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 du ring hyperacute rejection of allografts transplanted into sensitized patien ts and remains a major problem in experimental xenotransplantation. A simil ar process is also found in disorders of diverse etiology including atheros clerosis, vasculitis, and disseminated intravascular coagulation. Methods. We have previously constructed two membrane-tethered anticoagulant fusion proteins based on human tissue fatter pathway inhibitor and the lee ch anticoagulant hirudin and demonstrated their functional efficacy in vitr o. 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 cel ls isolated from the inferior vena cava of normal pigs, Results, In resting endothelial cells, fusion protein expression colocalize d with P-selectin and was confined to Weibel-Palade bodies. These cells had a procoagulant phenotype in recalcified human plasma. However, after activ ation 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 age nts for gene therapy in thrombotic disease and postangioplasty or for trans genic expression in animals whose organs may be used for clinical xenotrans plantation. Expression in vascular endothelial cells may be regulated by in clusion of P-selectin cytoplasmic sequence, to restrict cell surface expres sion to activated endothelium.