Journal of Thrombosis and Haemostasis, 13: 1084–1089
DOI: 10.1111/jth.12894
IN FOCUS
Generation of a stable thrombin-activatable fibrinolysis inhibitor deletion mutant exerting full carboxypeptidase activity without activation X . Z H O U and P . J . D E C L E R C K Department of Pharmaceutical and Pharmacological Sciences, Laboratory for Therapeutic and Diagnostic Antibodies, Katholieke Universiteit Leuven, Leuven, Belgium
To cite this article: Zhou X, Declerck PJ. Generation of a stable thrombin-activatable fibrinolysis inhibitor deletion mutant exerting full carboxypeptidase activity without activation. J Thromb Haemost 2015; 13: 1084–9. See also Plug T, Meijers JCM. New clues regarding the mysterious mechanism of activated thrombin-activatable fibrinolysis inhibitor selfdestruction. This issue, pp 1081–3.
Summary. Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a zymogen that can be activated to form activated TAFI (TAFIa) (Ala93–Val401) through removal of the N-terminal activation peptide (Phe1– Arg92). TAFIa is thermally unstable, and the role of the activation peptide in the activity and stability of TAFI zymogen remains unclear. Objectives: To better understand the role of the activation peptide in the activity and stability of TAFI. Methods: We constructed a deletion mutant, TAFI-CIIYQ-Δ1–73, in which the first 73 amino acids of the activation peptide are absent. The intrinsic activity and functional stability were determined with a chromogenic assay. The activation of TAFI-CIIYQ-Δ1–73 by TAFI activators was evaluated with western blot analysis. Results: In comparison with TAFI-CIIYQ, the deletion mutant exerted high intrinsic activity (‘full’ apparent TAFIa activity) without cleavage by TAFI activators. TAFI-CIIYQ-Δ1–73 was cleavable by thrombin. However, in the presence of thrombomodulin, the thrombin-mediated cleavage of TAFI-CIIYQ-Δ1–73 was not accelerated. TAFI-CIIYQ-Δ1–73 showed a similar functional stability profile to that of TAFI-CIIYQ. Full cleavage by thrombin did not affect the apparent carboxypeptidase activity of TAFI-CIIYQ-Δ1–73, but resulted in a significant loss of functional stability. Conclusions: A stable deletion mutant of TAFI with full carCorrespondence: Paul Declerck, Department of Pharmaceutical and Pharmacological Sciences, Laboratory for Therapeutic and Diagnostic Antibodies, Katholieke Universiteit Leuven, Campus Gasthuisberg, O&N2, PB 820, Herestraat 49, Leuven B-3000, Belgium. Tel.: +32 16 32 34 31; fax: +32 16 32 34 60. E-mail: [email protected] Received 7 May 2014 Manuscript handled by: T. Lisman Final decision: P. H. Reitsma, 24 February 2015
boxypeptidase activity without activation is described. The segment Ala74–Arg92 in the activation peptide contributes significantly to the role of the activation peptide in stabilization of the catalytic moiety in TAFI zymogen. Keywords: carboxypeptidase U; proteins; thrombin-activatable zymogens.
