Protein S as Cofactor for TFPI

ATVB In Focus Tissue Factor: Past, Present, and Future Series Editor: Nigel Mackman and Mark B. Taubman Protein S as Cofactor for TFPI Tilman M. Hackeng, Jan Rosing Abstract In the last decades evidence was obtained that protein S not only acts as cofactor of activated protein C (APC) in the downregulation of coagulation, but also expresses anticoagulant activity in the absence of APC. The search for the mechanism(s) underlying the APC-independent anticoagulant activity of protein S was hampered by the fact that protein S exhibited 2 seemingly identical anticoagulant activities in model systems and in plasma. Later it was shown that the anticoagulant activity of purified protein S in model systems was dependent on the concentration of phospholipid vesicles and was explained by low amounts of protein S multimers generated during purification that effectively inhibited phospholipid-dependent coagulation reactions via competition for phospholipid binding sites. Plasma does not contain multimers, and the anticoagulant activity of protein S in plasma was not affected by the phospholipid concentration but was dependent on the amount of tissue factor (TF) used for initiation of thrombin generation. This led to the discovery that protein S acts as cofactor of tissue factor pathway inhibitor (TFPI) which stimulates the inhibition of factor Xa by TFPI 10-fold. The current review describes the background of the TFPI-cofactor activity of protein S as well as the rationale for the observation that the TFPI/protein S system particularly inhibits the TF pathway at low procoagulant stimuli. (Arterioscler Thromb Vasc Biol. 2009;29:2015-2020.) Key Words: TFPI proteins thrombin generation extrinsic pathway anticoagulant mechanisms Anticoagulant Activities Protein S Protein S is a vitamin K-dependent plasma protein (Mr 75 kda) that is synthesized in the liver and in endothelial cells and which circulates in plasma both in a free form (150 nmol/l) and in complex with C4b-binding protein (200 nmol/l; for a review see 1 ). It is well known that protein S acts as a cofactor of activated protein C (APC) in the proteolytic inactivation of blood coagulation factors Va and VIIIa, thus providing a negative feedback on coagulation and making the APC/protein S pathway essential for normal hemostasis. 2 5 Homozygous deficiencies in either protein C or protein S result in severe neonatal procoagulant phenotypes, whereas heterozygous deficiencies are associated with a 10-fold increased risk of venous thrombosis. 6 8 More recently it has been shown that protein S also effectively inhibits thrombin generation in plasma in the absence of APC. 9 This anticoagulant property of protein S was especially observed at low TF concentrations ( 1 pmol/l) leading to the concept that protein S took part in regulating the initiation phase of coagulation and effectively counteracts ongoing procoagulant processes in the circulation. 10 Interestingly, protein S had no effect on thrombin generation when coagulation was initiated through the intrinsic route, regardless of the amount of trigger used. 5 Because of the dependence of the anticoagulant activity of protein S on the TF concentration, a new role of protein S was identified as a cofactor for TFPI in the inhibition of FXa. 11 In contrast to investigations in plasma, many studies were performed in model systems using purified protein S preparations that also led to reports about APC-independent anticoagulant properties of protein S. 12 16 Although the anticoagulant activities in plasma and in model systems containing purified proteins initially looked mechanistically similar, the activity of purified protein S in model systems could for the greater part be allotted to in vitro generated protein S multimers. Protein S multimers are absent in plasma 17,18 and hence are not involved in the APC-independent anticoagulant activity of protein S in plasma. Intermezzo: Protein S Multimers When protein S was first reported to exhibit anticoagulant activity in the absence of APC, 19 subsequent experiments supporting this observation were performed in model systems using purified proteins. 