Inhibition of tissue factor-dependent and -independent coagulation by cellsurface expression of novel anticoagulant fusion proteins

Background Thrombotic vascular occlusion occurs in disorders of diverse eti ology, including atherosclerosis, vasculitis, and disseminated intravascula r coagulation. The same process results in hyperacute rejection of renal al lografts 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 proper ties. They are based on two naturally occurring soluble anticoagulant prote ins, human tissue factor pathway inhibitor (hTFPI) and the leech protein hi rudin, which act early and late in the clotting cascade, respectively. We r eport the expression of human hTFPI-CD4 on the surface of immortalized porc ine endothelial cells (IPEC), and show that it functions across the species divide as evidenced by the binding of membrane-expressed porcine tissue fa ctor (pTF)-human factor VIIa complexes. Results. Using a human plasma recalcification clotting assay, we distinguis hed between pTF-dependent and pTF-independent fibrin generation, and we hav e demonstrated that expression of hTFPI-CD4 on IPEC effectively prevented p TF-dependent clotting. Moreover, we show that when hTFPI-CD4 was co-express ed with the hirudin construct, the procoagulant properties of in vitro cult ured, 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 exp ression in animals whose organs may be used for clinical xenotransplantatio n.