The clinical phenotype of severe protein C deficiency in neonatal purpura fulminans implies that APC exerts multiple physiologically essential activities, including potent anticoagulant and antiinflammatory actions (Chap. 130). Recent advances establish that APC’s antiinflammatory actions are but one manifestation of its ability to interact directly with cell receptors to provide multiple cytoprotective activities.7,8,110,111 These two distinct types of activities of APC—intravascular anticoagulant activity and initiation of cell signaling—are mediated by different sets of molecular interactions, and both types of activities are clinically relevant.
ACTIVATED PROTEIN C ANTICOAGULANT ACTIVITY
Mechanisms for APC’s direct anticoagulant activity involve factors V and VIII, the two homologous coagulation cofactors that circulate as inactive molecules and that are converted to active cofactors by limited proteolysis (see Chap. 113, Figs. 113–11 and 113–13). APC circulates at 40 pM (picomolars) in normal humans, and there is an inverse correlation between fibrinopeptide A, the product that is cleaved from fibrinogen by thrombin, and APC levels in healthy nonsmoking adults, suggesting APC is a significant regulator of basal thrombin activity.15,158
Factors V and VIII are synthesized as large single-chain precursor coagulation cofactors of Mr 330,000, consisting of three homologous A domains (A1, A2, and A3) and two homologous C domains (C1 and C2) with a very large intervening, generally nonhomologous domain, designated the B domain, that connects the A2 and A3 domains (Chap. 113). Activation of the inactive precursor form of the two cofactors V and VIII involves limited proteolysis.23,159,160,161,162,163,164 Factor V activation involves cleavages at Arg709, Arg1018, and Arg1545 by thrombin, factor Xa, or other proteases.23,164,165,166,167,168 Cleavage at Arg1545 is the key step for generating factor Va activity because this proteolysis releases the B domain that blocks binding of factor Xa to factor Va.164,169 The various forms of factor Va (see Chap. 113, Fig. 113–11) are composed of two polypeptide chains, one bearing the A1-A2 domains and the other bearing the A3-C1-C2 domains. Although generally similar to factor V activation, factor VIII activation (see Fig. 113–13) involves formation of a heterotrimer of polypeptide chains containing the A1 domain, the A2 domain, and the A3-C1-C2 domains, respectively. In contrast to heterodimeric factor Va, heterotrimeric factor VIIIa is intrinsically unstable as a consequence of spontaneous dissociation of the A2 domain.170
Factors Va and VIIIa as Substrates for Activated Protein C
Irreversible proteolytic inactivation of factors Va and VIIIa by APC can be accomplished by proteolysis at Arg506 and Arg306 in factor Va and Arg562 and Arg336 in factor VIIIa (see Chap. 113, Figs. 113–11 and 113–13).23,171,172,173 Currently, the most common identifiable venous thrombosis risk factor involves a mutation of Arg506 to Gln in factor V that results in APC resistance (Chap. 130). The complexities of APC-dependent inactivation of factor Va and VIIIa are compounded by the number of different molecular forms of Va and VIIIa that can be generated by limited proteolysis by a variety of proteases and by their differing susceptibilities to APC and to the different APC cofactors.
Activated Protein C Resistance
APC resistance is defined as an abnormally reduced anticoagulant response of a plasma sample to APC (Chap. 130) and can be caused by many potential abnormalities in the protein C anticoagulant pathway. Such abnormalities could include defective APC cofactors, defective APC substrates, or other molecules that interfere with the normal functioning of the protein C anticoagulant pathway (e.g., autoantibodies against APC, APC cofactors, or APC substrates).
A report of familial venous thrombosis associated with APC resistance without any identifiable defect in four Swedish families174 led to an intensive search for a genetic explanation that was soon found to involve replacement of G by A at nucleotide 1691 in exon 10 of the factor V gene which causes the amino acid replacement of Arg506 by Gln.175,176,177 This factor V variant, like the prothrombin variant nt G20210A, arose in a single white founder some 18,000 to 29,000 years ago178,179 and is known as Gln506-factor V or factor V Leiden. This mutation is currently a common, but not the only, cause of APC resistance (Chap. 130).
The molecular mechanism for APC resistance of Gln506-factor V is based on the fact that the variant molecule is inactivated 10 times slower than normal Arg506-factor Va.23,177,180,181,182 The variant factor Va exhibits only a partial resistance to APC because cleavage at Arg306 in factor Va also occurs, causing complete loss of factor Va activity.
