13 Branson HE, Katz J, Marble R, Griffin JH. Inherited protein C ... 26 Domotor E, Benzakour O, Griffin JH, Yule D, Fukudome K, Zlokovic. BV. Activated protein C ...
Journal of Thrombosis and Haemostasis, 5 (Suppl. 1): 73–80
INVITED REVIEW
Activated protein C J . H . G R I F F I N , J . A . F E R N A´ N D E Z , A . J . G A L E and L . O . M O S N I E R Division of Translational Vascular Medicine, Department of Molecular and Experimental Medicine (MEM-180), The Scripps Research Institute, La Jolla, CA, USA
To cite this article: Griffin JH, Ferna´ndez JA, Gale AJ, Mosnier LO. Activated protein C. J Thromb Haemost 2007; 5 (Suppl. 1): 73–80.
Introduction Summary. Protein C is a vitamin K-dependent plasma protein zymogen whose genetic mild or severe deficiencies are linked with risk for venous thrombosis or neonatal purpura fulminans, respectively. Studies over past decades showed that activated protein C (APC) inactivates factors (F) Va and VIIIa to downregulate thrombin generation. More recent basic and preclinical research on APC has characterized the direct cytoprotective effects of APC that involve gene expression profile alterations, anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. These actions generally require endothelial cell protein C receptor (EPCR) and protease activated receptor1. Because of these direct cytoprotective actions, APC reduces mortality in murine endotoxemia and severe sepsis models and provides neuroprotective benefits in murine ischemic stroke models. Furthermore, APC reduces mortality in patients with severe sepsis (PROWESS clinical trial). Although much remains to be clarified about mechanisms for APCÕs direct effects on various cell types, it is clear that APCÕs molecular features that determine its antithrombotic action are partially distinct from those providing cytoprotective actions because we have engineered recombinant APC variants with selective reduction or retention of either anticoagulant or cytoprotective activities. Such APC variants can provide relatively enhanced levels of either cytoprotective or anticoagulant activities for various therapeutic applications. We speculate that APC variants with reduced anticoagulant action but normal cytoprotective actions hold the promise of reducing bleeding risk because of attenuated anticoagulant activity while reducing mortality based on direct cytoprotective effects on cells. Keywords: activated protein C, endothelial protein C receptor, protease activated receptor-1, protein C, sepsis, stroke.
Correspondence: John H. Griffin, Department of Molecular and Experimental Medicine (MEM-180), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, USA. Tel.: +1 858 784 2220; fax: +1 858 784 2243; e-mail: jgriffin@ scripps.edu Received 2 February 2007, accepted 16 February 2007 Ó 2007 International Society on Thrombosis and Haemostasis
A vitamin K-dependent plasma protein was named ÔProtein CÕ when Stenflo purified it from bovine plasma [1]. Activated protein C (APC) is generated from the protein C zymogen by proteolytic activation by thrombin (Fig. 1A); notably, APC, designated autoprothrombin II-A where ÔAÕ indicated anticoagulant (Fig. 1B), had been studied by Seegers and colleagues, who thought it was a derivative of prothrombin. Protein C concentration in plasma is 70 nM, while its activated form, APC, is 40 pM [2]. Mature human protein C contains 419 amino acids, and the human gene (PROC) on chromosome 2 (2p13-14) contains 9 exons. Key post-translational modifications include ß-hydroxylation at Asp71, N-linked glycosylation at residues 97, 248, 313 and 329 and gamma-carboxylation of nine glutamic acid residues giving rise to nine Gla residues in the amino terminus (the ÔGla domainÕ). The domains of protein C comprise the Gla domain (residues 1–37), two epidermal growth factor (EGF)like regions (EGF-1, residues 46–92 and EGF-2, residues 93–136), an activation peptide (residues 158–169), and the enzymatic serine protease domain (residues 170–419) (Fig. 2A). Thrombin cleavage of the zymogen at Arg169 removes the activation peptide and generates APC, which is a trypsin-like serine protease with a typical serine protease active site triad, His211, Asp257 and Ser360, corresponding to chymotrypsinÕs His57, Asp102 and Ser195 active site triad. This active site triad is characteristic of all the plasma clotting enzymes. In contrast to the various procoagulant plasma factors, APC has anticoagulant and cytoprotective activities (Fig. 1). The Gla domain mediates binding to lipids to express anticoagulant activity and binding to endothelial protein C receptor (EPCR) to express cytoprotective activities (Figs 1 and 2A) [3–5]. Amino acid sequences on APCÕs surface that are remote from the active site, termed ÔexositesÕ, mediate APCÕs interactions with cofactors and substrates (Fig. 2). Studies identified positively charged exosites in the protease domain that are critical for factor (F) Va inactivation [6–11], and genetic engineering of APC variants has provided molecules with selective reduction of either anticoagulant or cytoprotective activities (see below).
74 J. H. Griffin et al A
B
Protein C activation
APC Anticoagulant activity
C
APC Cytoprotective activity
InActivation of fVa and fVllla
TM APC PC
APC
IIa EPCR
EPCR Cytoplasm
APC APC
Activation of PAR-1
fVi and fVllli
protein cofactors lipid cofactors
Cytoplasm
EPCR
PAR-1
Pleiotropic cytoprotective effects
Fig. 1. Protein C activation and activated protein C (APC) activities. (A) The endothelial cell receptors, thrombomodulin (TM) and endothelial cell protein C receptor (EPCR), are required for efficient activation of protein C by thrombin (IIa). Dissociation of APC from EPCR allows expression of APCÕs anticoagulant activity (B), whereas retention of APC bound to EPCR allows APC to express multiple direct cellular activities (C). (B) APC conveys its anticoagulant activity when bound to cell membrane surfaces by cleaving the activated cofactors Va (FVa) and VIIIa (FVIIIa) to yield the inactivated cofactors, FVi and FVIIIi. (C) Beneficial cytoprotective activities of activated protein C (APC) that involve direct effects of APC on cells require the cellular receptors EPCR and PAR-1. These activities include APC-mediated alteration of gene expression, profiles, anti-inflammatory activities, anti-apoptotic activities and protection of endothelial barrier functions. This figure is modified from a figure originally published in Blood: Mosnier, Zlokovic and Griffin. The cytoprotective protein C pathway, 207; 109: 3161–72. ÓThe American Society of Hematology.
