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The M358R variant of α1-proteinase inhibitor inhibits coagulation factor VIIa
Biochemical and Biophysical Research Communications 470 (2016) 710e713
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
The M358R variant of a1-proteinase inhibitor inhibits coagulation factor VIIa William P. Sheffield a, b, *, Varsha Bhakta a a b
Canadian Blood Services, Centre for Innovation, Hamilton, Ontario, Canada Department of Pathology and Molecular Mediciney, McMaster University, Hamilton, Ontario, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 December 2015 Accepted 11 January 2016 Available online 13 January 2016
The naturally occurring M358R mutation of the plasma serpin a1-proteinase inhibitor (API) changes both its cleavable reactive centre bond to ArgeSer and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an ArgeSer reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparinindependent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 102 M1sec1. We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin. © 2016 Elsevier Inc. All rights reserved.
Keywords: FVIIa Alpha-1 proteinase inhibitor Antithrombin Serpins Heparin Blood coagulation
1. Introduction Members of the serpin superfamily such as a1-proteinase inhibitor (API) form stable, covalently bonded inhibitory complexes following target protease attack on the serpin reactive centre bond [1]. The naturally occurring API M358R variant (also called APIPittsburgh) demonstrates sharp alterations in inhibitory specificity due to conversion of its reactive centre bond from MeteSer to ArgeSer [2]. The mutant protein inhibits the preferred physiological target of API, neutrophil elastase, at rates three to four orders of magnitude slower than the wild-type protein [2], while demonstrating commensurately increased inhibition rates for other proteases such as thrombin [2], factor Xa [3], factor XIa, factor XIIa,
kallikrein [4], and activated protein C [5]. This altered inhibitory profile explains in part the bleeding tendencies of members of three unrelated families who have been found to carry this alteration of codon 358 of the API gene [2,6,7]. Two groups reported in 1993 that another serpin, antithrombin (AT), which, like API M358R, contains an ArgeSer (Arg393-Ser394) reactive centre, inhibits coagulation factor VIIa (FVIIa) [8,9]. Because API M358R is, like AT, a relatively promiscuous protease inhibitor, we hypothesized that it would inhibit FVIIa, a reaction not previously described in the biomedical literature. We report here that API M358R, unlike its wild-type counterpart, inhibits FVIIa in a reaction accelerated by tissue factor (TF) but unaffected by heparin. 2. Materials and methods
* Corresponding author. McMaster University, Department of Pathology and Molecular Medicine, HSC 4N66 1280 Main Street West, Hamilton, Ontario, L8S 4K1, Canada. E-mail address: sheffi[email protected] (W.P. Sheffield). http://dx.doi.org/10.1016/j.bbrc.2016.01.069 0006-291X/© 2016 Elsevier Inc. All rights reserved.
2.1. Proteins Plasma-derived purified AT and recombinant full-length TF were purchased from Hematologic Technologies (Essex Junction,
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711
VT, USA). Recombinant FVIIa (Niastase) was from Novo Nordisk, (Mississauga, Canada). Recombinant API M358R was expressed in N-terminally His6-tagged form under arabinose control in E. coli TOP10 cells (Thermo-Fisher Scientific, Waltham, MA, USA) and purified to homogeneity using nickel-chelate and ion exchange chromatography as previously described [10]. 2.2. Gel-based analysis of FVIIa inhibition Recombinant FVIIa was reacted with purified serpins (either AT or API M358R) in 25 mM HEPES pH 7.4, 100 mM NaCl, 5 mM CaCl2in the presence or absence of TF or 10 U/ml heparin (sodium salt from porcine intestinal mucosa, Grade 1A, 180 US Pharmacopeia units/ mg, SigmaeAldrich, St. Louis, MO, USA) at 37 C. At timed intervals aliquots were removed, quenched in SDS-PAGE sample buffer, and analyzed on 10% SDS-polyacrylamide gels stained after electrophoresis with Coomassie Brilliant Blue. 2.3. Kinetic comparison of FVIIa inhibition by AT or API M358R Inhibition of FVIIa-mediated amidolysis was monitored kinetically using chromogenic substrate S2288 (DiaPharma, West Chester, OH, USA) in discontinuous assays. Pseudo-first order conditions of excess serpin (500 nM AT or API M358R), 25 nM FVIIa, 25 nM TF in 25 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% (w/vol) BSA (HEPES reaction buffer, HRB) at 37 C were employed. Some reactions were supplemented with 1.0 U/ml heparin. At timed intervals, reaction aliquots were diluted 1:10 into HRB containing 1.0 mM S2288, and the residual FVIIa activity was determined by monitoring the change in absorbance at 405 nm using a plate reader. 