Protective mechanisms of activated protein C in severe inflammatory disorders

Abstract

The protein C system is an important natural anticoagulant mechanism mediated by activated protein C (APC) that regulates the activity of factors VIIIa and Va. Besides well-defined anticoagulant properties, APC also demonstrates anti-inflammatory, anti-apoptotic and endothelial barrier-stabilizing effects that are collectively referred to as the cytoprotective effects of APC. Many of these beneficial effects are mediated through its co-receptor endothelial protein C receptor, and the protease-activated receptor 1, although exact mechanisms remain unclear and are likely pleiotropic in nature. Increased insight into the structure-function relationships of APC facilitated design of APC variants that conserve cytoprotective effects and reduce anticoagulant features, thereby attenuating the risk of severe bleeding with APC therapy. Impairment of the protein C system plays an important role in acute lung injury/acute respiratory distress syndrome and severe sepsis. The pathophysiology of both diseases states involves uncontrolled inflammation, enhanced coagulation and compromised fibrinolysis. This leads to microvascular thrombosis and organ injury. Administration of recombinant human APC to correct the dysregulated protein C system reduced mortality in severe sepsis patients (PROWESS trial), which stimulated further research into its mechanisms of action. Several other clinical trials evaluating recombinant human APC have been completed, including studies in children and less severely ill adults with sepsis as well as a study in acute lung injury. On the whole, these studies have not supported the use of APC in these populations and challenge the field of APC research to search for additional answers.

Figure 1

The Anticoagulant APC Pathway. TF is the major initiator of the coagulation cascade and can be up-regulated under inflammatory conditions. The activation of the coagulation cascade leads to the formation of thrombin (IIa) and fibrin. Thrombin and fibrin are responsible for the pro-inflammatory effects of coagulation. Thrombin signals through PAR-1 to exert its detrimental effects. APC is generated from the circulating protein C zymogen. Thrombin bound to TM activates protein C. Binding of protein C to the EPCR is responsible for a more efficient activation. After dissociation from EPCR, APC can exert its anticoagulant function. APC cleaves the activated coagulation factors FVa and FVIIIa that become inactive. APC further inactivates the PAI-1 resulting in increased fibrinolysis. These anticoagulant and pro-fibrinolytic characteristics are responsible for the indirect protective effects of APC during inflammatory disorders. APC bound to EPCR is responsible for its direct cytoprotective effects involving PAR-1 signalling. APC, activated protein C; EPCR, endothelial protein C receptor; PAI-1, plasminogen activator inhibitor-1; PAR-1, protease-activated receptor-1; t-PA, tissue-type plasminogen activator; TF, tissue factor; TM, thrombomodulin.

Figure 2

The cytoprotective APC pathway. This figure provides an overview of the direct cytoprotective effects for anti-inflammatory and anti-apoptotic activity on leukocytes and endothelial cells. The anti-inflammatory effects of APC on endothelial cells include the inhibition of NF-κB activation with decreased expression of endothelial adhesion molecules and the reduced expression of pro-inflammatory cytokines. APC also inhibits the NF-κB and AP-1 transcription factors on leukocytes and reduces their production of pro-inflammatory mediators. The end result of these effects is a reduction in leukocyte chemotaxis and infiltration. APC exerts anti-apoptotic effects on both leukocytes and endothelial cells by reducing several pro-apoptotic signals. The direct protective effects of APC on endothelial cells are mediated in the presence of EPCR and PAR-1. The effects of APC on leukocytes are mediated by EPCR alone or in the presence of PAR-1. APC increases endothelial production of PGI₂ and MCP-1. The role of sEPCR, which is up-regulated in different inflammatory diseases, and PR3 on activated neutrophils are still not completely understood. PR3 binds both sEPCR and membrane-bound EPCR and may play a role in binding APC to neutrophils and/or inactivating the protein C pathway. The exact mechanism of APC signalling through PAR-1 is still not clear, and different models have been described in literature. AP-1, activator protein-1; APC, activated protein C; EPCR, endothelial protein C receptor; ICAM-1, intercellular adhesion molecule-1; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; NF-κB, nuclear factor-kappa B; PAR-1, protease-activated receptor-1; PGI₂, prostaglandin I₂; PR3, proteinase-3; sEPCR, soluble EPCR; TNF-α, tumour necrosis factor alpha; VCAM-1, vascular cell adhesion molecule-1.

Figure 3

Endothelial barrier protection. Activated protein C (APC) bound to endothelial protein C receptor (EPCR) and thrombin (IIa) at low concentrations activates sphingosine kinase-1 (SK1) through activation of protease-activated receptor-1 (PAR-1). This results in increased production of sphingosine-1/phosphate (S1P) and S1P signals through its receptor S1P receptor-1 (S1P₁) to exert its barrier-protective effects on the endothelium. The role of EPCR in activating the barrier-protective pathway downstream PAR-1 by APC is still not known. APC bound to EPCR may also transactivate S1P₁ depending on the PI3-kinase/Akt pathway. Activated S1P₁ mediates its barrier-protective effect through activation of Rac. This results in a reduction of stress fibre formation and a reduction of the tensile strengths on the intercellular junctions and reduces endothelial permeability. Platelet-derived growth factor-BB (PDGF-BB) is increased by APC and stimulates wound healing and endothelial cell migration.