12 14 At that time, the anticoagulant effect of protein S was explained by direct interactions of protein S with the components of the prothrombinase complex: FVa, FXa, or phospholipids that resulted in inhibition of prothrombin activation. 12 14 Soon the problem arose that the APC-independent anticoagulant activity was difficult to reproduce, and that protein S preparations purified by different purification procedures had different APC-independent anticoagulant activities (Figure 1). It was observed that the activity of protein S Received June 26, 2009; revision accepted July 29, 2009. From the Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University Maastricht, The Netherlands. Correspondence to Prof Dr Jan Rosing, Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University Maastricht, Maastricht, 6200 MD, The Netherlands. E-mail j.rosing@bioch.unimaas.nl 2009 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.108.177436 2015

2016 Arterioscler Thromb Vasc Biol December 2009 Figure 1. Inhibition of prothrombin activation by protein S multimers depends on the phospholipid concentration. FVa, FXa, and phospholipid vesicles (20/80 DOPS/DOPC) were incubated with or without purified protein S in 50 mmol/l Hepes buffered saline ph 7.4 containing 3 mmol/l CaCl 2 and 5 mg/ml BSA for 15 minutes at 37 C. Prothrombin was added and thrombin formation was followed with chromogenic substrate. 18 Final concentrations were 10 pmol/l FVa, 10 pmol/l FXa, 1.5 mol/l prothrombin, 0 nmol/l (100%), or 100 nmol/l protein S and 0.2 to 50 mol/l phospholipid vesicles. Inhibition of prothrombin activation by protein S1 (F) and protein S2 (E) is presented as function of the phospholipid concentration. No inhibition of prothrombin activation by protein S was observed at 50 mol/l phospholipids. At 1 mol/l phospholipid, protein S1 inhibited thrombin generation 40%, and protein S2 inhibited 90%. Inset, Western blot of native PAGE 17 of protein S1 (F) and protein S2 (E) showing comparable protein S monomers (Mo) in protein S1 and protein S2, but more protein S multimers (Mx) in protein S2 (E). Table. Kinetic Constants for the Inhibition of FXa by TFPI With or Without Protein S TFPI K i (nmol/l) K i * (nmol/l) PL/Ca 2 TFPI fl 14.6 32 ; 4.4 11 0.07 32 ; 0.05 11 PL/Ca 2 protein S TFPI fl 0.5 11 0.02 11 PL/Ca 2 TFPI 1 161 39.0 11 0.21 11 PL/Ca 2 protein S TFPI 1 161 42.3 11 0.10 11 PL indicates phospholipid vesicles 20/60/20 DOPS/DOPC/DOPE; TFPI fl, full-length TFPI; TFPI 1 161, TFPI that lacks the third Kunitz domain and the C terminus. preparations correlated with the affinity of protein S for phospholipid surfaces, 20 and eventually modified protein S forms (stable multimers) were identified which were formed in protein S preparations as result of the use of ion-exchange and affinity chromatography during purification. 17,18 The multimers represented between 2% to 5% of total purified protein S and were responsible for the observed APC-independent anticoagulant activity of protein S in model systems. Protein S multimers were reported to bind to negatively-charged phospholipid surfaces with a much higher affinity (K D 0.5 nmol/l) than protein S monomers (K D 250 nmol/l), and the APC-independent activity of protein S preparations was explained by competition between protein S multimers and components of the prothrombinase complex for phospholipid binding sites. 17 Hence, the anticoagulant activity of purified protein S could be overcome by addition of high concentrations of phospholipid vesicles (Figure 1). Furthermore, differences in activities between purified protein S preparations were explained by different protein S multimer content. 17 APC-independent anticoagulant activity of protein S was also observed in plasma in which protein S was neutralized by monoclonal antibodies. 9 At first it was assumed that the mechanistic basis of anticoagulant activities of protein S in model systems and in plasma was identical, but later this was shown not to be the case. In the first place, we were not able to detect protein S multimers in plasma, 17,18 although this is subject to debate. 