2.4. Determination of the second order rate constant of FVIIa inhibition Second order rate constants (k2) were determined for the reaction of 500 nM API M358R with 25 nM FVIIa/25 nM TF in HRB, with or without supplementation with 1.0 U/ml heparin, by dividing the pseudo-first order rate constant k1 by the API M358R initial concentration. Values for k1 were obtained by determining the slope of the plot of the natural logarithm of the ratio uninhibited FVIIa amidolysis to its inhibited counterpart after t seconds of inhibition by API M358R versus time, analogously to our previous approach to k2 determination of thrombin inhibition [10]. 2.5. Statistical methods Statistical comparisons were made with the aid of GraphPad Instat software (version 3.1, GraphPad Software, La Jolla, CA, USA). 3. Results and discussion We first confirmed AT-mediated inhibition of FVIIa using gelbased assays (Fig. 1). As expected based on earlier reports [8,9], incubation of equimolar AT with FVIIa in the presence of heparin resulted in the formation of an SDS-stable inhibitory complex. Complex formation was promoted in the presence of TF (Fig. 1, compare third and fourth lanes). Under reducing conditions it was clear that the heavy chain, which contains the FVIIa catalytic site, was partially consumed in forming the denaturation-resistant inhibitory complexes, as expected for a serpin-enzyme reaction product in which the N-terminal amino acid of the reactive centre and the active site serine of the protease are linked by an acyl ester bond [1]. The differential Coomassie Blue staining intensity of the FVIIa light chain relative to the heavy chain has been previously noted [11].
Fig. 1. Formation of SDS-stable complexes between AT and FVIIa. AT (5 mM), FVIIa (5 mM) and TF (1 mM) were combined in the presence of 10 U/ml heparin (HEP) for 5 min at 37 C (AT þ FVIIa þ TF, 4th lane), and compared to reactions lacking one (AT þ FVIIa) or two (AT) protein reactants or mixtures of FVIIa and TF (FVIIa þ TF) or TF alone. A 12% SDS-polyacrylamide gel electrophoresed under reducing conditions and stained with Coomassie Blue is shown. M refers to molecular mass markers of 200, 150, 120, 100, 85, 70, 60, 50 (darker intensity), 40, 30, and 25 kDa. The position of AT-FVIIa complexes (**), AT, TF, and the heavy (FVIIa-HC) and light (FVIIa-LC) chains of FVIIa are indicated, at right. Equivalent portions of reactions or control mixtures were electrophoresed in each lane.
SerpineFVIIa reactions were next analyzed under more physiologically likely conditions of excess serpin (Fig. 2). In the absence of TF, incubation of API M358R with FVIIa led to the slow formation of a 75 kDa AT-FVIIa heavy chain complex; addition of TF resulted in greater conversion of FVIIa into inhibitory complexes in 0.5e1.0 min than was observed in 30e60 min in its absence (compare Fig. 2A to B). A similar stimulatory effect of TF was noted for AT reactions, although heparin was an obligatory cofactor under these conditions (compare Fig. 2C to D). These observations were borne out by more quantitative assays employing amidolysis of a chromogenic substrate; the API M358R-FVIIa reaction appeared insensitive to heparin, while inhibition by AT under these conditions was completely heparin-dependent (Fig. 3, compare diamond to inverted triangle progress curves). Indeed, k2 values for FVIIa inhibition by API M358R were unaffected by the presence (7 ± 2 102 M1sec1) or absence of heparin (7.8 ± 0.8 102 M1sec1) (n ¼ 7e10, p ¼ 0.32, non-significant by two tailed unpaired t test). API M358R was found to inhibit FVIIa at a much slower rate than other serine proteases, even when the conformation of FVIIa was optimized by TF binding. Using API M358R produced in the same bacterial expression system as in the current study, we previously reported mean rate constants for the inhibition of thrombin, factor Xa, factor XIa, and factor XIIa of 4.7, 0.41, 2.6, and 0.26 105 M1sec1 [12], indicative of inhibition rates 33- to 600fold faster than that observed for FVIIa. Rates of AT-mediated inhibition of FVIIa similarly fall short of those mediated by this serpin on other proteases; in the absence of heparin, reported k2 values for AT-mediated FVIIa/TF inhibition only range from 0.33 [13] to 4.5 102 M1sec1 [8]. Heparin catalyzes a modest acceleration of inhibition of FVIIa by AT, into the range of 0.56 [8] to 1.5 104 M1sec1 [13], far less than the >4000-fold enhancement it confers on AT-mediated inhibition of thrombin [14]. Binding of heparin accelerates protease inhibition by some serpins via conformational changes transmitted to the reactive centre loop, via template effects if the protease also binds heparin, or via both mechanisms [1,15]. Since both AT and FVIIa bind heparin [16], some of the inhibition of FVIIa likely involves a template mechanism