Plasma and recombinant factor V can exist in two biochemically distinct forms, designated factor V1 and factor V2 that differ in N-linked carbohydrate on Asn2181, near the phospholipid binding region of the C2 domain as factor V2 has none.183,184 Because the N-linked carbohydrate appears to decrease the apparent affinity of factor V1 or Va1 for phospholipid, it reduces the specific clotting activity and susceptibility to APC. Normal plasma contains a mixture of factors V1 and V2. Removal of the carbohydrate attached to factor V increases the rate of inactivation of factor Va by APC, although the clinical significance of this phenomenon is unknown.185
APC resistance with no identifiable genetic or acquired abnormalities is well described in patients with venous and arterial thrombosis and, at least for research purposes, should be therefore examined in patients with a suspected thrombophilia. Further studies are needed to identify the causes of APC resistance in such patients.186,187,188 One major challenge involves defining the normal range for the clotting assays that are actually used to characterize APC resistance and the multiple plasma analytes or nonplasma assay components that are present in the assays. For example, activated partial thromboplastin time-based assays are not equivalently sensitive as are dilute tissue-factor-based assays to plasma high-density lipoprotein (HDL) levels or oral contraceptive use.189,190,191 Plasma variables, such as elevated prothrombin levels,192,193 may affect the response to APC by inhibiting APC anticoagulant actions. Endogenous thrombin potential assays involving dilute tissue factor as the procoagulant initiator provide additional tools for defining and characterizing APC resistance and extend the tools for shedding light on the gray area of APC resistance found in some thrombosis patients that is not linked to currently known factors.
ACTIVATED PROTEIN C ANTICOAGULANT COFACTORS
APC anticoagulant activity is enhanced by a number of factors that may be termed APC anticoagulant cofactors; these include Ca2+ ions; certain, but not all, phospholipids; protein S; factor V; certain glycosphingolipids; and HDL.
Phospholipids as Activated Protein C Cofactors
Certain phospholipids, such as phosphatidylserine, phosphatidylethanolamine, and cardiolipin, enhance the anticoagulant activity of APC. In addition, phosphatidylethanolamine and cardiolipin stimulate the APC anticoagulant pathway activities much more than they stimulate the procoagulant pathway activities.194,195,196,197
Protein S as Activated Protein C Cofactor
Protein S structure–activity relationships are informed by much biochemical work and the large number of mutations.71,198 Protein S, as an anticoagulant APC cofactor, forms a 1:1 complex with APC and enhances by 10- to 20-fold the rate of APC’s cleavage at Arg306 in factor Va but not the Arg506 cleavage.181,182 Part of the mechanism for this activity of protein S may be related to its ability to bring the active site of APC closer to the plane of the phospholipid membrane on which the APC–protein S complex is located when the complex is formed.199,200 Protein S also facilitates the action of APC against factor VIIIa.201 Protein S enhances APC’s action, in part at least, by ablating the ability of factor Xa to protect factor Va from APC.202 The GLA domain, thrombin-sensitive region, and EGF1 and EGF2 domains of protein S are implicated in binding APC for expression of anticoagulant activity by the APC–protein S complex.198,203,204,205,206 Cleavage of the thrombin-sensitive region by thrombin abolishes normal binding of protein S to phospholipid and its normal APC-cofactor anticoagulant activity.205,207
Factor V as Activated Protein C Cofactor
Factor V apparently can have anticoagulant as well as procoagulant properties because it enhances the anticoagulant action of APC against factors VIIIa and Va in a reaction in which protein S acts synergistically with factor V.23,208,209,210,211 Cleavage at Arg1545, which optimizes factor Va procoagulant activity, ablates the molecule’s anticoagulant cofactor activity. However, when factor V is cleaved at Arg506 by APC, its APC cofactor activity is increased 10-fold. This suggests that Gln506-factor V has two potential prothrombotic defects, namely, resistance of the variant factor Va to APC inactivation and resistance of the variant factor V to activation of its APC cofactor function.23,209,210,211
High-Density Lipoprotein as Activated Protein C Cofactor
HDL can exert antithrombotic activity through multiple mechanisms.212 HDL enhances the anticoagulant activity of APC both in plasma and in purified reaction mixtures, and this APC cofactor activity requires protein S and involves, at least in part, stimulation of APC’s cleavage at Arg306 in factor Va.189,190 HDL is heterogeneous in both protein and lipid composition, and the components responsible for this activity have not been identified, although large HDL, but not small HDL, possesses APC anticoagulant cofactor activity.190 Venous thrombosis in males and in subjects experiencing venous thrombosis recurrence are associated with a pattern of dyslipoproteinemia and low HDL, consistent with the hypothesis that deficiency of large HDL is a risk factor for venous thrombosis.213,214
Glycosphingolipids as Activated Protein C Cofactors
Although both procoagulant and anticoagulant reactions are markedly enhanced by the presence of negatively charged phospholipid surfaces in vitro, certain lipoproteins, for example, HDL,189 and certain lipids, for example, glycosphingolipids and sphingosine,215,216,217,218 selectively enhance anticoagulant reactions in plasma. Plasma glucosylceramide deficiency is a biomarker and may be a potential risk factor for venous thrombosis.215 Sphingosine and several of its common analogues are potent inhibitors of thrombin generation in plasma and on cell surfaces because they inhibit interactions between factors Va and Xa.218 Further studies are needed to characterize the anticoagulant or procoagulant properties of minor abundance plasma and their significance for clinical thrombotic events.
