Thrombin generation is characterized by the sequential activation of a series of serine proteases that can be broadly divided into initiation, propagation and amplification stages (Davie et al, 1991). Upon injury and consequent tissue factor exposure, the activation of factor VII leads to initial thrombin generation, which can then propagate further thrombin generation through activating components within the intrinsic pathway (Osterud & Rapaport, 1977). This leads to amplification in the coagulant process with the rapid and explosive conversion of fibrinogen to fibrin and the securing of haemostasis (Blombäck, 1996). Simultaneously, there is thrombin-mediated platelet activation through protease activated receptors (PARs) (Andersen et al, 1999) as well as thrombin cleavage of activated factor XIII (factor XIIIa) to stabilize fibrin formation (Sakata & Aoki, 1982; Shen & Lorand, 1983). Further clot consolidation is mediated by thrombin through activation of thrombin-activatable fibrinolysis inhibitor (TAFI), which retards clot lysis by removing binding sites for the attachment of tissue plasminogen activator (tPA) to fibrin for consequent lysis (Bajzar et al, 1996).
Whilst a burst in thrombin generation is crucial in achieving prompt haemostasis, uncontrolled thrombin generation may be harmful. Indeed, thrombin generation is kept in close check under physiological conditions with a short half-life of only 15 s (Jesty, 1986). It is rapidly inactivated by different inhibitors within the family of serine protease inhibitors (SERPINS), such as antithrombin (Rosenberg & Rosenberg, 1984), α1-antitrypsin (α1AT) and heparin co-factor II (Derechin et al, 1990), as well as by α2-macroglobulin (α2M) (Feinman et al, 1985). At the boundaries of tissue injury, any excess thrombin is then bound by the receptor thrombomodulin (TM) on intact endothelial surfaces. TM-bound thrombin can no longer express procoagulant activity (Esmon et al, 1982; Maruyama et al, 1985) but instead activates PC (Esmon & Owen, 1981). This rate of activation is increased significantly when PC is bound or complexed to the endothelial protein C receptor (EPCR) at the endothelial surface (Stearns-Kurosawa et al, 1996; Taylor et al, 2001). In the absence of EPCR, PC activation is reduced by 20-fold (Fukudome et al, 1998). The resulting APC can then express anticoagulant properties by cleaving and inactivating factors Va (Kisiel et al, 1977; Marlar et al, 1982) and VIIIa (Vehar & Davie, 1980) to reduce thrombin generation and procoagulant activity. As with other enzymes, APC has its own regulatory network consisting of PC inhibitor (Suzuki et al, 1987) and α1AT as first-line inactivators (Heeb & Griffin, 1988) with α2M as a secondary, calcium-dependent inhibitor (Hoogendoorn et al, 1991) (Scully et al, 1993).
Of interest is that APC anticoagulant activity in solution can be inhibited by soluble EPCR (sEPCR) (Fukudome et al, 1996). This is somewhat paradoxical as membrane-bound EPCR promotes anticoagulant activity. This apparent Janus type function is due to altered exposure of the APC active site upon conformational change in EPCR, as a result of cleavage at the cell surface (Esmon, 2003). sEPCR, when cleaved and released from the cell surface by tumour necrosis factor-α (TNF-α) converting enzyme (TACE) (Qu et al, 2007), is 4 kDa smaller than its membrane counterpart. Receptor shedding is upregulated by thrombin (Gu et al, 2000; Xu et al, 2000) and this apparent auto-regulatory mechanism of EPCR could theoretically modulate coagulant activity in the immediate vicinity of the cell surface. However, this alteration in APC within sEPCR could have other structure-function consequences given that it retains proteolytic activity towards small substrates (Regan et al, 1996).
Membrane-bound EPCR is homologous to the CD1/major histocompatibility class 1 family of molecules, most of which are involved in inflammation and antigen presentation (Fukudome & Esmon, 1995). Indeed the crystal structure of EPCR in complex with PC shares similarities with phospholipid binding within the antigen-presenting groove of the CD1 family (Oganesyan et al, 2002). Presentation and exchange of pathogen or cell-derived lipids may be involved in the processes by which these molecules modulate the inflammatory and immune response (Prigozy et al, 2001).