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Fig. 2. Reactions of API M358R and AT with FVIIa. Serpins (API M358R or AT, 10 mM) were reacted with FVIIa (1.0 mM) in the presence (panels B and D) or absence (panels A and C) of TF (1.0 mM) for times (in minutes) indicated above the lanes. Except where indicated, all AT reactions contained 10 U/ml heparin (HEP). In some reactions FVIIa was omitted (“-” below the lanes) or electrophoresed alone or in combination with TF. The position of denaturation-resistant API M358R-FVIIa (*) or AT-FVIIa (**) complexes is indicated, at right. M refers to molecular mass markers identical to those used in Fig. 1. Equivalent portions of reactions or control mixtures were electrophoresed in each lane.
Fig. 3. Inhibition of FVIIa amidolytic activity by serpins. API M358R or AT (500 nM) were combined with 25 nM FVIIa and 25 nM TF (20:1:1 M ratio) in 25 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% (w/vol) BSA for times indicated in minutes on the x axis. Reactions were diluted into chromogenic substrate S2288 in the same buffer, the rate of colour development was observed, and residual FVIIa activity was then determined as a percentage of the uninhibited activity and plotted on the y axis. Inhibitor and cofactor components are linked by arrows to their respective progress curves. The mean ± SD of 3e5 determinations is shown.
unavailable to API M358R due to its lack of heparin affinity. That neither serpin inhibits VIIa/TF at close to the maximal velocities reported for other serpin/protease pairs, even with the added impetus, in the case of AT, of heparin binding, suggests that characteristics unique to FVIIa may underlie this relatively slow inhibition. Binding of FVIIa to TF accelerates its rate of cleavage of natural substrates (e.g. factor X) by at least 8000-fold [17] but has less dramatic effects on amidolysis of small substrates [18]. Crystals of FVIIa-soluble TF complexes stabilized by p-aminobenzamidine
(PAB) in the FVIIa active site lacked the oxyanion hole, a key structural feature of functional serine proteases; it formed, however, following exchange of PAB for D-FPR-chloromethyl ketone in the active site by crystal soaking [19]. This observation led to the hypothesis that full conformational activation of FVIIa required not only TF binding, but also macromolecular substrate binding to FVIIa exosites, and induction of the oxyanion hole by cleavage site peptide engagement [19,20]. The crystallized encounter complex of API M358R with trypsin revealed contacts only between the serpin reactive centre loop and the protease active site [21]. Assuming that only these limited contacts also form with FVIIa, the absence of exosite binding would certainly constrain the rate of inhibition. Moreover, the cleavage site peptides in the API M358R (AIPR-S, where the dash indicates the scissile bond) [2] and AT (IAGR-S) [22] reactive centre loops differ considerably from FVIIa natural cleavage sites in either factor X (NLTR-I) [23] or factor IX (KLTR-A or DFTR-V) [24]. These differences could contribute both to the efficacy of reactive centre cleavage by FVIIa in the two serpins, as well as the propensity of these cleavage site peptides to induce oxyanion hole formation in TF/VIIa. Irrespective of the latter issue, the contribution of exosite contacts is underscored by the much more rapid rate of inhibition of FVIIa/TF by Tissue Factor Pathway Inhibitor (TFPI), which forms a ternary TFPI/FXa/FVIIa/TF inhibitory complex 12,820-fold faster than API M358R, judging from the reported k2 value of 1 107 M1sec1 [25]. In spite of the considerably faster reaction of FVIIa/TF with TFPI/ Xa than with AT, the plasma concentration of AT (1.9e2.4 mM [26]) is approximately 1000-fold greater than that of TFPI (1.3e3.6 nM [27]). Since the average k2 value for reported heparin-catalyzed inhibition of FVIIa/TF is 1000-fold lower than that for TFPI/Xa, the rate constant and concentration differences negate each other, and