fibrinolysis; mutant fibrinolysis inhibitor;
Introduction Thrombin-activatable fibrinolysis inhibitor (TAFI) (proCPU, EC 3.4.17.20) is a human plasma procarboxypeptidase that acts as an important regulator between the coagulation and fibrinolytic systems [1,2]. TAFI zymogen (56 kDa) consists of a heavily glycosylated Nterminal activation peptide (Phe1–Arg92; Asn22, Asn51, Asn63, and Asn86; 20 kDa) and a catalytic moiety (Ala93–Val401; 36 kDa). Crystal structures of TAFI indicate that the activation peptide forms a globular structure (Phe1–Leu76) with a linker region (Ala76– Arg92), which connects it to the catalytic moiety [3]. Within the globular structure, Ala74–Asp75 forms a bturn (type VIII) connecting it to the linker region (Ala76–Arg92). In the zymogen form, the activation peptide covers the catalytic pocket and prevents substrate access to the preformed active cleft within the catalytic domain [3,4]. TAFI is activated to its active form, activated TAFI (TAFIa), through cleavage of the Arg92–Ala93 peptide bond, either by thrombin, by plasmin, or, most efficiently, by the thrombin–thrombomodulin complex [2,5,6]. TAFIa attenuates fibrinolysis by removing C-terminal lysines from partially degraded fibrin that mediate positive feedback in the fibrinolytic cascade [2]. To date, no physio© 2015 International Society on Thrombosis and Haemostasis
Active TAFI deletion mutant 1085
logic inhibitors of TAFIa have been found, and the development of TAFIa inhibitors is hindered by the intrinsic instability of TAFIa. TAFIa is thermally unstable (t1/2 of 8–15 min at 37 °C) [7], owing to an unspecified conformational change and inactivation [8]. The structural mechanisms underlying this spontaneous and rapid loss of activity remain unclear. It has been speculated that the dynamics of a highly mobile segment comprising residues Phe296–Trp350 (termed the ‘dynamic flap’) in the catalytic domain contribute to the intrinsic instability of TAFIa [3]. Marx et al. [3] hypothesized that, in the TAFI zymogen form, the activation peptide stabilizes the dynamic flap region through hydrophobic interactions between Tyr341 and Leu35 and Val39. However, after TAFI activation, it is unclear whether the activation peptide is released or remains attached to the catalytic domain, and the role of the activation peptide in the activity and stability of TAFIa remains controversial [4,9,10]. It has been reported that TAFI zymogen itself exerts intrinsic carboxypeptidase activity, ranging from 5.6% [11] to less than 1% [9] as compared with TAFIa. Whether the intrinsic activity of TAFI plays a physiologic role in the regulation of fibrinolysis remains controversial [11–13]. A TAFI variant, TAFI-S305C-T325I-T329I-H333YH335Q (TAFI-CIIYQ), containing stabilizing mutations in the dynamic flap region, has been reported previously [14]. Its activated form, TAFIa-CIIYQ, has been reported to be a stable TAFIa variant with a 180-fold increased half-life (1140 66 min, 37 °C). In the current study, a deletion mutant of TAFI-CIIYQ, TAFI-CIIYQ-Δ1–73, in which the first 73 amino acids (which form the globular structure) of the activation peptide are lacking, is described. The activity, functional stability and activation of this deletion mutant by thrombin and thrombin– thrombomodulin were determined and compared with those of TAFI-CIIYQ. Materials and methods Construction, expression and purification of TAFI-CIIYQ-Δ1–73 and N-His-TAFI-CIIYQ-Δ1–73
The TAFI-CIIYQ-Δ1–73-coding sequence was generated by overlap extension PCR, based on the full-length cDNA of TAFI-CIIYQ in pcDNA5/FRT, yielding pcDNA/FRT-TAFI-CIIYQ-Δ1–73. The construct was transiently transfected into HEK293T cells. For efficient purification of TAFI-CIIYQ-Δ1–73 from the conditioned medium, an N-terminal hexahistidine tag was inserted in frame in the pcDNA/FRT-TAFI-CIIYQ-Δ1–73 construct. pcDNA/FRT-N-His-TAFI-CIIYQ-Δ1–73 was transiently transfected into HEK293T cells, and N-His-TAFI-CIIYQ-Δ1–73 was purified by standard immobilized metal affinity chromatography.
