Regulated inhibition of coagulation by porcine endothelial cells expressing P-selectin-tagged hirudin and tissue factor pathway inhibitor fusion proteins.
BACKGROUND
Thrombotic vascular occlusion resulting in infarction occurs during hyperacute rejection of allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation. A similar process is also found in disorders of diverse etiology including atherosclerosis, vasculitis, and disseminated intravascular coagulation.


METHODS
We have previously constructed two membrane-tethered anticoagulant fusion proteins based on human tissue factor pathway inhibitor and the leech anticoagulant hirudin and demonstrated their functional efficacy in vitro. These constructs have now been modified by the addition of a P-selectin sequence to the cytoplasmic tail to localize them in Weibel-Palade bodies. They have been transfected into Weibel-Palade body-positive endothelial cells isolated from the inferior vena cava of normal pigs.


RESULTS
In resting endothelial cells, fusion protein expression colocalized with P-selectin and was confined to Weibel-Palade bodies. These cells had a procoagulant phenotype in recalcified human plasma. However, after activation with phorbol ester the anticoagulant proteins were rapidly relocated to the cell surface where they specifically inhibited the clotting of human plasma.


CONCLUSIONS
Novel anticoagulant molecules may prove useful therapeutic agents for gene therapy in thrombotic disease and postangioplasty or for transgenic expression in animals whose organs may be used for clinical xenotransplantation. Expression in vascular endothelial cells may be regulated by inclusion of P-selectin cytoplasmic sequence, to restrict cell surface expression to activated endothelium.
Chen
D
D
Department of Immunology and MRC Clinical Sciences Centre Haemostasis Research Group, Imperial College School of Medicine, Hammersmith Hospital, London, UK.
Riesbeck
K
K
McVey
J H
JH
Kemball-Cook
G
G
Tuddenham
E G
EG
Lechler
R I
RI
Dorling
A
A
D000818:Animals; D015704:CD4 Antigens / Q000494:pharmacology; D004211:Disseminated Intravascular Coagulation / Q000503:physiopathology*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000494:pharmacology; D006629:Hirudins / Q000096:biosynthesis; D008074:Lipoproteins / Q000096:biosynthesis*; D008565:Membrane Proteins / Q000378:metabolism; D019007:P-Selectin / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013552:Swine; D013556:Swine, Miniature; D020302:Weibel-Palade Bodies / Q000378:metabolism
D015704:CD4 Antigens; D005343:Fibrinolytic Agents; D006629:Hirudins; D008074:Lipoproteins; D008565:Membrane Proteins; D019007:P-Selectin; D011993:Recombinant Fusion Proteins; C051928:lipoprotein-associated coagulation inhibitor