Human thrombin and FXa mediate porcine endothelial cell activation; modulation by expression of TFPI-CD4 and hirudin-CD4 fusion proteins

Aside from their critical role in thrombosis, activated coagulation factors also have inflammatory properties and these may be important during delaye d xenograft rejection (DXR). This study assessed whether porcine EC could b e activated by factor Xa (FXa) and thrombin (FIIa) and whether expression o f tissue factor pathway inhibitor (TFPI)-CD4 and hirudin-CD4 fusion protein s could prevent such activation. Incubation of porcine EC with human FXa an d FIIa induced cell surface expression of E-selectin, VCAM and tissue facto r (TF) in a time-dependent and concentration-dependent manner. In contrast, porcine EC transfected with a human TFPI-CD4 fusion protein were selective ly resistant to these pro-inflammatory effects of FXa but not FIIa. Likewis e, the transfectants expressing the hirudin-CD4 fusion protein were selecti vely resistant to the pro-inflammatory effects of FIIa but not those of FXa . When combined, the FXa and Flla had an additive effect on the activation of control EC. In contrast, coexpression of both hirudin-CD4 and TFPI-CD4 f usion proteins completely inhibited the upregulation of VCAM with the FXa/F IIa mix. These results indicate that expression of novel anticoagulant fusi on proteins on the surface of porcine EC can protect against EC activation induced by human coagulation factors FXa and FIIa. In vivo, we anticipate t hat 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 fur ther protecting the grafts from DXR.