The relatively long half-life of APC, at approximately 20 min (Okajima et al, 1990), maintains a circulating concentration of 38 pmol/l (Gruber & Griffin, 1992). This contrasts with the much shorter half-life of thrombin and is likely to relate to the importance of maintaining vascular patency following localization of clot formation. Thrombin needs to be rapidly inactivated in solution to ensure that its potent effects are not further disseminated. Clot-based thrombin, however, can still organize and consolidate the fibrin structure, and also trigger the repair/healing mechanisms following injury (Mann et al, 2003). It is not known if there are functionally significant concentrations of clot-based APC although leucocytes bearing TM (Grey & Hancock, 1996) and EPCR (Galligan et al, 2001) are entrapped within developing thrombi. APC has been associated with pro-fibrinolytic properties that have been ascribed to its ability to complex plasminogen activator inhibitor-1 (de Fouw et al, 1987; Rezaie, 2001). However, the kinetics of this reaction do not appear to be physiologically relevant and an indirect role via reducing thrombin activation of TAFI would seem to be more likely in explaining this pro-fibrinolytic role.
Thrombin has a number of cellular effects. In addition to promoting platelet activation (Davey & Lüscher, 1967) and stimulating release of their mediators (Hamberg et al, 1975), it exerts influence over monocytes, macrophages (Bar-Shavit et al, 1986) and neutrophils in processes related to tissue repair at the site of injury (Frenette et al, 1996; Goldsack et al, 1998) In vitro, thrombin increases the expression of genes influencing vasomotor tone (Tesfamariam et al, 1993; Hamilton et al, 2001), cell proliferation, inflammation and leucocyte adhesion (Wu & Aird, 2005). Mediation of the thrombin pro-inflammatory response on the endothelium is thought to occur through various pathways including nuclear factor-κB (NFκB), early growth response factor-1 and GATA binding proteins (Minami et al, 2004). Primarily, this involves signalling through the family of 7-transmembrane G-protein-coupled PARs (Coughlin, 2000) and in humans, this is predominantly PAR-1 and PAR-3 (Ishihara et al, 1997).
With increasing recognition of the pleiotropic nature of APC, it is not surprising that areas of overlap with thrombin have been discovered. Whilst an anti-inflammatory role for APC can be indirect by nature of its ability to reduce thrombin generation, APC also has direct anti-inflammatory properties. APC can cleave and activate PAR1-dependent cellular pathways (Riewald et al, 2002). Proteolytic cleavage of the extracellular N-terminal tail forms a ligand within the G-protein coupled-PAR-1 receptor to trigger intracellular signalling (Coughlin, 2000). Unlike thrombin however, free APC is unable to cleave PAR-1 and requires membrane-bound EPCR as a co-factor for this effect (Guo et al, 2004). A further distinction from the thrombin–PAR-1 interaction is that EPCR–APC mediated signalling characteristically leads to anti-inflammatory and anti-apoptotic cellular effects. Examples of this include phosphorylation of mitogen-activated protein kinase (MAPK) (Riewald et al, 2002), suppression of NFκB expression (Joyce et al, 2001), as well as downregulation of p53 and thrombospondin 1 (Riewald & Ruf, 2005). An endothelial barrier protective effect is also observed through mechanisms involving sphingosine 1-phosphate (Feistritzer & Riewald, 2005; Finigan et al, 2005). In addition, the APC–PAR-1 signalling pathway leads to the generation of microparticles that can present and disseminate anticoagulant-active APC in complex with EPCR (Perez-Casal et al, 2005). These respective effects contrast with those mediated by high-dose thrombin in both enhancing endothelial permeability (van Nieuw Amerongen et al, 1998) and generating pro-apoptotic microparticles (Sapet et al, 2006).
Non-PAR-1-dependent mechanisms for the anti-inflammatory properties of APC also exist. Many of these are EPCR-dependent and relate to EPCR expression on cells other than at the endothelial surface. These include neutrophils (Kurosawa et al, 2000), eosinophils (Feistritzer et al, 2003) and monocytes (Feistritzer et al, 2006). In vitro, APC on human neutrophils can inhibit chemotaxis triggered by interleukin-8 (Sturn et al, 2003). The binding of sEPCR to CD11b/CD18 and to activated neutrophils via proteinase-3 can interfere with leucocyte adhesion and neutrophil-signalling events (Kurosawa et al, 2000). These findings may be relevant to the observation of reduced neutrophil accumulation in response to rhAPC in an in vivo human model of acute pulmonary inflammation (Nick et al, 2004).