tissue-type plasminogen activator (t-PA) both bind to cultured human umbilical vein endothelial cells (HUVEC). After cleavage of t-PA by plasmin, "single-chain" t-PA (sct-PA) is converted into "two-chain" t-PA (tct-PA), which differs from the former in a number of respects. We compared binding of sct-PA and tct-PA to the surface of HUVEC. Removal of t-PA bound to HUVEC by a mild treatment with acid and a subsequent quantification of eluted t-PA both by activity- and immunoradiometric assays revealed that, at concentrations between 10 and 500 nM, HUVEC bind about 3-4 times more sct-PA than tct-PA. At these concentrations, both sct-PA and tct-PA remain active when bound to HUVEC. Mutual competition experiments showed that sct-PA and tct-PA can virtually fully inhibit binding of each other to HUVEC, but that an about twofold higher concentration of tct-PA is required to prevent half-maximal binding of sct-PA than visa versa. These results demonstrate that sct-PA and tct-PA bind with different affinities to the same binding sites on HUVEC.
Aerts
R J
RJ
Department of Molecular Biology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Gillis
K
K
Pannekoek
H
H
D000941:Antigens / Q000032:analysis; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D011485:Protein Binding; D012685:Sepharose; D010959:Tissue Plasminogen Activator / Q000276:immunology / Q000378:metabolism*
D000941:Antigens; D012685:Sepharose; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin