Characterization of the anti-apoptotic effect of activated protein C

Abstract number: OC298

Mosnier L. O., Gale A. J., Griffin J. H.

The Schripps Research Institute, USA

Activated protein C (APC) is a natural anti-coagulant that down regulates thrombin formation by inactivating the activated cofactors Va and VIIIa. Recent interest in the cellular functions of APC spiked after discovery of its beneficial effects on mortality in severe sepsis. To study structural requirements of the APC anti-apoptotic activity as well as its mode of action, a staurosporin induced apoptosis model was used. APC inhibited apoptosis in both kidney epithelial cells (K293) and endothelial cells (EA.hy.926) as determined by Apopercentage staining, YOPRO-1 uptake, annexin V labeling and induction of caspase-3 like activity (DEVD). Inhibition of apoptosis was dependent on both the APC concentration and the time of preincubation. In K293 cells, half maximal inhibition of apoptosis was observed at 35 nM APC, whereas in endothelial cells this was obtained at 1.7 nM APC using a 24-h preincubation. In endothelial cells, half maximal inhibition of apoptosis occurred after 46 min and maximal inhibition was obtained within 4 h of preincubation of 50 nM APC before applying the apoptotic stimuli. In addition, APC inhibited apoptosis by approximately 50% even in the absence of an apoptotic stimuli. Immuno-histochemical analysis indicated intracellular localization of APC and suggested a correlation between APC intracellular localization and protection against apoptosis. Inhibition of apoptosis by APC is specific for APC, as it was blocked by an antibody against APC and no anti-apoptotic effects were observed when APC was heat-inactivated. Furthermore, inhibition of apoptosis by APC required a functional active-site, since the zymogen was devoid of anti-apoptotic activity and also a mutation of the serine in the catalytic triad to alanine (S360A-APC) abrogated the anti-apoptotic activity. This demonstrated that the anti-apoptotic activity of APC was mediated by proteolysis. Indeed, blocking antibodies against the protease-activated receptor 1 (PAR-1) abolished the anti-apoptotic effect of APC. These results indicate that inhibition of apoptosis by APC is dependent on a functional APC active-site and involves APC-mediated activation of PAR-1. If APC-mediated PAR-1 activation is sufficient to account for the anti-apoptotic effect of APC or is merely a first step in a more complex mechanism is unknown, but the correlation between APC internalization and protection against apoptosis might suggest the latter.