The normal role of Activated Protein C in maintaining homeostasis and its relevance to critical illness

Thrombin is a multifunctional protein, with procoagulant, inflammatory and anticoagulant effects. Binding of thrombin to thrombomodulin results in activation of Protein C and initiation of the Activated Protein C anticoagulant pathway, a process that is augmented by the endothelial cell Protein C receptor (EPCR). Activated Protein C has demonstrated antithrombotic, anti-inflammatory, and profibrinolytic properties. Its antithrombotic activity is particularly important in the microcirculation, and Protein C deficiency is associated with microvascular thrombosis. Activated Protein C has also been shown to modulate inflammation. When the level of thrombomodulin or Protein C is reduced in sepsis there is a vicious cycle of coagulation and inflammation, with potentially lethal consequences. In vitro studies and animal models have shown that Activated Protein C blunts the inflammatory and coagulant response to sepsis through a variety of mechanisms.


Introduction
The initiation of coagulation, especially in severe sepsis, is probably mediated by the induction of tissue factor (TF) expression by endotoxin. This leads to the activation of factor X, which then combines with factor Va to convert prothrombin to thrombin. Although thrombin is usually considered to have purely procoagulant activity, it is in fact a multifunctional protein that has some important homeostatic anticoagulant effects ( Fig. 1) [1]. One of its main functions is to bind to thrombomodulin, which is expressed on the endothelial cell surface. Thrombomodulin is the major physiologic buffer for the procoagulant effects of thrombin in normal vessels [2,3]. Because thrombomodulin binds to the same site on thrombin that would normally bind to fibrinogen, platelets or factor V, all of these functions are blocked. Instead, the thrombinthrombomodulin complex activates Protein C (through a different site on the thrombin molecule), resulting in initiation of the Activated Protein C pathway [3]. This process is augmented by the EPCR [4,5]. Activated Protein C must dissociate from EPCR before it can bind to Protein S and function as an effective anticoagulant through the inactivation of factor Va.

Thrombomodulin and Activated Protein C in the microcirculation
by the number of endothelial cells that are in contact with the blood. Because the endothelial cell surface area per unit blood volume is much greater in the microcirculation (approximately 3000-5000 cm 2 of endothelium/ml blood) than in larger blood vessels (approximately 1.5 cm 2 /ml) ( Fig. 2) [6], there is a correspondingly high concentration of thrombomodulin in the normal microvasculature (approximately 500 nmol/l) as compared with the larger vessels (approximately 0.1-0.2 nmol/l). As a result, thrombin is rapidly removed from the microcirculation as it is bound to thrombomodulin. This suggests that the Activated Protein C system is uniquely poised to regulate coagulopathies in the microcirculation, and this has been confirmed in clinical studies [7]. Importantly, at least in some forms of sepsis, thrombomodulin can be downregulated, with resulting loss of ability to regulate thrombin, and hence clotting, in the microcirculation. Homozygous Protein C deficiency has a similar result, such that affected babies develop microvascular thrombosis (purpura fulminans) almost immediately after birth [3,8]. In contrast, antithrombin deficiencies cause largevessel thrombosis.

