Research

Various host defense systems act in concert in normal physiology. Coagulation pathways, fibrinolysis pathways and anticoagulant mechanisms prevent bleeding while avoiding harmful blood clots. The protein C pathway provides antithrombotic, anti-inflammatory and anti-apoptotic activities and is a focus for our research. 

DIRECT ANTI-APOPTOTIC EFFECTS OF ACTIVATED PROTEIN C ON CELLS

Activated protein C (APC) anti-apoptotic activity, first described in 2001, may provide cytoprotective activity that reduces cell death following a variety of cellular insults.  APC anti-apoptotic activity was studied using human brain microvascular endothelial cells (BEC) subjected to hypoxia as well as using kidney epithelial cells (K293) and endothelial cells (EA.hy.926) subjected to staurosporine-induced apoptosis. Inhibition of apoptosis by APC required a functional wnzyme active site since the zymogen was devoid of anti-apoptotic activity and a mutation of the serine in the catalytic triad to alanine (S360A-APC) abrogated the anti-apoptotic activity. This suggested that the anti-apoptotic activity of APC was mediated by proteolysis. Indeed, blocking antibodies against the protease-activated receptor 1 (PAR-1) as well as against endothelial protein C receptor (EPCR) abolished the anti-apoptotic effects of APC in each cell studied. In BEC studies, APC reduced the hypoxia-induced increases in levels of p53 and of Bax and APC blocked most of the hypoxia-induced reduction in Bcl-2 levels. These results demonstrate APC’s direct anti-apoptotic activity in the setting of brain endothelial ischemia through inhibition of hypoxia-induced p53 and Bax-2 upregulation resulting in a decrease in the pro-apoptotic Bax/Bcl-2 ratio, and show that APC’s anti-apoptotic activity on BEC requires PAR-1 and EPCR.

NEUROPROTECTIVE ACTIVITIES OF ACTIVATED PROTEIN C AND PROTEIN S

Stroke is a major cause of morbidity and mortality. Neuroprotective activities of the protein C pathway were studied in collaboration with Professor Berislav Zlokovic at theUniversity of Rochester. To characterize APC’s in vivo neuroprotection, APC was infused during focal ischemic stroke caused by a one-hour occlusion of the middle cerebral artery in mice. Intravenous infusions of recombinant murine or human APC significantly reduced brain infarction volumes and brain edema induced by ischemia. Following ischemic insult, the protective effects of APC were markedly reduced in mice with severe EPCR deficiency (< 10% of wild type) or in mice that had been pre-infused with an anti-PAR-1 blocking antibody 10 min before middle cerebral artery occlusion. To try to distinguish the in vivo contributions of APC antithrombotic action from APC anti-apoptotic effects, we determined the volumes of brain injury, changes in the cerebral blood flow, deposition of cerebrovascular fibrin and neutrophils, and behavioral neurologic scores in the presence of low dose (0.2 mg/kg) and high dose murine APC (2 mg/kg). High dose APC reduced the volume of brain injury and motor neurological score by 60% and 75%, respectively, associated with 34% improvement of the post-ischemic CBF and significant reductions in fibrin and neutrophils. In contrast, low dose murine APC reduced volume of injury and motor neurological score by > 30% and 56%, respectively, via mechanisms that appeared to be independent of APC’s anticoagulant activity as indicated by a lack of significant improvement in the post-ischemic cerebral blood flow and no significant changes in fibrin deposition. In summary, the in vivo neuroprotective effects of APC, like its in vitro anti-apoptotic activity, required EPCR and PAR-1, and APC in vivo effects appeared, in part, to be independent of its antithrombotic activity. The overall results strongly suggest that APC protects brain from ischemia in vivo by acting directly on brain cells. 

Protein S manifests anticoagulant and neuroprotective properties, apparently independent of APC. New studies demonstrated in vivo and in vitro neuroprotective activities of protein S in murine models of ischemic stroke and of neuronal hypoxia/reoxygenation injury. In the in vivo stroke model, protein S significantly improved motor neurological deficit, reduced infarction and edema volumes, improved post-ischemic cerebral blood flow, and reduced brain fibrin deposition and infiltration with neutrophils. Intracerebral bleeding was not observed with protein S treatment. Both plasma-derived and recombinant human protein S preparations were effective. Protein S protected ischemic neurons in vivo and cultured neurons in vitro from hypoxia/reoxygenation-induced apoptosis in a time- and dose-dependent manner. Thus, protein S is a significant neuroprotectant during ischemic brain injury with direct anti-apoptotic effects on neurons and with systemic antithrombotic effects. Hence, protein S could be a prototype of a new class of agents for ischemic stroke with combined direct neuronal protective effects and systemic antithrombotic and anti-inflammatory activities. 

CIRCULATING ACTIVATED PROTEIN C PHYSIOLOGY

Circulating APC, the active enzyme derived from the protein C zymogen, is a normal constituent of blood. APC rises in vivo in animals following thrombin infusions because thrombin converts protein C to APC. We found that atherosclerosis decreases thrombin-dependent generation of APC while platelet factor 4 increases APC generation. Interestingly, diet-induced regression of atherosclerosis restores normal generation of APC by thrombin.  We found that circulating APC is significantly lower inpatients who experience an ischemic stroke after an infection, in systemic lupus erythematosus patients and in smokers. Surprisingly, we found that APC levels are only modestly reduced in patients taking oral anticoagulants whereas protein C and clotting factors are markedly reduced. The increasing amount of clinical and animal data are consistent with the hypothesis that circulating APC is beneficial and that conditions that increase risk for atherothrombosis or venous thrombosis deleteriously decrease generation of APC.