© 2015 International Society on Thrombosis and Haemostasis
Evaluation of TAFI-CIIYQ-Δ1–73 and N-His-TAFI-CIIYQ-Δ1–73 antigen levels
Expression and secretion of TAFI-CIIYQ-Δ1–73 in the conditioned medium were evaluated by western blot (WB) analysis, with MA-TCK27A4 [15] as the primary antibody, and goat anti-mouse–horseradish peroxidase (HRP) as secondary antibody, followed by electrochemiluminescence (ECL) staining. Antigen levels of TAFICIIYQ-Δ1–73 in the conditioned medium or of purified N-His-TAFI-CIIYQ-Δ1–73 were quantified with in-house ELISA (MA-TCK27A4 for capture and MA-TCK28F8– HRP for detection). Analysis of the intrinsic activities of TAFI-CIIYQ and N-HisTAFI-CIIYQ-Δ1–73
The intrinsic activities of TAFI-CIIYQ and N-HisTAFI-CIIYQ-Δ1–73 were determined with a chromogenic assay, as described previously, with minor modifications [9,16]. Briefly, TAFI-CIIYQ (5.4 nM during substrate conversion) or N-His-TAFI-CIIYQ-Δ1–73 (3.0 nM during substrate conversion) was incubated with the synthetic substrate Hip-Arg (11.1 mM during substrate conversion). Substrate conversion was allowed to proceed for 1 h at 22 °C. Reactions were stopped, 6% cyanuric chloride (dissolved in 1,4-dioxane) was added, and the mixtures were vortexed and centrifuged (16 100 9 g, 2 min). The absorbance of the supernatant was measured and the intrinsic activity was calculated as described previously [9]. One unit (U) of intrinsic activity or carboxypeptidase activity is defined as the amount of enzyme converting 1 lmol min–1 at 22 °C. Evaluation of TAFI-CIIYQ and N-His-TAFI-CIIYQ-Δ1–73 fragmentation patterns generated upon treatment with thrombin or thrombin–thrombomodulin
Fragments of TAFI-CIIYQ or N-His-TAFI-CIIYQ-Δ1–73 upon treatment with thrombin or thrombin–thrombomodulin were evaluated with WB analysis, as described previously [9], with the following modifications. TAFI-CIIYQ (8.9 nM during activation) or N-His-TAFI-CIIYQΔ1–73 (5.0 nM) was treated with thrombin (100 nM) or thrombin–thrombomodulin (thrombin, 100 nM; thrombomodulin, 100 nM) and CaCl2 (5 mM) in 60 lL of HEPES buffer (25 mM HEPES, 137 mM NaCl, 3.5 mM KCl, pH 7.4, supplemented with 0.1% bovine serum albumin) at 37 °C for 5 min, 10 min, or 30 min. The reactions were stopped at the indicated time points by addition of SDS (1% final concentration). The samples were heated at 100 °C for 30 s, and subjected to SDS-PAGE (10–15% gradient), WB analysis with MA-T3D8 [17] as primary antibody, and goat anti-mouse–HRP as secondary antibody, and ECL staining.
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are expressed relative to the initial activity observed at day 0 prior to incubation at 37 °C.
Analysis of TAFIa activity upon thrombin treatment
Upon full cleavage by thrombin (100 nM, 37 °C for 4 h), the TAFIa activities of TAFIa-CIIYQ (8.9 nM during activation) and N-His-TAFIa-CIIYQ-Δ1–73 (5.0 nM during activation) were determined as described previously, with minor changes [9]. Substrate conversion was allowed to proceed for 1 h at 22 °C.
Results and conclusions The construct containing the TAFI-CIIYQ-Δ1–73-coding sequence was transiently transfected into HEK293T cells. Conditioned medium was collected 96 h post-transfection. WB analysis revealed a clear band at 40 kDa, representing TAFI-CIIYQ-Δ1–73 in the conditioned medium. According to ELISA, TAFI-CIIYQ-Δ1–73 was present at a concentration of 180 30 ng mL 1 (mean standard deviation [SD]; n = 3) in the conditioned medium. These levels are 10-fold lower than those found for TAFI-CIIYQ when the corresponding DNA construct was used. For efficient purification of TAFI-CIIYQ-Δ1–73 from the conditioned medium, an N-terminal hexahistidine tag was inserted in frame in the pcDNA/FRT-TAFI-CIIYQ-Δ1–73 construct. TAFI antigen levels of purified N-His-TAFI-CIIYQ-Δ1–73 were 400 ng mL 1 as determined with ELISA. Purified N-His-TAFI-CIIYQ-Δ1–73 showed high intrinsic activity; that is, at 22 °C, the intrinsic activity of N-HisTAFI-CIIYQ-Δ1–73 (54 2 U mg 1, mean SD, n = 4) was ~ 90-fold higher than that of purified TAFI-CIIYQ (0.6 0.2 U mg 1, mean SD, n = 4). The intrinsic activ-
Determination of the functional stability of TAFIa-CIIYQ and N-His-TAFIa-CIIYQ-Δ1–73