Protein C hereditary deficiency Physiologic roles of protein C are evident because massive, usually lethal, thrombotic complications arise in infants with severe homozygous protein C deficiency and because heterozygous-deficient adults present with increased risk for venous thrombosis [12,13]. Related to the latter risk, an APC-cleavage site mutation (Arg506Gln, FV Leiden) is widely appreciated [14–16]. Protein C knockout mice studies show perinatal lethality [17,18], while protein C levels of 1–18% in mice permit growth and birth. Mice with low levels of protein C suffer early onset thrombosis and inflammation [19]. Anticoagulant and cytoprotective protein C pathways Biochemical reactions on cell surfaces include protein C activation, expression of APC anticoagulant activity, and initiation of APCÕs cytoprotective actions (Fig. 1A–C). Normal activation of protein C by thrombin requires two membrane receptors, thrombomodulin and EPCR [20,21]. APCÕs anticoagulant actions involve the irreversible proteolytic inactivation of FVa and FVIIIa (Fig. 1B). Multiple APCcofactors, comprising both proteins and lipids such as protein S, FV, high density lipoprotein, anionic phospholipids (e.g. phosphatidylserine, cardiolipin, etc.) and glycosphingolipids (e.g. glucosylceramide), mediate anticoagulation as previously reviewed [20,22,23]. APCÕs multiple cytoprotective effects include: (i) alteration of gene expression profiles; (ii) anti-inflammatory activities; (iii) anti-apoptotic activity, and (iv) protection of endothelial barrier function (Fig. 1C) [24–28]. These cytoprotective effects often appear to require EPCR [20] and the G-protein coupled receptor, protease activated receptor-1 (PAR-1 [29]). Protease activated receptors (PARs) are derived from four genes in the human genome. PAR-1 is a G-protein coupled receptor that can be activated by proteolysis [30]. In reactions
requiring EPCR, APC can activate endothelial PAR-1 [25– 28,31] and inhibits hypoxia/hypoglycemia or staurosporineinduced apoptosis in various endothelial cell types [24,25,27]. These same two receptors mediate multiple in vivo antiinflammatory and neuroprotective effects of pharmacologic doses of APC in various murine injury models [25,32–34]. PAR-1 seems a promiscuous receptor as it can be either proinflammatory or anti-inflammatory, depending on activating protease, the dose of protease, and the cell type [35]. Notably, gene expression profile studies showed certain PAR1-dependent effects of APC differed from those of thrombin (e.g. down-regulation of p53 and thrombospondin-1 expression) [31]. Although clear evidence for a requirement for PAR1 in the beneficial actions of pharmacologic doses of APC has been provided, the importance of PAR-1 for physiologic APCÕs in vivo effects has been debated [36–38]. Molecular distinction of APCÕs anticoagulant and cytoprotective activities The accumulation of insights into APCÕs cytoprotective actions in recent years raises fundamental questions about the relative importance of the enzymeÕs anticoagulant actions vs. its cytoprotective actions. For example, for APCÕs protective effects in humans with severe sepsis and in various animal injury models, what molecular mechanisms explain APCÕs in vivo actions? Is the anticoagulant activity of APC both essential for success in the PROWESS trial and also for the increased risk for serious bleeding in severe sepsis patients, especially during the 4-day period of APC infusion [39,40]? Alternatively, does one or more of APCÕs cytoprotective actions reduce mortality in sepsis? And in various kinds of animal injury models, which activities of APC are critical, which are dispensable and which are deleterious? To help answer some of these questions, initial efforts in our laboratory used site-directed mutagenesis to engineer APC variants with Ó 2007 International Society on Thrombosis and Haemostasis
Activated protein C 75
A
Ca2+ loop
B
fVa APC
37 loop
active site
C
wt-APC
D
5A-APC
EGF2 A P C
EGF1
Gla domain
E P C R
Fig. 2. Activated protein C (APC) exosite structures mediating APC activities. Interactions of APC with its macromolecular substrates and cofactors are dictated by exosite interactions, and APC protease domain surface loops specifically recognize various APC substrates. (A) The serine protease domain of APC is shown at the top of panel A, with the active site residues His211, Asp257 and Ser360 in green, the 37-loop residues Lys191, Lys192 and Lys193 and the calcium-binding loop residues Arg229 and Arg230 in blue. APC anticoagulant activity requires binding of the APC Gla-domain to negatively charged phospholipids on cell and/or platelet membranes or microparticles, whereas APC cytoprotective activity requires binding of the APC Gla-domain to endothelial cell protein C receptor (EPCR) (purple). (B) Exosite specificity is schematically illustrated by the juxtaposition of both 37-loop and calciumbinding loop positively charged residues (blue) of APC near negatively charged residues of FVa in this APC:FVa complex model (from Pellequer et al. [80]) and these positively charged APC amino acids are required for normal cleavage of FVa at Arg506 and thus for normal anticoagulant activity. (C) and (D) The surface contours with positive (blue) and negative (red) side-chains shown for wt-APC (C) and for the 5A-APC variant (containing 5 Ala substitutions at Lys191, Lys192, Lys193, Arg229 and Arg230). Comparison of the protease domain for 5A-APC (D) with that in (C) shows the loss of five positively charged surface residues from an exosite that is required for normal recognition of FVa. The model of full length APC [80] is based on the serine protease domain structure of APC (Protein Data Bank entry 1AUT [42]). The Gla-domain of the model of full length APC was aligned with the protein C Gladomain peptide crystallized in complex with soluble EPCR (1LQV) [80,81]. The model of 5A-APC was generated from the serine protease domain structure of APC 1AUT [42] using Modeller [82].
