EXPRESSION OF HUMAN THROMBOMODULIN COFACTOR ACTIVITY IN PORCINE ENDOTHELIAL-CELLS

Background. Xenograft rejection may predispose to vascular thrombosis because of putative cross-species' functional incompatibilities betwee n natural anticoagulants present on the donor endothelium and host act ivated coagulation factors. For example, porcine thrombomodulin expres sed on porcine aortic endothelial cells (PAEC) does not provide the ex pected thrombomodulin (TM)-cofactor activity for human protein C in th e presence of human thrombin. In addition, TM may be down-regulated af ter cellular activation, Our aim was to express human TM cofactor acti vity in PAEC and to study the proinflammatory effect of tumor necrosis factor-alpha (TNF-alpha) on stable expressed human thrombomodulin in vitro. Methods and Results. Retroviral transduction of PAEC with the g ene encoding for human thrombomodulin (hTM) resulted in expression of high levels of specific TRI cofactor activity on PAEC (0.62 mu g/ml ac tivated protein C/10(5) cells). High-level expression of hTM resulted in a 620-fold higher activation of human protein C in the presence of human thrombin when compared with mock-transduced PAEC (0.0001 mu g/ml / 10(5) cells; P<0.001), Transduced PAEC expressing hTM also bound mor e human thrombin than control PAEC, as determined by inhibition of thr ombin-induced platelet activation (P<0.05). We noted that exposure. to TNF-alpha significantly reduced exogenous hTM cofactor activity on tr ansduced PAEC in a time- and dose-dependent fashion; this occurred des pite the relatively stable expression of hTM mRNA and hTM antigen In t hese cells, Treatment of transduced PAEC with selected antioxidants co uld protect against the loss of hTM cofactor activity directly associa ted with the oxidative stress induced by TNF-alpha activation response s. Conclusions, Our data show that the functional deficiency of the an ticoagulant protein C pathway in PAEC may be corrected by viral transd uction of these cells. As analysis of the hTM function showed modulati on under conditions of cellular activation, me suggest that expression of hTM mutants resistant to oxidation may have greater therapeutic ut ility in the genetic modification of porcine xenografts.