The functional stability of N-His-TAFI-CIIYQ-Δ1–73 and TAFI-CIIYQ, before and after thrombin treatment (100 nM, 37 °C for 4 h), was measured as described previously, with some modifications [9]. N-His-TAFI-CIIYQΔ1–73 and TAFI-CIIYQ, either as such or after thrombin treatment (4 h at 37 °C, followed by incubation with Dchloromethylketone), phenylalanyl-L-prolyl-L-arginine were incubated at 37 °C for 7 days. At each time point indicated in Fig. 2B, aliquots were placed on ice until measurement of residual intrinsic activity (i.e. without thrombin pretreatment; substrate conversion, 1 h at 22 °C) or residual TAFIa activity (i.e. with thrombin pretreatment; substrate conversion, 1 h at 22 °C). All values
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Fig. 1. Western blot (WB) analysis of fragmentation patterns of TAFI-CIIYQ and N-His-TAFI-CIIYQ-Δ1–73 upon thrombin and thrombin–thrombomodulin treatment. Upon cleavage for 5 min, 10 min, or 30 min, samples were subjected to SDS-PAGE, followed by WB analysis with MA-T3D8 as primary antibody. (A) TAFI-CIIYQ (8.9 nM, lane 1) was fully activated to TAFIa after thrombin–thrombomodulin treatment for 5 min (lane 3). The cleavage was not complete after thrombin treatment for 30 min (lane 6). (B) N-His-TAFI-CIIYQ-Δ1–73 (5 nM, lane 1) was cleaved to a similar extent, after both thrombin–thrombomodulin and thrombin treatment for 5 min (lanes 2 and 3), 10 min (lanes 4 and 5), or 30 min (lanes 6 and 7). Intact TAFI-CIIYQ (56 kDa) is indicated by the arrow, N-His-TAFI-CIIYQ-Δ1–73 (40 kDa) by the arrowhead, and activated TAFI-CIIYQ (TAFIa-CIIYQ) by the dashed arrow. TAFI, thrombin-activatable fibrinolysis inhibitor; TAFIa, activated thrombin-activatable fibrinolysis inhibitor; T, thrombin; T/M, thrombin–thrombomodulin. © 2015 International Society on Thrombosis and Haemostasis
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ity of this deletion mutant could be fully inhibited by potato tuber carboxypeptidase inhibitor (PTCI) (data not shown). WB analysis of the fragmentation patterns of N-HisTAFI-CIIYQ-Δ1–73 after thrombin or thrombin–thrombomodulin treatment revealed that the deletion mutant could be cleaved by either thrombin or thrombin–thrombomodulin (Fig. 1B; the primary antibody, MA-T3D8, reacted specifically with the catalytic moiety of TAFI [17]). Full cleavage by thrombin–thrombomodulin or thrombin was not achieved even after treatment for 30 min at 37 °C (Fig. 1B; lanes 6 and 7). In contrast to the activation of TAFI-CIIYQ by thrombin–thrombomodulin and thrombin (Fig. 1A, lanes 2–7), the presence of thrombomodulin did not accelerate the cleavage of NHis-TAFI-CIIYQ-Δ1–73 by thrombin (Fig. 1B, lanes 2–7). These results are in line with the data reported by Plug
et al. [18] and Wu et al. [19], demonstrating that several positively charged residues (Arg12 [18] and Lys42–Lys44 [19]) within the globular structure of the activation peptide might be important for the activation of TAFI by thrombin–thrombomodulin. Indeed, lack of the globular structure in the deletion mutant prevents efficient interaction with thrombomodulin, thereby abolishing the cofactor function. It is notable that, after cleavage of N-His-TAFI-CIIYQ-Δ1–73, two bands (~ 36 kDa) clearly appeared (Fig. 1B, lanes 2–7) on SDS-PAGE. When reducing reagent was applied prior to gel electrophoresis, this double-band migration pattern disappeared, and activated N-His-TAFI-CIIYQ-Δ1–73 migrated as a single band (data not shown), which may suggest that the deletion mutant also exists with partially oxidized cysteines (i.e. fewer than three disulfide bonds are formed). N-His-TAFICIIYQ-Δ1–73
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Fig. 2. Determination of the functional stability before and after cleavage by thrombin. (A) Evaluation of the cleavage levels of TAFI-CIIYQ and N-His-TAFI-CIIYQ-Δ1–73 upon cleavage by thrombin, prior to incubation at 37 °C. Western blot analysis (with MA-T3D8 as primary antibody) showed that TAFI-CIIYQ and N-His-TAFI-CIIYQ-Δ1–73 (lanes 1 and 3, respectively) were completely cleaved after thrombin treatment (lanes 2 and 4, respectively). Intact TAFI-CIIYQ (56 kDa) is indicated by the arrow, N-His-TAFI-CIIYQ-Δ1–73 (40 kDa) by the arrowhead, and TAFIa-CIIYQ by the dashed arrow. (B) Functional stability (37 °C) of N-His-TAFI-CIIYQ-Δ1–73 (■, □) and of TAFI-CIIYQ (●, ○) before (■, ●) and after (□, ○) thrombin treatment. All values are expressed relative to the