Activated Protein C (APC) is an endogenous anticoagulant that regulates thrombin generation. Its physiological importance is reflected by the thrombotic phenotype of PC deficient individuals and those with APC resistance due to mutation at the Factor V cleavage site. At a molecular level, thrombin binding to the receptor thrombomodulin (TM) converts endothelial protein C receptor (EPCR) bound PC into APC. APC has a negative feedback role in curbing thrombin generation by inactivating coagulation factors V and VIII. Clinically, acquired PC deficiency occurs in conditions such as severe sepsis as a result of mechanisms involving consumption, increased degradation by proteolytic enzymes and reduced hepatic synthesis. The initial success of APC in sepsis (PROWESS Trial) over other anticoagulants is thought to relate to anti-inflammatory and cyto-protective properties The interaction of EPCR-APC with protease activated receptor-1(PAR-1}, the prototypical thrombin receptor appears to be relevant in vivo. Intravenous injection of APC into mice leads to PAR-1-dependent gene induction and interestingly gene expression profiling of cytokine-stimulated endothelial cells demonstrates marked differences between APC and thrombin signaling. Cardiovascular dysfunction occurs frequently in patients with sepsis, increasing the mortality rate significantly from 20% to 70-90%. Key observations from the PROWESS Trial highlighted a possible link between APC and cardiovascular recovery. For those with cardiovascular dysfunction at baseline, APC treatment led to a significantly greater likelihood of organ resolution. Furthermore, in the most critically ill patients with multiple organ dysfunction, time to resolution of cardiac dysfunction was significantly shorter for APC treated patients with improvements linked to overall mortality reduction. The precise cause of cardiac dysfunction in sepsis remains unclear. Endothelial activation has been implicated as has decreased contractility and impaired myocardial compliance. Postulated mechanisms include cardiac myofibrillar dysfunction with altered calcium sensitivity and the influence of myocardial depressant factors such as pro-inflammatory cytokines and nitric oxide. The potential effects of APC on the cardiovascular system could be multifactorial. This thesis sets out to examine if the anti-inflammatory and cytoprotective properties of APC may be involved. In the first experimental chapter, the effect of APC in free or microparticulate form on human coronary artery endothelial cells was examined. An anti-inflammatory and cytoprotective profile is induced although evidence of reversing the pro-inflammatory effect of tumour necrosis factor alpha (TNFo) was inconclusive. In the second experimental chapter the effect of APC on cardiomyocyte contraction was studied using rat ventricular cardiomyocytes and this showed the potential role of EPCR and PAR-1. In view of the high efficiency of PAR-1 activation by thrombin, the third experimental chapter looked at whether any APC effect would continue to be relevant in the presence of thrombin. The APC response was observed at clinically relevant concentrations, without significant modification of calcium handling and despite the presence of thrombin. This led to the final experimental chapter where PAR-1 mediated effects appear to be regulated by downstream protein kinase C alpha (PKCa) providing mechanistic insight in to the findings This thesis concludes that APC has functional relevance with respect to cardiac cells both at the endothelial and contractile level. The results offer a framework to delineate distinct pathways for improved therapeutic manipulation in the treatment of myocardial sepsis and depression.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:631571 |
| Date | January 2012 |
| Creators | Dutt, Tina |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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