Activated Protein C Function on in vitro and in vivo Cellular Microparticles.
Lead Research Organisation:
University of Liverpool
Department Name: Department of Haematology
Abstract
The risk of severe infections or septicaemia is increasing and causes over 1,000 deaths each year in England and Wales. Recently, activated protein C (APC) has been approved for treating septicaemia because it can reduce the death rate by 20%. Severe bleeding is a problem due to the APC action of preventing blood clotting. However, APC can also dampen inflammation on cells lining blood vessels and this is thought to be its more beneficial property in treating septicaemia. This anti-inflammation effect is dependent on APC binding to the endothelial protein C receptor (EPCR) at the cell surface.
Recently, we at the University of Liverpool have discovered that APC on EPCR can be released from the vessel wall surface into the circulation. Early studies show that this is a way of spreading its anti-inflammation property, especially to areas in the body where EPCR is not present. Our project aims to deepen this knowledge through a combination of studies using complete cells and genetic material on material generated in the laboratory as well as from patients. We speculate that the knowledge gained could advance treatment strategies in septicaemia by optimising beneficial anti-inflammation properties and minimising the bleeding risks associated with current treatment.
Recently, we at the University of Liverpool have discovered that APC on EPCR can be released from the vessel wall surface into the circulation. Early studies show that this is a way of spreading its anti-inflammation property, especially to areas in the body where EPCR is not present. Our project aims to deepen this knowledge through a combination of studies using complete cells and genetic material on material generated in the laboratory as well as from patients. We speculate that the knowledge gained could advance treatment strategies in septicaemia by optimising beneficial anti-inflammation properties and minimising the bleeding risks associated with current treatment.
Technical Summary
Activated protein C (APC) is an endogenous anticoagulant with anti-inflammatory properties. Therapeutic administration of APC has emerged as the first treatment to significantly reduce the relative risk of death in patients with severe sepsis. How APC mediates its protective effects is not fully understood, but treatment carries serious bleeding risks. We have made the novel observation that APC induces endothelial microparticle (MP) release into the circulation of septic patients being treated with this agent. These released MPs contain APC bound to the endothelial protein C receptor (EPCR) and are capable of localised anticoagulant function. In addition, preliminary data suggests that these MPs are more effective in inhibiting cell inflammation and apoptosis than is APC alone.
My hypothesis is that the above mechanism of disseminating APC could be harnessed into more efficient strategies for delivering APC to cell surfaces so as to prevent overlying thrombus formation and to protect endothelial cell integrity, especially under inflammatory conditions. The aim of the project will be to assess the role of APC on MP-associated EPCR in disseminating anti-inflammatory properties, with the specific objectives being to determine if (1) APC on MP-EPCR can manifest anti-inflammatory properties, even at vascular sites that are deficient in EPCR, and (2) whether APC on MP-EPCR is more efficient than free APC in inducing cytoprotective signalling via protease activated receptor (PAR)-1. The methodological approach will involve using in vitro-derived MP- EPCR with confirmation of in vivo relevance using clinically derived MP-EPCR. Firstly, gene microarray, protein expression and function studies will be performed comparing the effect of free APC versus APC at equal amounts on MP-EPCR. Secondly, PAR-1 deficient cells will be transfected with PAR-1 alone and together with EPCR so as to investigate whether the APC-MP-EPCR complex can still induce PAR-1 dependent cell signalling effects in cells that do not express EPCR on their surfaces. The further determination of whether APC signalling is limited by the extent of cell surface EPCR will involve over-expressing EPCR in relation to PAR-1 on human umbilical vein endothelial cells using lentiviral expression systems.
The potential scientific and medical opportunities of this molecular-cell biological approach using MP-EPCR directly from patients, would include
Improved understanding of functional events at the blood/endothelial interface that account for the beneficial effects of APC treatment in severe sepsis.
Development of therapeutic strategies that optimise the benefit over risk properties of APC.
Improving outcome for patients with sepsis.
My hypothesis is that the above mechanism of disseminating APC could be harnessed into more efficient strategies for delivering APC to cell surfaces so as to prevent overlying thrombus formation and to protect endothelial cell integrity, especially under inflammatory conditions. The aim of the project will be to assess the role of APC on MP-associated EPCR in disseminating anti-inflammatory properties, with the specific objectives being to determine if (1) APC on MP-EPCR can manifest anti-inflammatory properties, even at vascular sites that are deficient in EPCR, and (2) whether APC on MP-EPCR is more efficient than free APC in inducing cytoprotective signalling via protease activated receptor (PAR)-1. The methodological approach will involve using in vitro-derived MP- EPCR with confirmation of in vivo relevance using clinically derived MP-EPCR. Firstly, gene microarray, protein expression and function studies will be performed comparing the effect of free APC versus APC at equal amounts on MP-EPCR. Secondly, PAR-1 deficient cells will be transfected with PAR-1 alone and together with EPCR so as to investigate whether the APC-MP-EPCR complex can still induce PAR-1 dependent cell signalling effects in cells that do not express EPCR on their surfaces. The further determination of whether APC signalling is limited by the extent of cell surface EPCR will involve over-expressing EPCR in relation to PAR-1 on human umbilical vein endothelial cells using lentiviral expression systems.
The potential scientific and medical opportunities of this molecular-cell biological approach using MP-EPCR directly from patients, would include
Improved understanding of functional events at the blood/endothelial interface that account for the beneficial effects of APC treatment in severe sepsis.
Development of therapeutic strategies that optimise the benefit over risk properties of APC.
Improving outcome for patients with sepsis.
