title: Thrombin decreases the urokinase receptor and surface-localized fibrinolysis in cultured endothelial cells.
abstract: The endothelial cell (EC) urokinase receptor plays an important role in the localization and receptor-mediated activation of EC-bound plasminogen and hence surface-localized fibrinolysis. Thrombin induced a rapid (< 5 minute), time- (0 to 30 minutes) and dose- (0.1 to 8 U/mL) dependent decrease in the specific binding of 125I-labeled two-chain urokinase-type plasminogen activator (tcu-PA) or diisopropylfluoro-phosphate-tcu-PA to urokinase-type plasminogen activator receptor (u-PAR) in cultured ECs from various sources (range, 21% to 50%). The thrombin receptor activation peptide but not control peptide showed a similar but reduced decrease in the specific binding of 125I-labeled tcu-PA to u-PAR. Incubation of thrombin-treated cultures (10 to 12 hours) in complete medium restored 125I-labeled tcu-PA ligand binding to normal levels. u-PAR mRNA levels rapidly (1 hour) increased and peaked 10 to 12 hours after thrombin treatment as analyzed by reverse transcriptase-polymerase chain reaction. Decreased thrombin-induced 125I-labeled tcu-PA binding correlated with the time-dependent decrease in surface-localized plasmin generation, as measured by the direct activation of 125I-labeled Glu-plasminogen and quantification of the 20-kD light chains of 125I-labeled plasmin. After incubation with thrombin, plasmin generation was decreased 50% to 56% (125 to 152 fmol/3 to 3.5 x 10(4) cells). Isolation of metabolically labeled 35S-labeled u-PAR from the media of thrombin and phospholipase C-treated human aortic cultures yielded approximately 10- and approximately 12-fold more 55-kD M(r) and approximately 6-fold more 35-kD M(r) 35S-labeled u-PAR forms than control cultures, respectively. The u-PAR antigen forms (M(r), 54 kD) and the glycosyl-phosphatidylinositol-anchored protein CD59 (M(r), 20 kD) were also simultaneously identified by immunoprecipitation in the media of thrombin-treated cultures. This suggests that thrombin may release u-PAR and decrease u-PA ligand binding through a common pathway involving phospholipase C. These results establish a novel interrelation between thrombin and EC fibrinolysis and suggest that thrombin may also have an additional regulatory role in the net expression of surface-localized EC fibrinolytic activity.
authors.affiliation: Department of Medicine, University of Alabama at Birmingham 35294-2170, USA.
authors.forename: X N
authors.initials: XN
authors.lastname: Li
authors.forename: V K
authors.initials: VK
authors.lastname: Varma
authors.forename: J M
authors.initials: JM
authors.lastname: Parks
authors.forename: R L
authors.initials: RL
authors.lastname: Benza
authors.forename: J C
authors.initials: JC
authors.lastname: Koons
authors.forename: J R
authors.initials: JR
authors.lastname: Grammer
authors.forename: H
authors.initials: H
authors.lastname: Grenett
authors.forename: E M
authors.initials: EM
authors.lastname: Tabengwa
authors.forename: F M
authors.initials: FM
authors.lastname: Booyse
mesh_terms: D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002621:Chemistry; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005342:Fibrinolysis / Q000187:drug effects*; D006801:Humans; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D013917:Thrombin / Q000494:pharmacology*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
chemical_list: D015335:Molecular Probes; C524898:PLAUR protein, human; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D013917:Thrombin; D014568:Urokinase-Type Plasminogen Activator