Inhibition of tissue factor-dependent and -independent coagulation by cell surface expression of novel anticoagulant fusion proteins.
BACKGROUND
Thrombotic vascular occlusion occurs in disorders of diverse etiology, including atherosclerosis, vasculitis, and disseminated intravascular coagulation. The same process results in hyperacute rejection of renal allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation.


METHODS
We have previously described the design and expression of several genetic constructs encoding novel fusion proteins with anticoagulant properties. They are based on two naturally occurring soluble anticoagulant proteins, human tissue factor pathway inhibitor (hTFPI) and the leech protein hirudin, which act early and late in the clotting cascade, respectively. We report the expression of human hTFPI-CD4 on the surface of immortalized porcine endothelial cells (IPEC), and show that it functions across the species divide as evidenced by the binding of membrane-expressed porcine tissue factor (pTF)-human factor VIIa complexes.


RESULTS
Using a human plasma recalcification clotting assay, we distinguished between pTF-dependent and pTF-independent fibrin generation, and we have demonstrated that expression of hTFPI-CD4 on IPEC effectively prevented pTF-dependent clotting. Moreover, we show that when hTFPI-CD4 was co-expressed with the hirudin construct, the procoagulant properties of in vitro cultured, activated IPEC were almost completely abolished.


CONCLUSIONS
These results suggest that these novel anticoagulant molecules may prove useful therapeutic agents for gene therapy or for transgenic expression in animals whose organs may be used for cliniCal xenotransplantation.
Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, London, UK.
D
D
Chen
K
K
Riesbeck
G
G
Kemball-Cook
J H
JH
McVey
E G
EG
Tuddenham
R I
RI
Lechler
A
A
Dorling
D000818:Animals; D000925:Anticoagulants; D001777:Blood Coagulation; D015704:CD4 Antigens / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000502:physiology*; D015942:Factor VIIa / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006629:Hirudins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007865:Leeches; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013552:Swine; D013925:Thromboplastin / Q000502:physiology*; D014162:Transfection / Q000379:methods
D000925:Anticoagulants; D015704:CD4 Antigens; D006629:Hirudins; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins; C051928:lipoprotein-associated coagulation inhibitor; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa