Microparticle-associated endothelial protein C receptor and the induction of cytoprotective and anti-inflammatory effects.
BACKGROUND
The endothelial protein C receptor plays an important role within the protein C pathway in regulating coagulation and inflammation. Recently, we described that endothelial protein C receptor can be released in vitro in microparticulate form from primary endothelial cells by exogenous activated protein C. Activated protein C bound to this endothelial protein C receptor retains anticoagulant activity and we hypothesize that this microparticulate endothelial protein C receptor-activated protein C complex can also cleave endothelial protease-activated receptor 1 to modulate inflammation and increase cell survival. Our main objective was, therefore, to study the effect that microparticle-associated endothelial protein C receptor-activated protein C has on endothelial function.


METHODS
Mini-arrays were used and probed with cDNA obtained from endothelial cells after treatment with microparticle-associated endothelial protein C receptor-activated protein C and results were confirmed by real time polymerase chain reaction. The functional relevance of changes at gene level were further analyzed by endothelial apoptosis and permeability assays, in the presence and absence of specific blockade of endothelial protein C receptor, protein C and protease-activated receptor 1.


RESULTS
Gene profiling of endothelial cells stimulated by 40 nmol/L activated protein C on microparticles showed significant changes in anti-apoptotic and inflammatory pathways. This was accompanied by protease-activated receptor 1-dependent anti-apoptotic and barrier protective effects, the latter of which also involved sphingosine 1-phosphate receptor and vascular endothelial growth factor receptor-2/ kinase insert domain receptor. Protein C blockade reversed these effects showing specificity for activated protein C on microparticles. Furthermore, confocal microscopy and enzyme-linked immunosorbent assay of plasma obtained from septic patients during recombinant activated protein C treatment showed evidence of their presence in vivo.


CONCLUSIONS
Activated protein C on microparticle-associated endothelial protein C receptor release can induce protease-activated receptor 1-dependent endothelial effects. The mechanisms underlying barrier protection involve sphingosine 1-phosphate receptor and kinase insert domain receptor.
Pérez-Casal
Margarita
M
Department of Haematology, Royal Liverpool University Hospital, Prescot Street, Liverpool L7 8XP, United Kingdom.
Downey
Colin
C
Cutillas-Moreno
Beatriz
B
Zuzel
Mirko
M
Fukudome
Kenji
K
Toh
Cheng Hock
CH
D015703:Antigens, CD / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D019610:Cytoprotection / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D000074283:Endothelial Protein C Receptor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006801:Humans; D007249:Inflammation / Q000517:prevention & control; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D020411:Oligonucleotide Array Sequence Analysis; D010316:Particle Size; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism* / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D049349:Receptors, Lysosphingolipid / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018805:Sepsis / Q000517:prevention & control; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
D015703:Antigens, CD; D000074283:Endothelial Protein C Receptor; D004791:Enzyme Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011486:Protein C; D011956:Receptors, Cell Surface; D049349:Receptors, Lysosphingolipid; D040301:Vascular Endothelial Growth Factor Receptor-2; C478370:PROCR protein, human