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leave inhibition by either inhibitor equally likely under physiological conditions. The relative importance of the two FVIIa-inhibitory reactions in vivo is unknown, but AT-FVIIa complexes can be detected in the plasma of both normal controls and patients with thrombosis [28]. As to whether or not inhibition of FVIIa by API M358R could have contributed to the bleeding diathesis manifested by the rare individuals expressing this protein, the upper level of API M358R concentration in the index case reached 40 mM during the acute phase [2]. The approximately 20-fold excess of API M358R over AT in the patient circulation would effectively balance the similarly greater inhibition rate of heparin-AT over API M358R. It is therefore conceivable that API M358R inhibition of FVIIa could have contributed to the hemorrhagic pathology observed in this case and in the two other API M358R kindreds, in particular given the role of FVIIa/TF as the principal physiological initiator of coagulation [29]. The index case of API M358R was a boy who suffered from a lifelong and ultimately fatal bleeding tendency reported in 1983 [2]; characterization of the specificity of the mutant inhibitor was carried out over the next few years [4,5]. That the reactivity of API M358R with FVIIa was not investigated at that time likely arose due to the low nM levels of coagulation factor VII in plasma and the associated difficulty in its purification. Recombinant FVIIa became available for clinical and research use in the 1990s and has facilitated many investigations including the current report [30]. 4. Addendum W. Sheffield and V. Bhakta participated in designing and/or performing the research, analyzed data, and participated in writing the manuscript. W. Sheffield wrote the manuscript. 5. Conflicts of interest WPS and VB have no conflicts of interest.
[7] [8]
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Acknowledgements Funding for this study was made possible by a grant from the Heart and Stroke Foundation of Ontario (000267) and from a research contract between Canadian Blood Services and Health Canada, a division of the federal government of Canada. As a condition of the latter funding, this report must contain the statement, “The views expressed herein do not necessarily represent the views of the federal government [of Canada].”
[19]
[20] [21]
[22]
Transparency document
[23]
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.01.069.
[24]
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
The M358R variant of a1-proteinase inhibitor inhibits coagulation factor VIIa William P. Sheffield a, b, *, Varsha Bhakta a a b
Canadian Blood Services, Centre for Innovation, Hamilton, Ontario, Canada Department of Pathology and Molecular Mediciney, McMaster University, Hamilton, Ontario, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 December 2015 Accepted 11 January 2016 Available online 13 January 2016
The naturally occurring M358R mutation of the plasma serpin a1-proteinase inhibitor (API) changes both its cleavable reactive centre bond to ArgeSer and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an ArgeSer reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparinindependent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 102 M1sec1. We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin. © 2016 Elsevier Inc. All rights reserved.
Keywords: FVIIa Alpha-1 proteinase inhibitor Antithrombin Serpins Heparin Blood coagulation
1. Introduction Members of the serpin superfamily such as a1-proteinase inhibitor (API) form stable, covalently bonded inhibitory complexes following target protease attack on the serpin reactive centre bond [1]. The naturally occurring API M358R variant (also called APIPittsburgh) demonstrates sharp alterations in inhibitory specificity due to conversion of its reactive centre bond from MeteSer to ArgeSer [2]. The mutant protein inhibits the preferred physiological target of API, neutrophil elastase, at rates three to four orders of magnitude slower than the wild-type protein [2], while demonstrating commensurately increased inhibition rates for other proteases such as thrombin [2], factor Xa [3], factor XIa, factor XIIa,
kallikrein [4], and activated protein C [5]. This altered inhibitory profile explains in part the bleeding tendencies of members of three unrelated families who have been found to carry this alteration of codon 358 of the API gene [2,6,7]. Two groups reported in 1993 that another serpin, antithrombin (AT), which, like API M358R, contains an ArgeSer (Arg393-Ser394) reactive centre, inhibits coagulation factor VIIa (FVIIa) [8,9]. Because API M358R is, like AT, a relatively promiscuous protease inhibitor, we hypothesized that it would inhibit FVIIa, a reaction not previously described in the biomedical literature. We report here that API M358R, unlike its wild-type counterpart, inhibits FVIIa in a reaction accelerated by tissue factor (TF) but unaffected by heparin. 2. Materials and methods
* Corresponding author. McMaster University, Department of Pathology and Molecular Medicine, HSC 4N66 1280 Main Street West, Hamilton, Ontario, L8S 4K1, Canada. E-mail address: sheffi[email protected] (W.P. Sheffield). http://dx.doi.org/10.1016/j.bbrc.2016.01.069 0006-291X/© 2016 Elsevier Inc. All rights reserved.