ACTIVATED PROTEIN C DIRECT CELLULAR ACTIVITIES
As noted in Chap. 113, control of coagulation reactions does not occur in the absence of an integrated host defense system that involves a number of biologic processes involving multiple overlapping and integrated pathways. Reactions of the innate and acquired immune system including inflammatory processes, blood coagulation reactions, fibrinolysis, and thrombotic processes are intertwined in vivo via multiple molecular and cellular mechanisms.5,7,8,81,86,87,212,219,220 In addition to its anticoagulant activity, APC acts directly on cells to cause multiple cytoprotective effects. Cytoprotective actions of APC include antiapoptotic and antiinflammatory activities, beneficial changes in gene-expression profiles, and endothelial barrier stabilization. These cytoprotective activities of APC generally require EPCR, involve APC’s ability to activate PAR-1, and may also require additional receptors such as PAR-3, sphingosine-1-phosphate receptor 1, integrin CD11b/CD18, apoER2, EGF receptor, and/or Tie2.7,8,86,110,221,222,223,224,225,226,227
Pharmacologic APC infusions showed benefits in numerous animal injury model systems, with the most informative animal studies to date being in sepsis models and in neuroprotection experiments.7,8,110,111,226,227 Protein engineering permitted the molecular dissection of APC’s anticoagulant activity from its cytoprotective activities7,8,37,40,41,42,43,44,228 and led to proof of principle that APC’s cell signaling activities are both necessary and most likely sufficient for reducing lethality in murine septic shock models42,229 and for providing neuroprotective effects in ischemic stroke models.226,227,230,231,232 Notably, recombinant APC mutants that have little anticoagulant activity (<10 percent) but normal cell-signaling activity are able to convey beneficial effects in multiple injury and disease models with diminished risks for bleeding that would be anticipated with wild-type APC therapy.8,40,41,42,43,44
Activated Protein C Neuroprotective Effects
Neuroprotective effects of APC have been convincingly demonstrated in rodent ischemic stroke models and N-methyl-D-aspartate (NMDA) excitotoxic injury models.8,223,226,227,231,233–242 APC not only provides direct cytoprotection in vitro and in vivo for brain endothelium against ischemic injury but also directly protects neurons against NMDA-induced excitotoxic injury both in vivo and in vitro. APC mutants with reduced anticoagulant activity were as neuroprotective as wild-type APC, and certain cellular receptors were required for APC’s neuroprotection, strongly implying that neuroprotection by APC involves its actions directly on the endothelium and on neurons. Remarkably, in the ischemic penumbra in a murine stroke model, APC caused neovascularization and neurogenesis.223,240,243,244,245 The extensive preclinical studies on APC’s neuroprotective effects paved the pathway for translation of the 3K3A-APC variant to potential neuroprotective therapy for acute ischemic stroke.246,247 Because of the greatly reduced anticoagulant activity of 3K3A-APC (<10 percent of normal), high-dose bolus dosing in healthy volunteers can achieve circulating APC levels that are 100-fold higher than those used in the PROWESS or PROWESS-SHOCK sepsis trials without notable anticoagulant effects.46,49,246
Cellular Receptors for Physiologic Effects of Activated Protein C on Cells
The ability of exogenously administered APC to alter gene-expression profiles of cultured endothelial cells, to stabilize endothelial barriers, to reduce lethality caused by endotoxin in murine sepsis models, to prevent apoptosis of stressed endothelial cells, and to provide neuroprotection requires EPCR and PAR-1, strongly supporting the EPCR–PAR-1 cell-signaling pathway as key for APC’s pharmacologic benefits (see Fig. 114–4).7,8,40,41,42,43,221,225,229,233,234,236,237,244,248,249,250,251