intrinsic or TAFIa activity observed at day 0 prior to incubation at 37 °C. At day 0, without thrombin treatment, the intrinsic activity of TAFI-CIIYQ without thrombin treatment was 0.6 0.2 U mg 1, whereas the intrinsic activity of N-His-TAFI-CIIYQ-Δ1–73 was ~ 85-fold higher, i.e. 51 3 U mg 1. After thrombin treatment, the TAFIa activity of TAFI-CIIYQ was 42 2 U mg 1, whereas that of N-His-TAFI-CIIYQ-Δ1–73 was 50 5 U mg 1. Data are presented as mean standard deviation, n = 4. TAFI, thrombin-activatable fibrinolysis inhibitor; TAFIa, activated thrombin-activatable fibrinolysis inhibitor; T, thrombin. © 2015 International Society on Thrombosis and Haemostasis
1088 X. Zhou and P. J. Declerck
The functional stability of N-His-TAFI-CIIYQ-Δ1–73 before and after thrombin treatment was measured and compared with that of TAFI-CIIYQ before and after thrombin treatment. Full cleavage of N-His-TAFI-CIIYQΔ1–73 and TAFI-CIIYQ to TAFIa after thrombin treatment was confirmed by WB analysis (Fig. 2A). At day 0, the intrinsic activity (without thrombin treatment) of N-HisTAFI-CIIYQ-Δ1–73 was comparable to the TAFIa activity (after thrombin treatment) of N-His-TAFIa-CIIYQ-Δ1–73 (51 3 U mg 1; 50 5 U mg 1; P > 0.05). At day 0, the intrinsic activity of TAFI-CIIYQ (without thrombin treatment) was 0.6 0.2 U mg 1, whereas the TAFIa activity of TAFIa-CIIYQ was 42 2 U mg 1. As illustrated in Fig. 2B, without thrombin treatment, N-HisTAFI-CIIYQ-Δ1–73 showed similar stability to that of TAFI-CIIYQ, with 75% residual intrinsic activity after incubation for 7 days at 37 °C. After thrombin treatment, N-His-TAFI-CIIYQ-Δ1–73 and TAFI-CIIYQ were activated to N-His-TAFIa-CIIYQ-Δ1–73 and TAFIa-CIIYQ, respectively. As expected, the activity of TAFIa-CIIYQ resulting from the activation of TAFI-CIIYQ decreased to 35% at day 2 and to almost 0% after 7 days, owing to heat inactivation at 37 °C (half-life, 33 1 h; mean SD, n = 4). NHis-TAFIa-CIIYQ-Δ1–73 resulting from the activation of NHis-TAFI-CIIYQ-Δ1–73 showed a similar time-dependent stability pattern; that is, the activity of thrombin-treated NHis-TAFI-CIIYQ-Δ1–73 dropped after incubation at 37 °C. However, thrombin-treated N-His-TAFI-CIIYQ-Δ1–73 showed slightly higher TAFIa stability (half-life, 40 2 h; mean SD, n = 4) and an apparent residual TAFIa activity of ~ 30% after incubation for 7 days. Also, this residual activity could be inhibited by PTCI. Overall, these data indicate that thrombin treatment of N-His-TAFI-CIIYQ-Δ1–73 resulted in cleavage of the Arg92–Ala93 peptide bond, leading to the formation of an ‘activated’ form that was less stable than the uncleaved deletion mutant. Thus, removal of the short linker region Ala74–Arg92 by thrombin results in a concomitant loss of stability. Therefore, these results suggest that the segment Ala74–Arg92 may also contribute to the role of the activation peptide in stabilization of the catalytic moiety in TAFI zymogen, apart from the hydrophobic interactions between Tyr341 and Leu35 and Val39, as hypothesized by Marx et al. [3]. The stability of TAFI zymogen may be attributed to a combined effect of the stability of the dynamic flap mediated by Leu35, Val39, and Tyr341, and the interactions between segment Ala74–Arg92 and the catalytic moiety. In conclusion, we generated a stable deletion mutant of TAFI exerting full TAFIa activity prior to cleavage of the Arg92–Ala93 bond. This deletion mutant can be cleaved by thrombin. However, the cleavage of N-His-TAFI-CIIYQΔ1–73 by thrombin was not accelerated by thrombomodulin. Strikingly, this deletion mutant does not behave as a zymogen, as the intrinsic activity is not further increased by cleavage. The availability of this stable, active TAFI deletion mutant may facilitate further elucidation of the TAFIa
structure and the mechanisms of TAFIa destabilization. Most importantly, it can be used as a potent biochemical tool to screen TAFIa inhibitors, and may pave the way for the development of new profibrinolytic drugs that target TAFI or TAFIa to prevent thrombosis. Addendum X. Zhou performed the research, analyzed and interpreted the data, performed statistical analysis, and wrote the manuscript. P. J. Declerck conceived and designed the study, coordinated the experiments, and reviewed the manuscript. Both authors read and approved the final manuscript.
