Binding of recombinant variants of human tissue-type plasminogen activator (t-PA) to human umbilical vein endothelial cells.
Endothelial cells synthesize and secrete hemostatic components like tissue-type plasminogen activator (t-PA) which is thought to be the major determinant of fibrinolytic activity in the blood. Most recently, a receptor protein for t-PA on human umbilical vein endothelial cells (HUVEC) in culture has been described (1); there are, however, in addition low affinity binding sites for t-PA on HUVEC. The sites of binding are of particular interest, because they are potential regulators of t-PA activity and clearance. We analysed the low affinity binding of recombinant t-PA (rt-PA) to normal diploid HUVEC and to the permanent human cell lines Jurkat, Daudi, HL 60 and K562 by flow cytometry applying t-PA specific monoclonal antibodies. Using this test system binding of both recombinant glycosylated human t-PA produced in Chinese hamster ovary cells (CHO-t-PA) and of nonglycosylated t-PA, produced in E. coli (BM 06.021) was investigated. Analysis of the binding pattern to HUVEC and other cell lines revealed that deglycosylation of full length rt-PA increases non-specific binding. Additionally, we investigated the binding properties of an unglycosylated t-PA deletion variant which comprises the kringle 2 and the protease domains (BM 06.022). Data obtained show that deletion of these domains most drastically reduces non-specific binding to HUVEC and other human cell lines.
Stockinger
H
H
Boehringer Mannheim GmbH, Biochemical Research Center, Penzberg, Germany.
Kubbies
M
M
Rudolph
R
R
Stern
A
A
Kohnert
U
U
Fischer
S
S
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D005434:Flow Cytometry; D006031:Glycosylation; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*; D014471:Umbilical Veins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010450:Endopeptidases; D010959:Tissue Plasminogen Activator