Although few details are known about intracellular mechanisms for APC’s multiple cytoprotective actions, some mechanistic details for APC’s cell signaling have become clear, as depicted in Fig. 114–6.8 Multiple considerations help explain how PAR-1 can mediate thrombin’s disruption of endothelial barrier leading to vascular leakage while, paradoxically, the same receptor mediates APC’s endothelial barrier protection, preventing vascular leakage.249,250 First, PAR-1–mediated APC signaling occurs in caveolae microdomains that contain EPCR whereas PAR-1–mediated thrombin signaling is not limited to caveolae (Fig. 114–6).252,253 Second, different cleavages in the extracellular N-terminus of PAR-1, either at the canonical Arg41 thrombin-cleavage site (i.e., widely recognized as the essential thrombin cleavage site) or at the novel Arg46 APC-cleavage site, results in very different signaling initiated by different tethered N-terminal peptide sequences which begin at either residue 42 or residue 47.124,254,255 Third, following thrombin cleavage at Arg41, PAR-1 initiates signaling involving G proteins, extracellular signal-regulated kinase (ERK)1/2 and RhoA, whereas, following APC cleavage at Arg46, PAR-1 initiates signaling involving β-arrestin-2, phosphatidylinositide 3′-kinase (PI3K)/Akt, and Rac1.256 Fourth, peptides mimicking the N-terminus of cleaved PAR-1 are peptide agonists with pharmacologic effects resembling those of the respective proteases that cleave PAR-1 differentially. For example, “thrombin receptor activating peptides (TRAPs)” that begin with Ser 42 promote G-protein–mediated signaling similar to thrombin. In contrast, peptides that begins with Asn 47 (TR47) promote APC-like signaling.254 TRAP but not TR47 promotes ERK1/2 phosphorylation on endothelial cells whereas TR47 but not TRAP promotes Akt phosphorylation.254 The different and opposite induction of signaling pathways is also mirrored in different and opposite functional effects, as thrombin peptide and TRAP cause endothelial barrier disruption and proinflammatory effects whereas APC and the TR47 peptide cause barrier-protective and antiinflammatory effects. Thus, PAR-1 displays biased signaling depending on the activation cleavage sites and the generated tethered-ligand with absolutely opposing outcomes for the cell, the tissue, and the host depending on which coagulation system protease, thrombin or APC, is cleaving PAR-1.
Biased protease-activated receptor (PAR)-1 signaling dependent on activation by thrombin or activated protein C (APC). Activation of PAR-1 by thrombin results in endothelial barrier-disruptive signaling (A) but activation of PAR-1 by APC in caveolae that also contain endothelial cell protein C receptor (EPCR) results in endothelial barrier-protective signaling (B).252,253 The different PAR-1 signaling induced by thrombin and APC are caused by different proteolysis cleavage sites in PAR-1 for thrombin and APC.254 Thrombin activates PAR-1 by cleavage at Arg41 (C). Synthetic agonist peptides with the N-terminal tethered-ligand sequence beginning with residue 42 are known as TRAP (thrombin receptor-activating peptide) and cause thrombin-like effects on cells. APC activates PAR-1 by cleavage at Arg46 (C). A synthetic agonist peptide with the N-terminal tethered-ligand sequence beginning with residue 47 (TR47) causes APC-like effects on cells. Activation of PAR-1 by thrombin or TRAP induces PAR-1 conformations such that the intracellular loops of PAR-1 preferentially interact with G proteins (termed “G-protein biased”) resulting in G-protein–dependent signaling whereas activation of PAR-1 by APC or TR47 induces PAR-1 conformations that preferentially interact with β-arrestin-2 (termed “β-arrestin biased”) resulting in β-arrestin-2–dependent signaling (D).254,256 The implication of biased PAR-1 signaling are evident by the differences in phosphorylation of extracellular signal-regulated kinase (ERK)1/2 compared to Akt because TRAP, but not TR47, induces phosphorylation of ERK1/2, whereas TR47 but not TRAP induces phosphorylation of Akt (E).254 (Reproduced with permission of Griffin JH, Zlokovic BV, Mosnier LO: Activated protein C: Biased for translation. Blood 125(19):2898–2907, 2015.)
Other receptors are recognized that may also play key roles for APC’s beneficial signaling effects, including PAR-3 and sphingosine-1-phosphate receptor-1.249,250,257,258 ApoER2 can initiate Disabled-1-dependent pathway activation of the PI3K-Akt cell-survival pathway, which may ultimately help explain additional aspects of APC’s cytoprotection.259
Although most studies demonstrating the cell-signaling activities of APC have focused on pharmacologic levels of APC, several reports of murine injury models demonstrate the physiologic importance of cell signaling by endogenous APC,260,261,262 implying that defects in APC’s endogenous cytoprotective actions might have pathophysiologic relevance. Future investigations on APC cellular receptors and on intracellular mechanisms involved in the protein C cellular pathway will likely provide novel clinical insights with diagnostic and therapeutic potential.