Vicious cycle of coagulation and inflammation
In addition to its procoagulant and anticoagulant effects, thrombin can also be involved in the process of inflammation ( Fig. 1) [1]. That is, it can activate endothelial cells to express P-selectin, causing neutrophil and monocyte adhesion. It is also chemotactic for polymorphonuclear leukocytes and is a potent inducer of endothelial platelet-activating factor formation. Platelet-activating factor is a major activator of neutrophils, which have been demonstrated to play an important role in the pathogenesis of sepsis.
The inflammatory response also modulates the coagulation system, including downregulation of the expression of thrombomodulin (Fig. 3) [9]. Inflammation also increases levels of α 1 -antitrypsin, which is an acute-phase response protein and an important inhibitor of the Protein C pathway. Acute inflammatory mediators such as tumor necrosis factor (TNF)-α and endotoxin amplify the production of TF, particularly by monocytes, triggering further coagulation. Complement activation by endotoxin increases exposure of clot-promoting membrane phospholipids to blood, and thereby propagates the coagulation response. In certain forms of inflammation, levels of both fibrinogen and plasminogen activator inhibitor (PAI)-1 (an inhibitor of fibrinolysis) are increased.
If there were no mechanisms to disrupt the amplification of coagulation by inflammation, and vice versa, then the ensuing vicious cycle would have a catastrophic effect, leading ultimately to death (Fig. 4) [10]. It was hypothesized, therefore, that endogenous Activated Protein C interferes with these amplification loops by modulating both coagulation and inflammation, which differentiates it from other types of anticoagulants, and that Protein C depletion may occur in severe sepsis [11].
A primate model was used in the initial study undertaken to investigate this possibility [11]. In animals infused with a lethal Escherichia coli dose alone, Protein C and fibrinogen levels fell, liver damage occurred (Fig. 5a), and the animals died within 24-32 h. In contrast, organ damage Cellular and humoral responses to nM concentrations of thrombin. EC, endothelial cell; PAF, platelet-activating factor; PDGF, platelet-derived growth factor; PMN, polymorphonuclear leukocyte; TGF, transforming growth factor. Adapted from Esmon [1].

Figure 2
Endothelial cell surface area per ml of blood. Adapted from Busch et al [6]. was prevented in animals given a lethal dose of E. coli plus an infusion of Activated Protein C (Fig. 5b). Importantly, it was shown that animals could still be rescued if Activated Protein C was given 2-3 h after the lethal E. coli dose was administered. Similarly, when animals were given a sublethal dose of E. coli and Protein C activation was blocked with an antibody, mimicking the clinical situation in a patient with sepsis and low Protein C levels, the sublethal bacterial dose became lethal, with complete fibrinogen consumption, a disseminated intravascular coagulation-like syndrome, organ failure, and a full-blown septic shock-like response (Fig. 6) [11].
Amplification of the inflammatory mediators in this setting aggravates the process still further. C4-binding protein (C4BP) is an acute-phase protein that is known to bind and inhibit Protein S, a vital cofactor for the Activated Protein C system. In another primate study [12], it was shown that administration of C4BP together with a The inflammation-coagulation autoamplification loop. Adapted from Esmon et al [10].  Roles of inflammatory mediators in the coagulation response. Adapted from Esmon [9]. normally sublethal dose of E. coli resulted in complete fibrinogen consumption and death. In the same study, it was shown that, whereas either sublethal E. coli doses or C4BP alone had no appreciable effect on TNF-α production, administration of both substances led to a circulating TNF-α level that approached that seen with a lethal dose of E. coli (Fig. 7), with rapid consumption of fibrinogen, systemic organ damage, and eventual death. With coadministration of Protein S, however, TNF-α levels remained similar to those seen in animals administered a sublethal dose of E. coli alone (Fig. 7), and all of these animals survived. This suggests that reversal of the inhibitory effects of inflammatory cytokines on the Activated Protein C pathway can control the inflammatory response.
Activated Protein C can promote fibrinolysis by at least two mechanisms. One involves the ability of Activated Protein C to form a tight complex with PAI-1. Once the complex with Activated Protein C forms, PAI-1 (the major inhibitor of tissue plasminogen activator) can no longer inhibit tissue plasminogen activator [13]. Because of the ability of Activated Protein C to limit thrombin generation, it can also reduce the activation of a precarboxypeptidase B commonly referred to as thrombin activatable fibrinolysis inhibitor [14,15]. Thrombin activatable fibrinolysis inhibitor functions by removing lysine residues from the fibrin clot that would normally play a role in augmenting plasminogen activation and the fibrinolytic activity of plasmin.
The potentially protective effect of Activated Protein C has also been shown in two rat studies [16,17], both of which suggested inhibition of leukocyte activation as an important mechanism. In one of these studies [16], Activated Protein C was shown to reduce motor disturbance resulting from spinal cord injury by reducing local levels of TNF-α, thereby inhibiting neutrophil accumulation. In the other study [17], animals treated with Activated Protein C were found to have a reduction in ischemia/reperfusioninduced renal injury, which was associated with reductions in renal levels of TNF-α, interleukin-8 and myeloperoxidase, with resulting inhibition of leukocyte activation.