21 Furthermore, the anticoagulant activity of protein S (multimers) in model systems containing purified proteins was dependent on the phospholipid concentration, and was completely abolished at high phospholipid concentrations (ie, conditions at which sufficient phospholipid surface was available for both protein S multimer binding and prothrombin activation 15,18 ; Figure 1). In contrast, the APCindependent anticoagulant activity of protein S in plasma was not affected by the phospholipid concentration, 10 but was strongly dependent on the TF-concentration and was particularly observed at low TF concentrations. 10 This eventually pointed us in the direction of the TFPI-cofactor activity of protein S. 11 TFPI TFPI is a 276-aa Kunitz-type inhibitor (Mr 42 kda) consisting of a negatively charged N terminus, 3 consecutive Kunitz domains, and a positively charged C-terminal tail. 22 The main site of synthesis of TFPI is the endothelium. 23 The first Kunitz domain binds to and inhibits FVIIa, the second binds to and inhibits FXa, and the C-terminal tail was proposed to bind to heparin-like structures on the vessel wall. 24 Kunitz-3 contains a heparin binding site, 25 the function of which is unknown. However, in various truncated forms of TFPI, Kunitz-3 is involved in cross-disulfide bonding between TFPI and low-density lipoprotein. 26 Of total TFPI, 80% is attached to the vessel wall, whereas 20% circulates in plasma (review 27 ). From the 20% plasma TFPI, 80% is bound to various lipoproteins and only 10% ( 0.25 nmol/l) circulates as free full-length TFPI. TF activity is regulated by TFPI via a 2-step feedback mechanism which involves formation of a bimolecular FXa/ TFPI complex that subsequently interacts with TF/FVIIa, yielding an inactive quaternary complex and resulting in termination of TF/FVIIa-catalyzed FX activation. 24,28 The fact that full-length TFPI is most effective in this reaction is attributable to an intact C-terminal basic tail that interacts with anionic membrane surfaces. 29,30 In this negative feedback mechanism, the initial formation a binary TFPI-FXa complex is a prerequisite for the inhibition of TF/FVIIa by TFPI. 24 The concentration of full-length free TFPI in plasma is low (10% of total plasma TFPI: 0.25 nmol/l), 31 and because TFPI is a slow tight binding inhibitor of which the K i of the initial inhibitory complex with FXa (4 to 15 nmol/l 32 ; Table) is much higher than the plasma TFPI concentration, this would imply that TFPI theoretically would be a weak inhibitor of TF-induced thrombin generation in plasma. Dependence of TFPI on Protein S In 2006 we reported that protein S acts as cofactor of TFPI 11 and that the TFPI cofactor activity explains the APC-

Hackeng and Rosing Protein S as Cofactor for TFPI 2017 Figure 2. TFPI anticoagulant activity during thrombin generation and its dependence of protein S. Thrombin generation in TFPI-depleted plasma was initiated with 1.4 pmol/l TF, 10 mol/l phospholipid vesicles, and 16 mmol/l CaCl 2 (final concentrations) in the presence of 30 g/ml corn trypsin inhibitor (CTI, Hematologic Technologies) and followed with the fluorogenic substrate Z-Gly-Gly-Arg-AMC.HCl. 11 A, Reconstitution of full-length TFPI in TFPIdepleted plasma at indicated final concentrations (0 to 3.2 nmol/l). B, Reconstitution of full-length TFPI in TFPI-depleted plasma to which inhibitory antiprotein S antibodies (Zebra Bioscience, Enschede) were added. independent anticoagulant activity of protein S in plasma. This discovery was based on the following observations: (1) in the absence of APC, protein S inhibited thrombin generation initiated via the extrinsic pathway, but not via the intrinsic pathway, (2) protein S only expressed APCindependent anticoagulant activity at low TF concentrations, (3) protein S did not express APC-independent anticoagulant activity in TFPI-deficient plasma, and (4) the anticoagulant activity of TFPI was greatly impaired in protein S-deficient plasma. The discovery that protein S acts as a cofactor for full-length TFPI in the formation of the initial bimolecular FXa/TFPI complex explains why TFPI is an effective inhibitor in plasma. 