Disclosure of Conflict of Interests The authors state that they have no conflict of interest.
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Active TAFI deletion mutant 1089 11 Valnickova Z, Thogersen IB, Potempa J, Enghild JJ. Thrombinactivable fibrinolysis inhibitor (TAFI) zymogen is an active carboxypeptidase. J Biol Chem 2007; 282: 3066–76. 12 Willemse JL, Heylen E, Hendriks DF. The intrinsic enzymatic activity of procarboxypeptidase U (TAFI) does not significantly influence the fibrinolysis rate: a rebuttal. J Thromb Haemost 2007; 5: 1334–6. 13 Foley JH, Kim P, Nesheim ME. Thrombin-activable fibrinolysis inhibitor zymogen does not play a significant role in the attenuation of fibrinolysis. J Biol Chem 2008; 283: 8863–7. 14 Ceresa E, Peeters M, Declerck PJ, Gils A. Announcing a TAFIa mutant with a 180-fold increased half-life and concomitantly a strongly increased antifibrinolytic potential. J Thromb Haemost 2007; 5: 418–20. 15 Mishra N, Vercauteren E, Develter J, Bammens R, Declerck PJ, Gils A. Identification and characterisation of monoclonal antibodies that impair the activation of human thrombin activatable fibrinolysis inhibitor through different mechanisms. Thromb Haemost 2011; 106: 90–101.
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16 Mosnier LO, von dem Borne PA, Meijers JC, Bouma BN. Plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation. Thromb Haemost 1998; 80: 829–35. 17 Guimaraes AH, Barrett-Bergshoeff MM, Gils A, Declerck PJ, Rijken DC. Migration of the activation peptide of thrombin-activatable fibrinolysis inhibitor (TAFI) during SDS-polyacrylamide gel electrophoresis. J Thromb Haemost 2004; 2: 780–4. 18 Plug T, Kramer G, Meijers JC. A role for arginine-12 in thrombin–thrombomodulin-mediated activation of thrombin-activatable fibrinolysis inhibitor. J Thromb Haemost 2014; 12: 1717–25. 19 Wu C, Kim PY, Manuel R, Seto M, Whitlow M, Nagashima M, Morser J, Gils A, Declerck P, Nesheim ME. The roles of selected arginine and lysine residues of TAFI (Pro-CPU) in its activation to TAFIa by the thrombin–thrombomodulin complex. J Biol Chem 2009; 284: 7059–67.
DOI: 10.1111/jth.12894
IN FOCUS
Generation of a stable thrombin-activatable fibrinolysis inhibitor deletion mutant exerting full carboxypeptidase activity without activation X . Z H O U and P . J . D E C L E R C K Department of Pharmaceutical and Pharmacological Sciences, Laboratory for Therapeutic and Diagnostic Antibodies, Katholieke Universiteit Leuven, Leuven, Belgium
To cite this article: Zhou X, Declerck PJ. Generation of a stable thrombin-activatable fibrinolysis inhibitor deletion mutant exerting full carboxypeptidase activity without activation. J Thromb Haemost 2015; 13: 1084–9. See also Plug T, Meijers JCM. New clues regarding the mysterious mechanism of activated thrombin-activatable fibrinolysis inhibitor selfdestruction. This issue, pp 1081–3.
