Binding of mutant tissue-type plasminogen activators to human endothelial cells and their extracellular matrix.
We previously demonstrated that tissue-type plasminogen activator (t-PA) specifically bound to its receptor (t-PAR) on human umbilical vein endothelial cells (HUVEC). In addition to analyses of t-PA binding to plasminogen activator inhibitor-1 (PAI-1) in the extracellular matrix (ECM) and to the t-PAR, we further evaluated the binding of three t-PA mutants, deltaFE1X t-PA lacking finger (F), epidermal growth factor-like (E) domains and one sugar chain at Asn177 thus comprising two kringles (K1 and K2) and protease (P) domains, deltaFE3X t-PA with three glycosylation sites deleted at Asn117, 184, and 448, and deltaFEK1 t-PA comprising K2 and P domains without glycosylation. Wild-type t-PA bound to ECM with high affinity, which was completely blocked by anti-PAI-1 IgG. Wild-type t-PA, deltaFE1X t-PA and deltaFEK1 t-PA bound to two classes of binding sites with high and low affinities on monolayer HUVEC. However, all t-PAs bound to a single class of binding site in the presence of anti-PAI-1 IgG. DeltaFEK1 t-PA bound t-PAR maximally among these t-PAs. These results suggested that the high affinity binding of t-PA mainly occurred with PAI-1 on ECM while the low affinity binding was with t-PAR. The deletion of F, E domains and sugar chains had no effect on binding with t-PAR. However, since only K1-missing t-PA (deltaFEK1) exhibited significantly increased binding sites among these t-PAs, it was suggested that the binding to t-PAR was mediated mainly by K2 domain and that the increase of binding was due to direct exposure of K2 domain.
Fukao
H
H
Department of Physiology, Kinki University School of Medicine, Osakasayama city, Osaka, Japan. fukao@med.kindai.ac.jp
Ueshima
S
S
Okada
K
K
Matsuo
O
O
D015119:Aminocaproic Acid / Q000494:pharmacology; D000818:Animals; D000933:Antifibrinolytic Agents / Q000494:pharmacology; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects
D000933:Antifibrinolytic Agents; D007457:Iodine Radioisotopes; D017395:Plasminogen Activator Inhibitor 1; D010959:Tissue Plasminogen Activator; D015119:Aminocaproic Acid