11 It was shown that protein S decreased the K i for FXa/TFPI complex formation from 4.5 to 0.5 nmol/l. Thus, protein S makes TFPI a more efficient inhibitor by bringing the K i of FXa inhibition in the range of the plasma concentration of full-length TFPI. 11 It was concluded that through stimulation of TFPI/FXa complex formation, protein S would also enhance the formation of the quaternary TF/FVIIa/TFPI/FXa complex. 11 However, more recently it was suggested that the TFPI-cofactor activity of protein S is only relevant for inhibition of free factor Xa, and not for the inhibition of TF/FVIIa by TFPI/FXa (ie, not for quaternary complex formation 33 ; see also below). It was reported that C-terminally truncated TFPI was a weak inhibitor of factor Xa 30,34 (Table). In addition, truncated TFPI (1 161) was not stimulated by protein S. 11 This strongly suggests that the interaction between protein S and TFPI is mediated through TFPI Kunitz-3 and the C-terminal tail. For both protein S and full-length TFPI activity it is crucial that they can bind to negatively charged phospholipids through their Gla domain and C-terminal tail, respectively. 13,29,35 In addition, FXaprotein S interactions have been reported. 36,37 Together with the observation that the C terminus of TFPI directly interacts with the Gla domain of FXa, 38 this implies that during inhibition of FXa by TFPI/protein S at a certain moment a trimolecular complex between FXa, TFPI, and protein S on phospholipids will exist. In this review, the abbreviation TFPI will be used for full-length TFPI, unless otherwise indicated. Supporting evidence for direct interactions between TFPI and protein S is the observation by Castoldi and coworkers that protein S and TFPI circulate as a complex in plasma (Castoldi, Simioni, Tormene, Rosing, and Hackeng, unpublished data, 2009), which could explain the covariance of protein S and TFPI in normals 39 and the low levels of TFPI in protein S-deficient patients (Castoldi et al, ISTH 2009, abstract # 2338). An illustrative example of the importance of the TFPI cofactor activity of protein S is shown in Figure 2 in which the effect of TFPI on thrombin generation in TFPI-deficient plasma and in combined TFPI-protein S-deficient plasma were compared. Reconstitution of TFPI-depleted plasma with TFPI shows a dose-dependent inhibition of thrombin generation, which is fully inhibited at 3.2 nmol/l TFPI. However, when protein S is neutralized by addition of antibodies against protein S, TFPI anticoagulant activity is severely impaired resulting in substantial thrombin generation ( 50%) at 3.2 nmol/l TFPI (Figure 2). TFPI is a Slow Tight Binding Inhibitor Inhibition of FXa by TPFI proceeds through a 2-step process. In the first step, rapidly a small amount of loose complex between Kunitz-2 of TFPI and FXa is formed (FXa TFPI, equation a) which results in rapid inactivation of part of the FXa. A subsequent slow rearrangement results in formation of the final tight enzyme-inhibitor complex 24 (FXa TFPI*, equation a). The dissociation constant of the first rapid equilibrium is represented by K i (K i k -1 /k 1 [FXa].[TFPI]/[FXa TFPI]) and after the subsequent slow isomerization has taken place the overall equilibrium constant becomes K i *(K i * [FXa].[TFPI]/{[FXa TFPI] [FXa TFPI*]}), which is several orders of magnitude lower than K i (Table). The fact that TFPI is a slow inhibitor of FXa has important implications for the downregulation of the TF pathway by TFPI in plasma. TFPI anticoagulant activity in thrombin generation was only observed at low concentrations of TF

2018 Arterioscler Thromb Vasc Biol December 2009 Figure 3. Effect of full-length TFPI on thrombin generation in the absence (A) and presence (B) of APC. Thrombin generation in recalcified citrated plasma was continuously followed with fluorogenic substrate I-1140 (Z-Gly-Gly-Arg-AMC.HCl) in the presence of 20 mol/l phospholipids vesicles (20/60/20 DOPS/DOPC/ DOPE), 16 mmol/l CaCl 2 (final concentrations), and 30 g/ml corn trypsin inhibitor (CTI, Hematologic Technologies). Thrombin generation was initiated with 14 pmol/l TF in the absence (A) or presence of 20 nmol/l APC (B) in protein S and TFPI-depleted plasma reconstituted with (F) 350 nmol/l protein S, or (E) 350 nmol/l protein S and 0.5 nmol/l full-length TFPI. Averages of duplicate measurements are shown. Adapted with permission from Hackeng et al. 27 ( 1 pmol/l), whereas at high concentrations of TF ( 10 pmol/l) thrombin generation was not effectively regulated by TFPI anymore. 