Effects of thrombin on RPE cells are mediated by transactivation of growth factor receptors.
OBJECTIVE
To determine the expression of blood coagulation factors and thrombin receptors in retinal pigment epithelial (RPE) cells and whether the effects of thrombin on the chemotaxis and the secretion of VEGF are mediated by transactivation of growth factor receptors.


METHODS
Gene expression in acutely isolated and cultured human RPE cells was evaluated by RT-PCR. Alterations in gene expression and secretion of VEGF were determined by real-time RT-PCR and ELISA, respectively. Chemotaxis was examined with a Boyden chamber assay.


RESULTS
RPE cells expressed the mRNA of the protease-activated receptors PAR1 and -3 and of various coagulation factors (III, V, VII, VIII, and X). Thrombin stimulated the expression and secretion of VEGF-A from RPE cells, decreased the expression of VEGFD, and increased the gene expression of VEGFR-1 (FLT1). The effects on the secretion of VEGF-A and the increase in FLT1 expression were mediated by stimulation of the secretion of TGF-beta1 and activation of the TGF-beta activin receptor-like kinase. Thrombin stimulated the chemotaxis of RPE cells, and this effect was mediated by transactivation of the PDGF receptor tyrosine kinase.


CONCLUSIONS
The expression of different coagulation factors suggests that RPE cells provide a procoagulant surface for the formation of thrombin from prothrombin via the extrinsic coagulation pathway. Thrombin stimulates the secretion of VEGF-A, the expression of FLT1, and the chemotaxis of RPE cells via transactivation of TGF-beta and PDGF receptors, respectively.
Department of Ophthalmology and Eye Hospital, Center of Clinical Research (IZKF), University of Leipzig, Leipzig, Germany. hollbm@medizin.uni-leipzig.de
Margrit
M
Hollborn
Carola
C
Petto
Anja
A
Steffen
Susanne
S
Trettner
Andrea
A
Bendig
Peter
P
Wiedemann
Andreas
A
Bringmann
Leon
L
Kohen
D001779:Blood Coagulation Factors / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics* / Q000378:metabolism; D018179:Receptors, Thrombin / Q000235:genetics / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013917:Thrombin / Q000494:pharmacology*; D015533:Transcriptional Activation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism
D001779:Blood Coagulation Factors; D012333:RNA, Messenger; D018179:Receptors, Thrombin; D018125:Receptors, Transforming Growth Factor beta; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C501162:FLT1 protein, human; D017479:Receptors, Platelet-Derived Growth Factor; D040281:Vascular Endothelial Growth Factor Receptor-1; D013917:Thrombin