Thrombin induces endothelial cell-surface exposure of the plasminogen receptor annexin 2.
Cell-surface annexin 2 (A2) and its ligand p11 have been implicated in fibrinolysis because of their ability to accelerate tissue plasminogen activator (tPA)-mediated activation of plasminogen to plasmin. Because thrombin is a potent cell modulator obligately produced at the site of clot formation, we hypothesized that the amount of cell-surface A2 and p11 might be altered by thrombin with consequent effects on plasmin generation. In support of this hypothesis, immunofluorescence microscopy and hydrophilic biotinylation experiments showed that both A2 and p11 were significantly increased on the surface of human umbilical vein endothelial cells (HUVECs) treated with thrombin (0.8-8 nM) for 5 minutes followed by 1 hour at 37 degrees C. Intracellular immunofluorescence microscopy and immunoblot analyses of whole cell extracts revealed increased p11 but unchanged A2 in response to thrombin, suggesting that transbilayer trafficking of A2 might be controlled by p11. The thrombin receptor-activating peptide (TRAP) similarly affected cells, demonstrating that cell signaling at least involved the type-1 protease activated receptor (PAR-1). An effect on the fibrinolysis pathway after treatment of HUVECs with thrombin was shown by increased fluorescein-labeled plasminogen binding to cells, which was inhibited by an antibody specific for p11. This was confirmed by observing that thrombin pretreatment of HUVECs increased biotin-modified plasminogen binding. Utilizing a chromogenic assay, pretreatment of HUVECs by thrombin further enhanced activation of the Glu and Lys forms of plasminogen by tPA. These data suggest a novel mechanism that links the coagulation and fibrinolysis pathways by thrombin-mediated feedback.
Canadian Blood Services, R&D Department, 1800 Alta Vista Drive, Ottawa, ON K1G 4J5, Canada.
Erica A
EA
Peterson
Michael R
MR
Sutherland
Michael E
ME
Nesheim
Edward L G
EL
Pryzdial
D017306:Annexin A2 / Q000187:drug effects / Q000378:metabolism*; D000906:Antibodies / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005342:Fibrinolysis / Q000187:drug effects / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D010958:Plasminogen / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D044463:Receptor, PAR-1 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000378:metabolism; D009418:S100 Proteins / Q000187:drug effects / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000378:metabolism / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D000099248:S100 Calcium Binding Protein A10
D017306:Annexin A2; D000906:Antibodies; D010958:Plasminogen; D044463:Receptor, PAR-1; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D009418:S100 Proteins; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D000099248:S100 Calcium Binding Protein A10; C472822:ANXA2 protein, human