10 Consequently, the TFPI-cofactor activity of protein S was also lost and hence, protein S does not express APC-independent anticoagulant activity at high TF concentrations. 10 However, at 14 pmol/l of TF the plasma concentration of free full-length TFPI (0.3 nmol/l) is still in 20-fold excess of TF. As the Ki* for FXa-inhibition by TFPI was reported to be 0.05 nmol/l (Table), 11,32 one would also expect efficient inhibition of FXa and thrombin generation by TFPI at TF concentrations as high as 14 pmol/l. That this is not the case is caused by the fact that TFPI is not only a tight but also a slow-binding inhibitor of factor Xa. 40 In other words, only when FXa formation is low and slow does TFPI get sufficient time to block the TF pathway before thrombin generation and clotting can occur. At high TF concentrations, the rate of FX activation becomes so high that the threshold concentration of FXa required for prothrombin activation and clotting is exceeded within the time frame that TFPI requires to shut down the TF pathway. Interestingly, there are a few examples of effective modulation of procoagulant responses by TFPI/protein S at high TF concentrations. One of these is the modulation of the effect of APC on thrombin generation at high TF-concentrations. In 1997 a thrombin generation-based APC resistance test was published 41 which appeared to be very sensitive for both hereditary (FV Leiden ) and acquired APC resistance (eg, oral contraceptive use). In this assay thrombin generation was measured at a high TF concentration in the absence and presence of APC. Thrombin generation in the absence of APC was not affected by TFPI, whereas TFPI appeared to be a major determinant of thrombin generation in the presence of APC. 42,43 This is illustrated by Figure 3 which shows that TFPI in the absence of APC has no effect on thrombin generation at high TF (14 pmol/l; Figure 3A), but significantly decreases thrombin generation in the presence of APC (Figure 3B). Because of the downregulation of thrombin generation by APC/protein S, an appreciable part of the reduction of thrombin generation by APC (shown by the gray area) is actually caused by the TFPI/protein S pathway. 27 Hence, in the presence of APC the TFPI/protein S system regains a grip on FXa-regulation even at high TF. A second example of TFPI activity at high TF is the observation that the APC-independent activity of protein S in clotting plasma, which we now know represents the ability of protein S to enhance the anticoagulant activity of TFPI, became particularly apparent in the presence of heparin. 44 These two examples share a common denominator, which is that both APC and heparin delay the onset of coagulation and hence give TFPI ample time to inhibit TF-FVII and downregulate thrombin formation. These observations suggest that the contribution of TFPI to the downregulation of coagulation also becomes more important in patients receiving anticoagulant treatment. TFPI: FXa Inhibitor, TF-FVIIa Inhibitor, or Both? TFPI is an inhibitor of coagulation that has 2 targets, FXa and TF-FVIIa, and theoretically it is possible that the TFPI/ protein S system downregulates thrombin formation and coagulation at 2 levels (ie, FXa and TF-FVIIa). It is generally accepted that inhibition of TF-FVIIa by TFPI is physiologically important, whereas virtually no information is available as to whether inhibition of FXa by TFPI also contributes to the downregulation of thrombin formation by TFPI. Although incorporation of FXa into a phospholipid-bound FX-FVa (prothrombinase) complex had no effect on the inhibition of FXa by TFPI in the absence of prothrombin, 31 TFPI appeared to be a poor inhibitor of the prothrombinase complex in the presence of physiological concentrations prothrombin. 45 As such, TFPI is not different from other anticoagulant proteins (antithrombin and APC) whose anticoagulant activities are also greatly reduced when their targets (FXa and FVa) are incorporated into the prothrombinase complex. 