decreased anticoagulant activity and normal cytoprotective activity [41], while later efforts were made to decrease cytoprotective activity while retaining anticoagulant activity. The basic initial idea was to make potentially safer APC variants with reduced risk of bleeding because of reduced anticoagulant activity that retained normal cytoprotection; Ó 2007 International Society on Thrombosis and Haemostasis
thus, we sought to alter FVa binding exosites in APC without affecting exosites that recognize PAR-1. APCÕs anticoagulant activity involves cleavage at Arg506 in FVa (Fig. 1B); this cleavage depends on positively charged residues in surface loops on APCÕs protease domain, including loop 37 (protein C residues 190–193, equivalent to chymotrypsin (CHT) residues
76 J. H. Griffin et al Anti-apoptotic activity
S360A
Relative apoptosis (%)
100
Anticoagulant activity Ratio wt-APC
1/1
75 229/230-APC
50
7/1
229/230 3K3A wt
No STS
25
3K3A-APC
25/1
0 0
0.1
1
10 APC (nM)
100
Fig. 3. Anticoagulantly impaired APC variants retain normal antiapoptotic activity. The dose–response for activated protein C (APC)Õs anti-apoptotic activity was determined in staurosporine (STS)-induced apoptosis assays using human endothelial cells. APC variants with Ala substitutions for Arg229 and Arg230 and Lys191, Lys192 and Lys193 (see Fig. 2A), designated 229/230-APC and 3K3A-APC, had normal activity compared with wild-type (wt) APC. The active site APC mutant, S360AAPC, had no activity. This shows that APC variants with reduced anticoagulant activity retain normal anti-apoptotic activity. This figure is taken with permission from Mosnier et al., Blood. 2004;104:1740–1744. ÓThe American Society of Hematology.
36–39), the Ca++-binding loop (residues 225–235, CHT residues 70–80) and the autolysis loop (residues 301–316, CHT residues 142–153) (Fig. 2C) [6,8–11,42,43]. We made two APC variants with Ala substitutions for Arg229 and Arg230 and Lys191, Lys192 and Lys193 (Fig. 2A) designated 229/230APC and 3K3A-APC, and these had greatly attenuated anticoagulant activity (5 to 13% compared with wild-type APC) while each retained normal anti-apoptotic activity which still required PAR-1 and EPCR (Fig. 3) [41]. Calculation of the cytoprotective to anticoagulant activity ratio for these APC variants gave 7/1 and 25/1 (Fig. 4). When another APC variant containing the five Ala substitutions of these two variants (designated 5A-APC) was studied, we found that human 5AAPC had < 3% anticoagulant activity of wild-type APC but normal anti-apoptotic activity (see Mosnier et al., ISTH 2007 Congress) and the ratio was 36/1 (Fig. 4). Thus, we show that APC variants can be engineered to reduce anticoagulant activity while preserving the enzymeÕs direct cytoprotective effects, which require EPCR and PAR-1. Our second and complementary goal in engineering APC variants with selectively altered activities was to make a molecule that had reduced cytoprotective activity but normal anticoagulant activity. For this purpose, we found that a single mutation of Glu149 to Ala (E149A-APC) gives an APC variant with increased anticoagulant activity but greatly reduced anti-apoptotic potency (see Mosnier et al., ISTH 2007 Congress). As indicated in Fig. 4, E149A-APC has a ratio of 1/72 for its cytoprotective to anticoagulant activity ratio. Thus, we show that APC variants can be engineered to reduce selectively the enzymeÕs cytoprotective activity while preserving or actually increasing the enzymeÕs anticoagulant activity.
5A-APC 36/1
E149A-APC
1/72
0
25
50
75
100
400
Activity (% wt-APC)
Fig. 4. Activated protein C (APC) variants with greatly reduced anticoagulant activity relative to cytoprotective activity and vice versa. For wild-type-APC (wt-APC) and APC variants, the values for anti-apoptotic activity and anticoagulant activity are shown and the ratio of these activities is given numerically on the left side of the figure. The values for each activity are shown on the abscissa where the values for wt-APC are defined as 100%. These data highlight important distinctions between structural requirements for APCs anticoagulant and cytoprotective functions. The availability of APC variants with greatly reduced anticoagulant activity relative to cytoprotective activity (e.g. 1/36 ratio) and vice versa (e.g. 72/1 ratio) permits proof of principle studies in animal models to distinguish the relative importance of APCs cytoprotective vs. anticoagulant activities for APCÕs in vivo benefits in sepsis, stroke or other injuries.