Unique properties of the Activated Protein C pathway
Activated Protein C has a number of properties that differentiate it from other anticoagulants, such as heparin or   TNF-α production in response to sublethal or lethal E. coli infusion, with or without C4BP and Protein S (PS) supplementation. C4BP and PS were administered at doses of 20 mg/kg and 3 mg/kg, respectively. SLEC, 10% of a lethal dose of E. coli; LEC, 100% lethal dose dose of E. coli. Adapted from Taylor et al [12]. antithrombin. First, the Activated Protein C system is geared to modulate coagulopathy within the microvasculature, and it is microvascular disease that lies at the root of sepsis. Second, the Activated Protein C system involves the EPCR, which was identified during research to discover possible receptors and pathways linked to the system [17]. The EPCR was found to be homologous to the major histocompatibility complex class I family of molecules [4], all of which are involved in the regulation of inflammation.
The Activated Protein C/EPCR-mediated anticoagulant pathway is illustrated in Fig. 8 [18]. The EPCR is synthesized on the endothelial cell surface and moves to specialized regions (caveolae) in the endothelium, through which it is able to enter the cell and then the cell nucleus. Once inside the nucleus, it is able to redirect the gene expression profiles within the cell. Unlike other plasma membrane receptors, EPCR is also capable of carrying Activated Protein C into the nucleus, with subsequent alterations to some of the gene products.
Furthermore, when thrombin or inflammatory cytokines come into contact with the endothelium, they induce shedding of EPCR from the cell surface, a process that is mediated by a metalloproteinase [5]. Soluble EPCR binds to proteinase 3, a serine proteinase that is found in neutrophils. This soluble EPCR-proteinase 3 complex, either alone or in a complex with Protein C, appears to interact with neutrophils via an integrin (Mac-1) to inhibit their adhesion to the endothelium, and as a result prevents the migration of the neutrophils into the tissues [19].
Several unique mechanisms by which Activated Protein C can affect inflammation and coagulation have been demonstrated in various in vitro studies [20][21][22][23]. In one study [20], it was shown that incubation of monocytes with Activated Protein C leads to a decrease in TNF-α production and blockade of the translocation and activation of the central proinflammatory transcription factor nuclear factor-κB in response to lipopolysaccharide exposure. The binding of Activated Protein C to a specific receptor on mononuclear phagocytes has also been shown to inhibit intracellular calcium signaling and monocyte-dependent T-cell proliferative responses [21]. Finally, EPCR-dependent TF suppression by Activated Protein C has been demonstrated in a human monoblastic leukemia cell line [22].

Conclusion
The sepsis cascade, which is associated with simultaneous activation of systemic inflammation and coagulation, along with altered fibrinolysis, leads to microvascular endothelial injury, acute organ dysfunction, and possibly death. Thrombin, with its procoagulant and anticoagulant effects, as well as its involvement in the process of inflammation, is thought to be central to the sepsis cascade. In vitro and animal models suggest that Activated Protein C, a protein that has antithrombotic, anti-inflammatory and profibrinolytic activities, may be an important modulator of the vicious cycle of coagulation and inflammation associated with the sepsis cascade, which if unchecked may ultimately lead to death. Several properties of Activated Protein C, including its unique positioning to regulate coagulopathies in the microvasculature and the ability of EPCR to facilitate Protein C activation, support the continued development of Activated Protein C-mediated strategies that are aimed at disrupting the process whereby inflammation initiates coagulation and coagulation amplifies inflammation.