Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and is neuroprotective

Abstract

Activated protein C (APC) is a systemic anti-coagulant and anti-inflammatory factor. It reduces organ damage in animal models of sepsis, ischemic injury and stroke and substantially reduces mortality in patients with severe sepsis. It was not known whether APC acts as a direct cell survival factor or whether its neuroprotective effect is secondary to its anti-coagulant and anti-inflammatory effects. We report that APC directly prevents apoptosis in hypoxic human brain endothelium through transcriptionally dependent inhibition of tumor suppressor protein p53, normalization of the pro-apoptotic Bax/Bcl-2 ratio and reduction of caspase-3 signaling. These mechanisms are distinct from those involving upregulation of the genes encoding the anti-apoptotic Bcl-2 homolog A1 and inhibitor of apoptosis protein-1 (IAP-1) by APC in umbilical vein endothelial cells. Cytoprotection of brain endothelium by APC in vitro required endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1), as did its in vivo neuroprotective activity in a stroke model of mice with a severe deficiency of EPCR. This is consistent with work showing the direct effects of APC on cultured cells via EPCR and PAR-1 (ref. 9). Moreover, the in vivo neuroprotective effects of low-dose mouse APC seemed to be independent of its anti-coagulant activity. Thus, APC protects the brain from ischemic injury by acting directly on brain cells.

Activated protein C promotes breast cancer cell migration through interactions with EPCR and PAR-1

“…In the PROWESS trial, sepsis patients that were treated with APC have a steady-state level of 45 ng/ ml [45]. However, our overall goal was to characterize the mechanism of APC to promote cell migration and we used similar APC concentrations as in previous in vitro studies (0.5-50 μg/ml) [20,26,29]. Interestingly, levels of APC used in HUVEC experiments are closer to physiological concentrations of APC (0.1-10 μg/ml) (data not shown).…”

“…Using zymogen PC, chemically inactivated DEGR-APC, and an active site mutant of APC (S195A) with the MDA-MB-231 cancer cells, one finding of our study showed that the active protease was needed to increase cell migration. Therefore, unlike the inhibitory role of APC with lymphocytes, the pro-migratory role of APC in the MDA-MB-231 cells requires the active site of the protease, most likely to bind and activate receptors, such as PAR-1 [20,21,26,30,31], and to activate extracellular matrix proteases, such as MMP-2 and MMP-9 [27,46,47]. It is possible that when bound to EPCR, APC may undergo modifications to its macromolecular substrate recognition.…”

“…The second goal was to study the interaction of APC with EPCR and PAR-1, receptors involved in mediating other non-hemostatic effects of APC on expression of apoptotic genes and cell proliferation [20,30,31]. The following results show that APC increases chemotaxis and invasion when incubated with the cancer cells, which requires active protease and is dependent on an interaction with both EPCR and PAR-1.…”

“…PAR-1 has been shown to be involved in mediating the nonhemostatic effects of APC [20,21,26,31]. With Western blots of cell lysates (Fig.…”

“…However, through in vitro and in vivo models, APC has been shown to not only regulate coagulation in the microvasculature but also affect inflammation, apoptosis, proliferation, and angiogenesis. APC inhibits apoptosis through upregulation of anti-apoptotic Bcl-2 [19][20][21], and downregulation of p53, Bax [20,21], and caspases 3 [20][21][22], 8, and 9 [21,22] all through interactions with EPCR and protease-activated receptors (PAR). Using a murine focal ischemic stroke model, human and mouse APC treatment restored blood flow, reduced infarct volume and edema, eliminated neutrophil infiltration, and reduced fibrin deposition [20][21][22][23][24], all mediated through EPCR [20], PAR-1 [20,21], and PAR-3 [21].…”