Overall, our data show that mutagenesis of protein C can effectively create APC variants with decreased anticoagulant activity and normal cytoprotective activity or vice versa. Furthermore, these data (Fig. 4) highlight important distinctions between structural requirements for APCs anticoagulant and cytoprotective functions. The availability of APC variants with greatly reduced anticoagulant activity relative to cytoprotective activity (e.g. 1:36 ratio) and vice versa (e.g. 72:1 ratio) (Fig. 4) will permit proof of principle studies in animal models to distinguish the relative importance of APCÕs cytoprotective vs. anticoagulant activities for APCÕs in vivo benefits in sepsis, stroke or other injuries. Such studies may ultimately help contribute to safer or better therapeutic APC variants. For example, safer APC variants with reduced bleeding risk may permit therapies using higher APC doses for shorter times. Higher doses of APC may stabilize stressed cells at risk for excessive inflammation or apoptosis and prevent organ failure. In recent collaborative studies, Dr Harmut Weiler and colleagues showed that recombinant murine 3K3A-APC and 5A-APC variants seem as active as wild-type murine APC in preventing death from endotoxemia in mice, and both EPCR and PAR-1 were required for mortality reduction by APC in the murine endotoxemia model [44,45]. These findings strongly Ó 2007 International Society on Thrombosis and Haemostasis
Activated protein C 77
imply a primary role for APCÕs cytoprotective in vivo activities in this endotoxemia model. Additional studies in various murine or other animal injury models using a selection of APC variants with altered properties (Fig. 4) should help clarify the extent to which either anticoagulant or cytoprotective activities of APC play key roles for APCÕs beneficial in vivo actions in reducing mortality in severe sepsis or in other injuries. In vivo role for APC cytoprotection Both anticoagulant and cytoprotective activities may contribute to APCÕs beneficial in vivo actions. For some time, researchers posited that APCÕs systemic anticoagulant action explained APCÕs anti-inflammatory effects via downregulation of thrombin generation because thrombin is proinflammatory. But recent findings dictate otherwise. In contrast to the PROWESS trial showing reduction of mortality in severe sepsis by APC, other anticoagulants such as antithrombin and tissue factor pathway inhibitor failed to reduce mortality [39,46,47]. Murine ischemic stroke model studies show that APCÕs neuroprotective effects require EPCR and PAR-1 and are, at least in part, independent of APCÕs anticoagulant activity [25,32–34]. Moreover, both EPCR and PAR-1 are required for mortality reduction in murine endotoxemia, and APC variants with < 10% anticoagulant activity but normal cytoprotective action are normally active in reducing death in murine endotoxemia (see below) [41,44,45]. Thus, we believe that APCÕs direct effects on cells are established as crucial for the life-saving effects of pharmacologic doses of APC in at least two animal injury models, namely sepsis and stroke. APC cytoprotection mechanistic considerations APCÕs direct effects on cells include: (i) alteration of gene expression profiles; (ii) anti-inflammatory activities; (iii) antiapoptotic activity; and (iv) endothelial barrier stabilization. One or more of these effects are termed APCÕs cytoprotective effects. Each of these activities is biologically distinct and may or may not involve the same intracellular mechanisms, and much remains to be clarified. However, mechanistic studies show that EPCR is essential for most if not all of APCÕs effects on cells while PAR-1 often appears essential. Gene expression profiles.
Gene expression profiling pattern studies revealed modulation of gene expression by APC for some major genes of inflammation and apoptosis, with the general effect of downregulation of proinflammatory and proapoptotic pathways and up-regulation of anti-inflammatory and anti-apoptotic pathways [24,25,28,31,32,48,49]. Notably, APC decreases proapoptotic p53 and Bax expression and increases expression of anti-apoptotic genes such as Bcl-2. For stressed endothelial cells, APC and thrombin, although both working via PAR-1 activation, differ significantly in their Ó 2007 International Society on Thrombosis and Haemostasis
transcriptome alteration effects. Thus, APC is capable of modulating gene expression by mechanisms that are, at least partially, different from those used by thrombin. APC anti-inflammatory activity
APCÕs anti-inflammatory effects on endothelial cells involve inhibition of inflammatory mediator release and of expression of vascular adhesion molecules with the net result of inhibiting leukocyte adhesion and leukocyte tissue infiltration. In addition, by helping to maintain endothelial barriers, APC reduces extravascular inflammatory processes [50–55]. Inflammatory mediator release by leukocytes or by endothelial cells is inhibited by APC [56–59]. EPCR exists on the surface of monocytes, CD56 + natural killer cells, neutrophils and eosinophils [55,60–62], and EPCR is critical for APCÕs anti-inflammatory effects on leukocytes [63–66]. Soluble EPCR binds the integrin CD11b/CD18 (Mac1) (aMb2) (CR3) on leukocytes, as does the neutrophil molecule, proteinase-3 [67]. To date, no evidence for a role of the EPCR:PR3:CD11b/CD18 has appeared, although this complex might help mediate APC cellular effects. Anti-apoptotic activity
Apoptosis is a widely known and key process that is based on efficient cell death programs [68–70]. For the intrinsic pathway of apoptosis that can be triggered by a variety of cellular stresses, the release of cytochrome c from mitochondria and subsequent procaspase 3 activation are central events and certain molecules such as the tumor suppressor protein, p53, and the Bcl-2 family of proteins, help to balance apoptotic signals and survival signals. The extrinsic apoptotic pathway, involving activation of initiator caspases, such as procaspase-8, requires membranebound death receptors and sensors of the extracellular environment. The extrinsic apoptotic pathway may also co-opt members of the intrinsic pathway via activation of the pro-apoptotic members of the Bcl-2 family or procaspase-3, which can be activated by either pathway, yielding caspase-3 that cleaves multiple substrates to advance apoptotic cell death [68–70]. APC exhibits anti-apoptotic activity both in vitro and in vivo via reactions requiring APCÕs enzymatic activity and EPCR and PAR-1 [24,25,27,33]. In murine injury models, APC inhibits apoptosis in the brain, and the intrinsic apoptosis pathway is blunted with decreases in p53 and Bax and with increases in Bcl-2. APC also appears capable of blunting extrinsic pathway apoptosis because it counteracts the neurotoxicity of tissue plasminogen activator (tPA) that exerts caspase 8-dependent, proapoptotic activity via the extrinsic pathway [33]. In murine endotoxemia models, apoptosis is reduced by APC in association with improved survival in sepsis [41,44,45]. In severe sepsis patients, recombinant APC (XIGRISTM) reduced apoptosis of circulating blood cells [71]. Thus, APCÕs anti-apoptotic effects are documented both in vitro and in vivo, and both murine injury models and clinical findings suggest that APCÕs anti-apoptotic activity may be very
78 J. H. Griffin et al
important for APCÕs pharmacologically beneficial effects in reducing mortality in sepsis [72,73].
angiogenesis and wound healing, and these indications have been discussed elsewhere in detail [79].
