title: Human thrombin and FXa mediate porcine endothelial cell activation; modulation by expression of TFPI-CD4 and hirudin-CD4 fusion proteins.
abstract: Aside from their critical role in thrombosis, activated coagulation factors also have inflammatory properties and these may be important during delayed xenograft rejection (DXR). This study assessed whether porcine EC could be activated by factor Xa (FXa) and thrombin (FIIa) and whether expression of tissue factor pathway inhibitor (TFPI)-CD4 and hirudin-CD4 fusion proteins could prevent such activation. Incubation of porcine EC with human FXa and FIIa induced cell surface expression of E-selectin, VCAM and tissue factor (TF) in a time-dependent and concentration-dependent manner. In contrast, porcine EC transfected with a human TFPI-CD4 fusion protein were selectively resistant to these pro-inflammatory effects of FXa but not FIIa. Likewise, the transfectants expressing the hirudin-CD4 fusion protein were selectively resistant to the pro-inflammatory effects of FIIa but not those of FXa. When combined, the FXa and FIIa had an additive effect on the activation of control EC. In contrast, coexpression of both hirudin-CD4 and TFPI-CD4 fusion proteins completely inhibited the upregulation of VCAM with the FXa/FIIa mix. These results indicate that expression of novel anticoagulant fusion proteins on the surface of porcine EC can protect against EC activation induced by human coagulation factors FXa and FIIa. In vivo, we anticipate that expression of these fusion proteins on the endothelium of transplanted xenografts, besides preventing intravascular thrombosis, will also protect against EC activation induced by trace amounts of FIIa and FXa, thereby further protecting the grafts from DXR.
authors.affiliation: Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK.
authors.forename: D
authors.initials: D
authors.lastname: Chen
authors.forename: K
authors.initials: K
authors.lastname: Riesbeck
authors.forename: J H
authors.initials: JH
authors.lastname: McVey
authors.forename: G
authors.initials: G
authors.lastname: Kemball-Cook
authors.forename: E G
authors.initials: EG
authors.lastname: Tuddenham
authors.forename: R I
authors.initials: RI
authors.lastname: Lechler
authors.forename: A
authors.initials: A
authors.lastname: Dorling
mesh_terms: D000818:Animals; D001777:Blood Coagulation; D015704:CD4 Antigens / Q000235:genetics / Q000502:physiology; D002448:Cell Adhesion; D002478:Cells, Cultured / Q000187:drug effects; D019040:E-Selectin / Q000096:biosynthesis / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D015951:Factor Xa / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006084:Graft Rejection / Q000517:prevention & control*; D006629:Hirudins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008074:Lipoproteins / Q000235:genetics / Q000502:physiology*; D011184:Postoperative Period; D011516:Prothrombin / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013552:Swine; D013917:Thrombin / Q000494:pharmacology*; D013925:Thromboplastin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013927:Thrombosis / Q000517:prevention & control*; D014162:Transfection; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis; D014682:Vena Cava, Inferior / Q000166:cytology
chemical_list: D015704:CD4 Antigens; D019040:E-Selectin; D006629:Hirudins; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins; D019010:Vascular Cell Adhesion Molecule-1; C051928:lipoprotein-associated coagulation inhibitor; D011516:Prothrombin; C025337:Factor IIa; D013925:Thromboplastin; D013917:Thrombin; D015951:Factor Xa