2.1. Proteins Plasma-derived purified AT and recombinant full-length TF were purchased from Hematologic Technologies (Essex Junction,
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VT, USA). Recombinant FVIIa (Niastase) was from Novo Nordisk, (Mississauga, Canada). Recombinant API M358R was expressed in N-terminally His6-tagged form under arabinose control in E. coli TOP10 cells (Thermo-Fisher Scientific, Waltham, MA, USA) and purified to homogeneity using nickel-chelate and ion exchange chromatography as previously described [10]. 2.2. Gel-based analysis of FVIIa inhibition Recombinant FVIIa was reacted with purified serpins (either AT or API M358R) in 25 mM HEPES pH 7.4, 100 mM NaCl, 5 mM CaCl2in the presence or absence of TF or 10 U/ml heparin (sodium salt from porcine intestinal mucosa, Grade 1A, 180 US Pharmacopeia units/ mg, SigmaeAldrich, St. Louis, MO, USA) at 37 C. At timed intervals aliquots were removed, quenched in SDS-PAGE sample buffer, and analyzed on 10% SDS-polyacrylamide gels stained after electrophoresis with Coomassie Brilliant Blue. 2.3. Kinetic comparison of FVIIa inhibition by AT or API M358R Inhibition of FVIIa-mediated amidolysis was monitored kinetically using chromogenic substrate S2288 (DiaPharma, West Chester, OH, USA) in discontinuous assays. Pseudo-first order conditions of excess serpin (500 nM AT or API M358R), 25 nM FVIIa, 25 nM TF in 25 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% (w/vol) BSA (HEPES reaction buffer, HRB) at 37 C were employed. Some reactions were supplemented with 1.0 U/ml heparin. At timed intervals, reaction aliquots were diluted 1:10 into HRB containing 1.0 mM S2288, and the residual FVIIa activity was determined by monitoring the change in absorbance at 405 nm using a plate reader. 2.4. Determination of the second order rate constant of FVIIa inhibition Second order rate constants (k2) were determined for the reaction of 500 nM API M358R with 25 nM FVIIa/25 nM TF in HRB, with or without supplementation with 1.0 U/ml heparin, by dividing the pseudo-first order rate constant k1 by the API M358R initial concentration. Values for k1 were obtained by determining the slope of the plot of the natural logarithm of the ratio uninhibited FVIIa amidolysis to its inhibited counterpart after t seconds of inhibition by API M358R versus time, analogously to our previous approach to k2 determination of thrombin inhibition [10]. 2.5. Statistical methods Statistical comparisons were made with the aid of GraphPad Instat software (version 3.1, GraphPad Software, La Jolla, CA, USA). 3. Results and discussion We first confirmed AT-mediated inhibition of FVIIa using gelbased assays (Fig. 1). As expected based on earlier reports [8,9], incubation of equimolar AT with FVIIa in the presence of heparin resulted in the formation of an SDS-stable inhibitory complex. Complex formation was promoted in the presence of TF (Fig. 1, compare third and fourth lanes). Under reducing conditions it was clear that the heavy chain, which contains the FVIIa catalytic site, was partially consumed in forming the denaturation-resistant inhibitory complexes, as expected for a serpin-enzyme reaction product in which the N-terminal amino acid of the reactive centre and the active site serine of the protease are linked by an acyl ester bond [1]. The differential Coomassie Blue staining intensity of the FVIIa light chain relative to the heavy chain has been previously noted [11].