Endothelial barrier stabilization
Conclusion
Endothelial barrier breakdown with infiltration of extravascular space by cells and inflammatory mediators contributes to the pathogenesis of inflammation. Specifically, increased endothelial permeability promotes edema and hypotension and thereby promotes inflammation, acute lung injury and organ failure (see [74–76]). Protective endothelial barrier effects are caused by APC acting via EPCR and PAR-1; in this context, APC can induce sphingosine kinase-1 and up-regulate sphingosine-1-phosphate (S1P) formation via sphingosine kinase [50,53]. The sphingolipid S1P acts to reduce endothelial permeability through the S1P receptor-1 (S1P1), a G-protein coupled receptor belonging to the endothelial differentiation gene (Edg) family. The actions of S1P1 that stabilize the cellular cytoskeleton are dependent on Rho family GTPases and mitogen-activated protein kinases (MAPK) (see [74,76,77]). Endothelial barrier protection by APC requires PAR-1. In contrast to APC, high concentrations of thrombin, acting via PAR-1, cause destabilization of endothelial barriers, whereas PAR-1 activation by APC stabilizes barriers [50]. It appears that endothelial barrier effects of APC and thrombin may depend on Rac (protective) and Rho (destabilizing) signaling, respectively [77], and, for APCÕs effects, there may be direct or indirect interactions between EPCR and S1P1 [53]. APCÕs barrier stabilization effects may be more effectively accomplished by endogenously generated APC than by exogenously added APC [78], supporting the idea that endogenous generation of APC might promote vascular integrity and limit bleeding when thrombin is locally generated. Strikingly, although APC is appropriately recognized as a potent anticoagulant, APC can also potently prevent bleeding in the brain! In murine ischemic stroke models where tPA administration induces brain hemorrhage, co-administration of APC reduces tPA-induced bleeding [25,32–34]. These antihemorrhagic effects of APC in stroke models require PAR-1 and may be partially based on both direct and indirect stabilization of endothelial barriers. APCÕs direct and immediate effects may involve its ability to stabilize the endothelial cytoskeleton while its indirect effects, in this setting, involve attenuation of tPAinduced up-regulation of MMP9, which itself promotes breakdown of the blood–brain barrier [25,32–34].
Inflammation, apoptosis and thrombosis are inextricably intertwined in host defense protective reactions. Protein C provides physiologic homeostasis via its antithrombotic and anti-inflammatory actions, while pharmacologically administered APC reduces mortality in severe sepsis in humans and in murine and animal injury models, including ischemic stroke. In animal injury models and in vitro experiments, APCÕs protective actions often require EPCR and PAR-1. Thus, basic preclinical and clinical studies heighten interest in the cytoprotective protein C pathway, which is distinct from the anticoagulant protein C pathway, suggesting the cytoprotective actions of APC are clinically very interesting. APC variants have been made with decreased anticoagulant activity but normal cytoprotective activity and vice versa. These APC variants are valuable not only for dissecting in vivo modes of action of APC but also for potentially reducing serious bleeding risks while retaining APCÕs beneficial therapeutic properties.
APCÕs in vivo effects Multiple beneficial in vivo effects of APC have been published and a detailed summary of them is beyond the scope of this review, although some key studies are cited in the text above. The multiple in vivo beneficial effects are no doubt derived from both anticoagulant and cytoprotective actions of APC, depending on the injury and the context of the study. Current and potential therapeutic applications for APC include severe sepsis, ischemic stroke, lung injury and inflammation, and
Acknowledgments Because of space limitations, we were unable to cite all of the interesting and relevant papers on APC, and we apologize to our colleagues for this limitation. We gratefully acknowledge stimulating discussions with Scripps colleagues X. Yang, S. Yegneswaran, W. Ruf, Z. Ruggeri and M. Riewald and with collaborators B. Zlokovic and H. Weiler. Support was provided in part by NIH grants HL21544, HL31950, HL52246 and HL63290 (J.H.G), HL082588 (A.J.G.) and HL087618 (L.O.M.) and a Basic Research Scholar Award from the American Society of Hematology (L.O.M.). Disclosure of Conflict of Interests JHG has carried out work on behalf of Socratech LLC. JAF, AJG and LOM state that they have no conflict of interests. References 1 Stenflo J. A new vitamin K-dependent protein. Purification from bovine plasma and preliminary characterization. J Biol Chem 1976; 251: 355–63. 2 Gruber A, Griffin JH. Direct detection of activated protein C in blood from human subjects. Blood 1992; 79: 2340–8. 3 Jhingan A, Zhang L, Christiansen WT, Castellino FJ. The activities of recombinant gamma-carboxyglutamic-acid-deficient mutants of activated human protein C toward human coagulation factor Va and factor VIII in purified systems and in plasma. Biochemistry 1994; 33: 1869–75. 4 Liaw PCY, Mather T, Oganesyan N, Ferrell GL, Esmon CT. Identification of the protein C/activated protein C binding sites on the endothelial cell protein C receptor. Implications for a novel mode of ligand recognition by a major histocompatibility complex class 1- type receptor. J Biol Chem 2001; 276: 8364–70.