46,47 However, TFPI appeared to be an effective inhibitor of prothrombin activation in model systems when FVa was substituted by FV 45 (ie, when FV had to be activated by FXa or traces of thrombin before the onset of prothrombin activation). Of course a possible contribution of direct inhibition of FXa by TFPI to the downregulation of thrombin formation should be put in the perspective of other FXa inhibitors in plasma (eg, antithrombin). At the plasma antithrombin concentration of 2.5 mol/l 48 and a rate constant of FXa inhibition 47 of 3.53 10 3 M 1 sec 1, antithrombin requires 1.3 minutes (t 2) to inhibit 50% of the FXa formed. Using 1 rate constants and K i s reported by Hackeng et al 11 it can be calculated that at the plasma-free TFPI concentration (0.25 nmol/l) the t 1 2 for inhibition of FXa by TFPI drops from 5

Hackeng and Rosing Protein S as Cofactor for TFPI 2019 believe that further studies are needed to unravel the precise mechanisms by which the TFPI/protein S system downregulates blood coagulation. Figure 4. Protein S dependent anticoagulant pathways of blood coagulation. Exposed TF binds FVIIa from the circulation to generate the transient TF/FVIIa complex, the physiological initiator of blood coagulation. (1) TF/FVIIa activates FX, and FXa will bind to its cofactor FVa on negatively charged phospholipid surfaces (ie, activated platelets) to generate the prothrombinase complex. Early thrombin generation will activate platelets, activate cofactors V and VIII, and in addition activates FXI from the propagation loop. (2) FXIa can activate FIX. FIXa and cofactor FVIIIa assemble on negatively charged phospholipids, and activate additional FX for participation in prothrombin activation and thus form sufficient thrombin to generate insoluble fibrin polymers to stabilize the primary platelet plug. Thrombin generation is tightly regulated by 2 protein S dependent feedback mechanisms (3, 4) and protease inhibitors that scavenge coagulation enzymes at large (5). The TFPI/protein S pathway inactivates FXa and subsequently shuts down TF/FVIIa. (3) Thrombomodulin-bound thrombin activates protein C, after which the APC/protein S pathway inactivates FVa and FVIIIa (4), both protein S-dependent pathways together limiting generation of the components of the prothrombinase complex, FXa and FVa. minutes in the absence of protein S to 1.1 minute in the presence of protein S, which is approximately the same as the t 1 2 of FXa inhibition by antithrombin. Hence, in the presence of protein S TFPI is as good as antithrombin in inhibiting the first FXa molecules that are formed during coagulation. However, it should be emphasized that because of its low plasma concentration, TFPI will not significantly contribute to FXa inhibition in the case of formation of large amounts of FXa. Two years after we reported that protein S is a cofactor of TFPI, 11 Ndonwi and Broze 33 confirmed that protein S enhances the inhibition of FXa by TFPI. However, they also showed that protein S does not stimulate the inhibition of TF-FVIIa by TFPI or by the TFPI-FXa complex and they proposed that the predominant pathway of inhibition of TF-FVIIa by TFPI involves the interaction of TFPI with a tertiary TF-FVIIa-FXa complex produced during FX activation. As the latter reaction sequence is not affected by protein S it was further hypothesized that the enhancing effect of protein S on FXa inhibition by TFPI is probably most important for the inhibition of FXa that escapes FVIIa-TF-Xa inhibition by TFPI. 33 This implies that the stimulatory effect of protein S in the inhibition of thrombin formation by TFPI is attributable to inhibition of FXa that has dissociated from the TF-FVIIa complex and that on its way to the prothrombinase complex is inhibited by TFPI. However, if this mechanism would apply, one would expect that the TFPI/ protein S system also inhibits intrinsically triggered thrombin formation, which appears not to be the case. 5 Hence, we Conclusions Novel insights in protein S anticoagulant activity resulted in the perception of a protein S dependent double-barrel negative feedback system that is constitutively active and that is aimed at limitation of prothrombinase activity (Figure 4). 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