Fig. 1. Formation of SDS-stable complexes between AT and FVIIa. AT (5 mM), FVIIa (5 mM) and TF (1 mM) were combined in the presence of 10 U/ml heparin (HEP) for 5 min at 37 C (AT þ FVIIa þ TF, 4th lane), and compared to reactions lacking one (AT þ FVIIa) or two (AT) protein reactants or mixtures of FVIIa and TF (FVIIa þ TF) or TF alone. A 12% SDS-polyacrylamide gel electrophoresed under reducing conditions and stained with Coomassie Blue is shown. M refers to molecular mass markers of 200, 150, 120, 100, 85, 70, 60, 50 (darker intensity), 40, 30, and 25 kDa. The position of AT-FVIIa complexes (**), AT, TF, and the heavy (FVIIa-HC) and light (FVIIa-LC) chains of FVIIa are indicated, at right. Equivalent portions of reactions or control mixtures were electrophoresed in each lane.
SerpineFVIIa reactions were next analyzed under more physiologically likely conditions of excess serpin (Fig. 2). In the absence of TF, incubation of API M358R with FVIIa led to the slow formation of a 75 kDa AT-FVIIa heavy chain complex; addition of TF resulted in greater conversion of FVIIa into inhibitory complexes in 0.5e1.0 min than was observed in 30e60 min in its absence (compare Fig. 2A to B). A similar stimulatory effect of TF was noted for AT reactions, although heparin was an obligatory cofactor under these conditions (compare Fig. 2C to D). These observations were borne out by more quantitative assays employing amidolysis of a chromogenic substrate; the API M358R-FVIIa reaction appeared insensitive to heparin, while inhibition by AT under these conditions was completely heparin-dependent (Fig. 3, compare diamond to inverted triangle progress curves). Indeed, k2 values for FVIIa inhibition by API M358R were unaffected by the presence (7 ± 2 102 M1sec1) or absence of heparin (7.8 ± 0.8 102 M1sec1) (n ¼ 7e10, p ¼ 0.32, non-significant by two tailed unpaired t test). API M358R was found to inhibit FVIIa at a much slower rate than other serine proteases, even when the conformation of FVIIa was optimized by TF binding. Using API M358R produced in the same bacterial expression system as in the current study, we previously reported mean rate constants for the inhibition of thrombin, factor Xa, factor XIa, and factor XIIa of 4.7, 0.41, 2.6, and 0.26 105 M1sec1 [12], indicative of inhibition rates 33- to 600fold faster than that observed for FVIIa. Rates of AT-mediated inhibition of FVIIa similarly fall short of those mediated by this serpin on other proteases; in the absence of heparin, reported k2 values for AT-mediated FVIIa/TF inhibition only range from 0.33 [13] to 4.5 102 M1sec1 [8]. Heparin catalyzes a modest acceleration of inhibition of FVIIa by AT, into the range of 0.56 [8] to 1.5 104 M1sec1 [13], far less than the >4000-fold enhancement it confers on AT-mediated inhibition of thrombin [14]. Binding of heparin accelerates protease inhibition by some serpins via conformational changes transmitted to the reactive centre loop, via template effects if the protease also binds heparin, or via both mechanisms [1,15]. Since both AT and FVIIa bind heparin [16], some of the inhibition of FVIIa likely involves a template mechanism
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Fig. 2. Reactions of API M358R and AT with FVIIa. Serpins (API M358R or AT, 10 mM) were reacted with FVIIa (1.0 mM) in the presence (panels B and D) or absence (panels A and C) of TF (1.0 mM) for times (in minutes) indicated above the lanes. Except where indicated, all AT reactions contained 10 U/ml heparin (HEP). In some reactions FVIIa was omitted (“-” below the lanes) or electrophoresed alone or in combination with TF. The position of denaturation-resistant API M358R-FVIIa (*) or AT-FVIIa (**) complexes is indicated, at right. M refers to molecular mass markers identical to those used in Fig. 1. Equivalent portions of reactions or control mixtures were electrophoresed in each lane.
Fig. 3. Inhibition of FVIIa amidolytic activity by serpins. API M358R or AT (500 nM) were combined with 25 nM FVIIa and 25 nM TF (20:1:1 M ratio) in 25 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% (w/vol) BSA for times indicated in minutes on the x axis. Reactions were diluted into chromogenic substrate S2288 in the same buffer, the rate of colour development was observed, and residual FVIIa activity was then determined as a percentage of the uninhibited activity and plotted on the y axis. Inhibitor and cofactor components are linked by arrows to their respective progress curves. The mean ± SD of 3e5 determinations is shown.