Ó 2007 International Society on Thrombosis and Haemostasis
Activated protein C 79 5 Zhang L, Jhingan A, Castellino FJ. Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity. Blood 1992; 80: 942–52. 6 Gale AJ, Heeb MJ, Griffin JH. The autolysis loop of activated protein C interacts with factor Va and differentiates between the Arg506 and Arg306 cleavage sites. Blood 2000; 96: 585–93. 7 Shen L, Villoutreix BO, Dahlba¨ck B. Interspecies loop grafting in the protease domain of human protein C yielding enhanced catalytic and anticoagulant activity. Thromb Haemost 1999; 82: 1078–87. 8 Friedrich U, Nicolaes GAF, Villoutreix BO, Dahlba¨ck B. Secondary substrate-binding exosite in the serine protease domain of activated protein C important for cleavage at Arg-506 but not at Arg-306 in factor Va. J Biol Chem 2001; 276: 23105–8. 9 Rezaie AR. Exosite-dependent regulation of the protein C anticoagulant pathway. Trends Cardiovasc Med 2003; 13: 8–15. 10 Gale AJ, Tsavaler A, Griffin JH. Molecular characterization of an extended binding site for coagulation factor Va in the positive exosite of activated protein C. J Biol Chem 2002; 277: 28836–40. 11 Gale AJ, Griffin JH. Characterization of a thrombomodulin binding site on protein C and its comparison to an activated protein C binding site for factor Va. Proteins 2004; 54: 433–41. 12 Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981; 68: 1370–3. 13 Branson HE, Katz J, Marble R, Griffin JH. Inherited protein C deficiency and coumarin-responsive chronic relapsing purpura fulminans in a newborn infant. Lancet 1983; 2: 1165–8. 14 Dahlba¨ck B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. PNAS 1993; 90: 1004–8. 15 Greengard JS, Sun X, Xu X, Ferna´ndez JA, Griffin JH, Evatt B. Activated protein C resistance caused by Arg506Gln mutation in factor Va. Lancet 1994; 343: 1361–2. 16 Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA, Reitsma PH. Mutations in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64–7. 17 Jalbert LR, Rosen ED, Moons L, Chan JCY, Carmeliet P, Collen D, Castellino FJ. Inactivation of the gene for anticoagulant protein C causes lethal perinatal consumptive coagulopathy in mice. J Clin Invest 1998; 102: 1481–8. 18 Castellino FJ. Gene targeting in hemostasis: protein C. Front Biosci 2001; 6: D807–19. 19 Lay AJ, Liang Z, Rosen ED, Castellino FJ. Mice with a severe deficiency in protein C display prothrombotic and proinflammatory phenotypes and compromised maternal reproductive capabilities. J Clin Invest 2005; 115: 1552–61. 20 Esmon CT. The protein C pathway. Chest 2003; 124: 26S–32S. 21 Stearns-Kurosawa DJ, Kurosawa S, Mollica JS, Ferrell GL, Esmon CT. The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex. Proc Natl Acad Sci USA 1996; 93: 10212–6. 22 Dahlba¨ck B, Villoutreix BO. Regulation of blood coagulation by the protein C anticoagulant pathway. Novel insights into structure-function relationships and molecular recognition. Arterioscler Thromb Vasc Biol 2005; 25: 1311–20. 23 Mosnier LO, Griffin JH. Protein C anticoagulant activity in relation to anti-inflammatory and anti-apoptotic activities. Front Biosci 2006; 11: 2381–99. 24 Joyce DE, Gelbert L, Ciaccia A, DeHoff B, Grinnell BW. Gene expression profile of antithrombotic protein C defines new mechanisms modulating inflammation and apoptosis. J Biol Chem 2001; 276: 11199–203. 25 Cheng T, Liu D, Griffin JH, Ferna´ndez JA, Castellino F, Rosen ED, Fukudome K, Zlokovic BV. Activated protein C blocks p53-mediated
Ó 2007 International Society on Thrombosis and Haemostasis
26
27
28
29
30 31
32
33
34
35 36 37 38
39
40
41
42
43
44
45
46
apoptosis in ischemic human brain endothelium and is neuroprotective. Nature Med 2003; 9: 338–42. Domotor E, Benzakour O, Griffin JH, Yule D, Fukudome K, Zlokovic BV. Activated protein C alters cytosolic calcium flux in human brain endothelium via binding to endothelial protein C receptor and activation of Protease Activated Receptor-1. Blood 2003; 101: 4797–801. Mosnier LO, Griffin JH. Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires protease activated receptor-1 and endothelial cell protein C receptor. Biochem J 2003; 373: 65–70. Riewald M, Petrovan RJ, Donner A, Mueller BM, Ruf W. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science 2002; 296: 1880–2. Vu TK, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057–68. Coughlin SR. Thrombin signaling and protease-activated receptors. Nature 2000; 407: 258–64. Riewald M, Ruf W. Protease-activated receptor-1 signaling by activated protein C in cytokine perturbed endothelial cells is distinct from thrombin signaling. J Biol Chem 2005; 280: 19808–14. Guo H, Liu D, Gelbard H, Cheng T, Insalaco R, Ferna´ndez JA, Griffin JH, Zlokovic BV. Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3. Neuron 2004; 41: 563–72. Liu D, Cheng T, Guo H, Ferna´ndez JA, Griffin JH, Song X, Zlokovic BV. Tissue plasminogen activator neurovascular toxicity is controlled by activated protein C. Nat Med 2004; 10: 1379–83. Cheng T, Petraglia AL, Li Z, Thiyagarajan M, Zhong Z, Wu Z, Liu D, Maggirwar SB, Deane R, Fernandez JA, LaRue B, Griffin JH, Chopp M, Zlokovic BV. Activated protein C inhibits tissue plasminogen activator-induced brain hemorrhage. Nat Med 2006; 12: 1278–85. Coughlin SR, Camerer E. PARticipation in inflammation. J Clin Invest 2003; 111: 25–7. Ruf W. Is APC activation of endothelial cell PAR1 important in severe sepsis?: Yes. J Thromb Haemost 2005; 3: 1912–4. Esmon CT. Is APC activation of endothelial cell PAR1 important in severe sepsis?: No. J Thromb Haemost 2005; 3: 1910–1. Ludeman MJ, Kataoka H, Srinivasan Y, Esmon NL, Esmon CT, Coughlin SR. PAR1 cleavage and signaling in response to activated protein C and thrombin. J Biol Chem 2005; 280: 13122–8. Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A, Steingrub JS, Garber GE, Helterbrand JD, Ely EW, Fisher CJ Jr. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699–709. Bernard GR, Macias WL, Joyce DE, Williams MD, Bailey J, Vincent JL. Safety assessment of drotrecogin alfa (activated) in the treatment of adult patients with severe sepsis. Crit Care 2003; 7: 155–63. Mosnier LO, Gale AJ, Yegneswaran S, Griffin JH. Activated protein C variants with normal cytoprotective but reduced anticoagulant activity. Blood 2004; 104: 1740–5. Mather T, Oganessyan V, Hof P, Huber R, Foundling S, Esmon CT, Bode W. The 2.8 A˚ crystal structure of Gla-domainless activated protein C. EMBO J 1996; 15: 6822–31. Shen L, Villoutreix BO, Dahlba¨ck B. Involvement of lys 62(217) and lys 63(218) of human anticoagulant protein C in heparin stimulation of inhibition by the protein C inhibitor. Thromb Haemost 1999; 82: 72–9. Kerschen EJ, Cooley BC, Castellino FJ, Griffin JH, Weiler H. Protective effect of activated protein C in murine endotoxemia: mechanism of action. Blood 2005; 106: 26. (abstract) Kerschen EJ, Cooley BC, Castellino FJ, Coughlin PB, Fernandez JA, Griffin JH, Weiler H. Mechanisms for mortality reduction by activated protein C in severe sepsis. Blood 2006; 108: 1.(abstract) Abraham E, Reinhart K, Opal S, Demeyer I, Doig C, Rodriguez AL, Beale R, Svoboda P, Laterre PF, Simon S, Light B, Spapen H, Stone J, Seibert A, Peckelsen C, De Deyne C, Postier R, Pettila V, Artigas A,
80 J. H. Griffin et al
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
Percell SR, et al. Efficacy and safety of tifacogin (recombinant tissue factor pathway inhibitor) in severe sepsis: a randomized controlled trial. JAMA 2003; 290: 238–47. Warren BL, Eid A, Singer P, Pillay SS, Carl P, Novak I, Chalupa P, Atherstone A, Penzes A, Kubler A, Knaub S, Keinecke HO, Heinrichs H, Schindel F, Juers M, Bone RC, Opal SM. High-dose antithrombin III in severe sepsis. A randomized controlled trial. JAMA 2001; 286: 1869–78. Joyce DE, Grinnell BW. Recombinant human activated protein C attenuates the inflammatory response in endothelium and monocytes by modulating nuclear factor-kappaB. Crit Care Med 2002; 30: S288– 93. Franscini N, Bachli EB, Blau N, Leikauf MS, Schaffner A, Schoedon G. Gene expression profiling of inflamed human endothelial cells and influence of activated protein C. Circulation 2004; 110: 2903–9. Feistritzer C, Riewald M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood 2005; 105: 3178–84. Murakami K, Okajima K, Uchiba M, Johno M, Nakagaki T, Okabe H, Takatsuki K. Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats. Blood 1996; 87: 642–7. Nick JA, Coldren CD, Geraci MW, Poch KR, Fouty BW, OÕBrien J, Gruber M, Zarini S, Murphy RC, Kuhn K, Richter D, Kast KR, Abraham E. Recombinant human activated protein C reduces human endotoxin-induced pulmonary inflammation via inhibition of neutrophil chemotaxis. Blood 2004; 104: 3878–85. Finigan JH, Dudek SM, Singleton PA, Chiang ET, Jacobson JR, Camp SM, Ye SQ, Garcia JG. Activated protein C mediates novel lung endothelial barrier enhancement: role of sphingosine 1-phosphate receptor transactivation. J Biol Chem 2005; 280: 17286–93. Zeng W, Matter WF, Yan SB, Um SL, Vlahos CJ, Liu L. Effect of drotrecogin alfa (activated) on human endothelial cell permeability and Rho kinase signaling. Crit Care Med 2004; 32: S302–8. Feistritzer C, Sturn DH, Kaneider NC, Djanani A, Wiedermann CJ. Endothelial protein C receptor-dependent inhibition of human eosinophil chemotaxis by protein C. J Allergy Clin Immunol 2003; 112: 375–81. Brueckmann M, Hoffmann U, De Rossi L, Weiler HM, Liebe V, Lang S, Kaden JJ, Borggrefe M, Haase KK, Huhle G. Activated protein C inhibits the release of macrophage inflammatory protein-1-alpha from THP-1 cells and from human monocytes. Cytokine 2004; 26: 106–13. Grey ST, Tsuchida A, Hau H, Orthner CL, Salem HH, Hancock WW. Selective inhibitory effects of the anticoagulant activated protein C on the responses of human mononuclear phagocytes to LPS, IFNgamma, or phorbol ester. J Immunol 1994; 153: 3664–72. White B, Schmidt M, Murphy C, Livingstone W, OÕToole D, Lawler M, OÕNeill L, Kelleher D, Schwarz HP, Smith OP. Activated protein C inhibits lipopolysaccharide-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) and tumour necrosis factor alpha (TNFalpha) production in the THP-1 monocytic cell line. Br J Haematol 2000; 110: 130–4. Yuksel M, Okajima K, Uchiba M, Horiuchi S, Okabe H. Activated protein C inhibits lipopolysaccharide-induced tumor necrosis factoralpha production by inhibiting activation of both nuclear factor-kappa B and activator protein-1 in human monocytes. Thromb Haemost 2002; 88: 267–73. Joyce DE, Nelson DR, Grinnell BW. Leukocyte and endothelial cell interactions in sepsis: relevance of the protein C pathway. Crit Care Med 2004; 32: S280–6. Galligan L, Livingstone W, Volkov Y, Hokamp K, Murphy C, Lawler M, Fukudome K, Smith O. Characterization of protein C receptor expression in monocytes. Br J Haematol 2001; 115: 408–14. Sturn DH, Kaneider NC, Feistritzer C, Djanani A, Fukudome K, Wiedermann CJ. Expression and function of the endothelial protein C receptor in human neutrophils. Blood 2003; 102: 1499–505.