unavailable to API M358R due to its lack of heparin affinity. That neither serpin inhibits VIIa/TF at close to the maximal velocities reported for other serpin/protease pairs, even with the added impetus, in the case of AT, of heparin binding, suggests that characteristics unique to FVIIa may underlie this relatively slow inhibition. Binding of FVIIa to TF accelerates its rate of cleavage of natural substrates (e.g. factor X) by at least 8000-fold [17] but has less dramatic effects on amidolysis of small substrates [18]. Crystals of FVIIa-soluble TF complexes stabilized by p-aminobenzamidine
(PAB) in the FVIIa active site lacked the oxyanion hole, a key structural feature of functional serine proteases; it formed, however, following exchange of PAB for D-FPR-chloromethyl ketone in the active site by crystal soaking [19]. This observation led to the hypothesis that full conformational activation of FVIIa required not only TF binding, but also macromolecular substrate binding to FVIIa exosites, and induction of the oxyanion hole by cleavage site peptide engagement [19,20]. The crystallized encounter complex of API M358R with trypsin revealed contacts only between the serpin reactive centre loop and the protease active site [21]. Assuming that only these limited contacts also form with FVIIa, the absence of exosite binding would certainly constrain the rate of inhibition. Moreover, the cleavage site peptides in the API M358R (AIPR-S, where the dash indicates the scissile bond) [2] and AT (IAGR-S) [22] reactive centre loops differ considerably from FVIIa natural cleavage sites in either factor X (NLTR-I) [23] or factor IX (KLTR-A or DFTR-V) [24]. These differences could contribute both to the efficacy of reactive centre cleavage by FVIIa in the two serpins, as well as the propensity of these cleavage site peptides to induce oxyanion hole formation in TF/VIIa. Irrespective of the latter issue, the contribution of exosite contacts is underscored by the much more rapid rate of inhibition of FVIIa/TF by Tissue Factor Pathway Inhibitor (TFPI), which forms a ternary TFPI/FXa/FVIIa/TF inhibitory complex 12,820-fold faster than API M358R, judging from the reported k2 value of 1 107 M1sec1 [25]. In spite of the considerably faster reaction of FVIIa/TF with TFPI/ Xa than with AT, the plasma concentration of AT (1.9e2.4 mM [26]) is approximately 1000-fold greater than that of TFPI (1.3e3.6 nM [27]). Since the average k2 value for reported heparin-catalyzed inhibition of FVIIa/TF is 1000-fold lower than that for TFPI/Xa, the rate constant and concentration differences negate each other, and
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leave inhibition by either inhibitor equally likely under physiological conditions. The relative importance of the two FVIIa-inhibitory reactions in vivo is unknown, but AT-FVIIa complexes can be detected in the plasma of both normal controls and patients with thrombosis [28]. As to whether or not inhibition of FVIIa by API M358R could have contributed to the bleeding diathesis manifested by the rare individuals expressing this protein, the upper level of API M358R concentration in the index case reached 40 mM during the acute phase [2]. The approximately 20-fold excess of API M358R over AT in the patient circulation would effectively balance the similarly greater inhibition rate of heparin-AT over API M358R. It is therefore conceivable that API M358R inhibition of FVIIa could have contributed to the hemorrhagic pathology observed in this case and in the two other API M358R kindreds, in particular given the role of FVIIa/TF as the principal physiological initiator of coagulation [29]. The index case of API M358R was a boy who suffered from a lifelong and ultimately fatal bleeding tendency reported in 1983 [2]; characterization of the specificity of the mutant inhibitor was carried out over the next few years [4,5]. That the reactivity of API M358R with FVIIa was not investigated at that time likely arose due to the low nM levels of coagulation factor VII in plasma and the associated difficulty in its purification. Recombinant FVIIa became available for clinical and research use in the 1990s and has facilitated many investigations including the current report [30]. 4. Addendum W. Sheffield and V. Bhakta participated in designing and/or performing the research, analyzed data, and participated in writing the manuscript. W. Sheffield wrote the manuscript. 5. Conflicts of interest WPS and VB have no conflicts of interest.
[7] [8]
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[15] [16]
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Acknowledgements Funding for this study was made possible by a grant from the Heart and Stroke Foundation of Ontario (000267) and from a research contract between Canadian Blood Services and Health Canada, a division of the federal government of Canada. As a condition of the latter funding, this report must contain the statement, “The views expressed herein do not necessarily represent the views of the federal government [of Canada].”
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[20] [21]
[22]
Transparency document
[23]
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.01.069.
[24]
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