63 Stephenson DA, Toltl LJ, Beaudin S, Liaw PC. Modulation of monocyte function by activated protein C, a natural anticoagulant. J Immunol 2006; 177: 2115–22. 64 Shu F, Kobayashi H, Fukudome K, Tsuneyoshi N, Kimoto M, Terao T. Activated protein C suppresses tissue factor expression on U937 cells in the endothelial protein C receptor-dependent manner. FEBS Lett 2000; 477: 208–12. 65 Shimizu S, Gabazza EC, Taguchi O, Yasui H, Taguchi Y, Hayashi T, Ido M, Shimizu T, Nakagaki T, Kobayashi H, Fukudome K, Tsuneyoshi N, DÕAlessandro-Gabazza CN, Izumizaki M, Iwase M, Homma I, Adachi Y, Suzuki K. Activated protein C inhibits the expression of platelet-derived growth factor in the lung. Am J Respir Crit Care Med 2003; 167: 1416–26. 66 Yuda H, Adachi Y, Taguchi O, Gabazza EC, Hataji O, Fujimoto H, Tamaki S, Nishikubo K, Fukudome K, DÕAlessandro-Gabazza CN, Maruyama J, Izumizaki M, Iwase M, Homma I, Inoue R, Kamada H, Hayashi T, Kasper M, Lambrecht BN, Barnes PJ, et al. Activated protein C inhibits bronchial hyperresponsiveness and Th2 cytokine expression in mice. Blood 2004; 103: 2196–204. 67 Kurosawa S, Esmon CT, Stearns-Kurosawa DJ. The soluble endothelial protein C receptor binds to activated neutrophils: involvement of proteinase-3 and CD11b/CD18. J Immunol 2000; 165: 4697–703. 68 Boatright KM, Salvesen GS. Mechanisms of caspase activation. Curr Opin Cell Biol 2003; 15: 725–31. 69 Reed JC. Proapoptotic multidomain Bcl-2/Bax-family proteins: mechanisms, physiological roles, and therapeutic opportunities. Cell Death Differ 2006; 13: 1378–86. 70 Green DR. Apoptotic pathways: ten minutes to dead. Cell 2005; 121: 671–4. 71 Bilbault P, Lavaux T, Launoy A, Gaub MP, Meyer N, Oudet P, Pottecher T, Jaeger A, Schneider F. Influence of drotrecogin alpha (activated) infusion on the variation of Bax/Bcl-2 and Bax/Bcl-xl ratios in circulating mononuclear cells: a cohort study in septic shock patients. Crit Care Med 2007; 35: 69–75. 72 Hotchkiss RS, Chang KC, Swanson PE, Tinsley KW, Hui JJ, Klender P, Xanthoudakis S, Roy S, Black C, Grimm E, Aspiotis R, Han Y, Nicholson DW, Karl IE. Caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte. Nat Immunol 2000; 1: 496–501. 73 Hotchkiss RS, Nicholson DW. Apoptosis and caspases regulate death and inflammation in sepsis. Nat Rev Immunol 2006; 6: 813–22. 74 McVerry BJ, Garcia JG. Endothelial cell barrier regulation by sphingosine 1-phosphate. J Cell Biochem 2004; 92: 1075–85. 75 Taylor FB Jr. Staging of the pathophysiologic responses of the primate microvasculature to Escherichia coli and endotoxin: examination of the elements of the compensated response and their links to the corresponding uncompensated lethal variants. Crit Care Med 2001; 29: S78– 89. 76 Burridge K, Wennerberg K. Rho and Rac take center stage. Cell 2004; 116: 167–79. 77 Singleton PA, Dudek SM, Chiang ET, Garcia JG. Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/ Rac1, and alpha-actinin. FASEB J 2005; 19: 1646–56. 78 Feistritzer C, Schuepbach RA, Mosnier LO, Bush LA, Di Cera E, Griffin JH, Riewald M. Protective signaling by activated protein C is mechanically linked to protein C activation on endothelial cells. J Biol Chem 2006; 281: 20077–84. 79 Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109: 3161–72. 80 Pellequer JL, Gale AJ, Getzoff ED, Griffin JH. Three-dimensional model of coagulation factor Va bound to activated protein C. Thromb Haemost 2000; 84: 849–57. 81 Oganesyan V, Oganesyan N, Terzyan S, Qu D, Dauter Z, Esmon NL, Esmon CT. The crystal structure of the endothelial protein C receptor and a bound phospholipid. J Biol Chem 2002; 277: 24851–4. 82 Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 1993; 234: 779–815. Ó 2007 International Society on Thrombosis and Haemostasis