Summary. Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a zymogen that can be activated to form activated TAFI (TAFIa) (Ala93–Val401) through removal of the N-terminal activation peptide (Phe1– Arg92). TAFIa is thermally unstable, and the role of the activation peptide in the activity and stability of TAFI zymogen remains unclear. Objectives: To better understand the role of the activation peptide in the activity and stability of TAFI. Methods: We constructed a deletion mutant, TAFI-CIIYQ-Δ1–73, in which the first 73 amino acids of the activation peptide are absent. The intrinsic activity and functional stability were determined with a chromogenic assay. The activation of TAFI-CIIYQ-Δ1–73 by TAFI activators was evaluated with western blot analysis. Results: In comparison with TAFI-CIIYQ, the deletion mutant exerted high intrinsic activity (‘full’ apparent TAFIa activity) without cleavage by TAFI activators. TAFI-CIIYQ-Δ1–73 was cleavable by thrombin. However, in the presence of thrombomodulin, the thrombin-mediated cleavage of TAFI-CIIYQ-Δ1–73 was not accelerated. TAFI-CIIYQ-Δ1–73 showed a similar functional stability profile to that of TAFI-CIIYQ. Full cleavage by thrombin did not affect the apparent carboxypeptidase activity of TAFI-CIIYQ-Δ1–73, but resulted in a significant loss of functional stability. Conclusions: A stable deletion mutant of TAFI with full carCorrespondence: Paul Declerck, Department of Pharmaceutical and Pharmacological Sciences, Laboratory for Therapeutic and Diagnostic Antibodies, Katholieke Universiteit Leuven, Campus Gasthuisberg, O&N2, PB 820, Herestraat 49, Leuven B-3000, Belgium. Tel.: +32 16 32 34 31; fax: +32 16 32 34 60. E-mail: [email protected] Received 7 May 2014 Manuscript handled by: T. Lisman Final decision: P. H. Reitsma, 24 February 2015
boxypeptidase activity without activation is described. The segment Ala74–Arg92 in the activation peptide contributes significantly to the role of the activation peptide in stabilization of the catalytic moiety in TAFI zymogen. Keywords: carboxypeptidase U; proteins; thrombin-activatable zymogens.
fibrinolysis; mutant fibrinolysis inhibitor;
Introduction Thrombin-activatable fibrinolysis inhibitor (TAFI) (proCPU, EC 3.4.17.20) is a human plasma procarboxypeptidase that acts as an important regulator between the coagulation and fibrinolytic systems [1,2]. TAFI zymogen (56 kDa) consists of a heavily glycosylated Nterminal activation peptide (Phe1–Arg92; Asn22, Asn51, Asn63, and Asn86; 20 kDa) and a catalytic moiety (Ala93–Val401; 36 kDa). Crystal structures of TAFI indicate that the activation peptide forms a globular structure (Phe1–Leu76) with a linker region (Ala76– Arg92), which connects it to the catalytic moiety [3]. Within the globular structure, Ala74–Asp75 forms a bturn (type VIII) connecting it to the linker region (Ala76–Arg92). In the zymogen form, the activation peptide covers the catalytic pocket and prevents substrate access to the preformed active cleft within the catalytic domain [3,4]. TAFI is activated to its active form, activated TAFI (TAFIa), through cleavage of the Arg92–Ala93 peptide bond, either by thrombin, by plasmin, or, most efficiently, by the thrombin–thrombomodulin complex [2,5,6]. TAFIa attenuates fibrinolysis by removing C-terminal lysines from partially degraded fibrin that mediate positive feedback in the fibrinolytic cascade [2]. To date, no physio© 2015 International Society on Thrombosis and Haemostasis
Active TAFI deletion mutant 1085
logic inhibitors of TAFIa have been found, and the development of TAFIa inhibitors is hindered by the intrinsic instability of TAFIa. TAFIa is thermally unstable (t1/2 of 8–15 min at 37 °C) [7], owing to an unspecified conformational change and inactivation [8]. The structural mechanisms underlying this spontaneous and rapid loss of activity remain unclear. It has been speculated that the dynamics of a highly mobile segment comprising residues Phe296–Trp350 (termed the ‘dynamic flap’) in the catalytic domain contribute to the intrinsic instability of TAFIa [3]. Marx et al. [3] hypothesized that, in the TAFI zymogen form, the activation peptide stabilizes the dynamic flap region through hydrophobic interactions between Tyr341 and Leu35 and Val39. However, after TAFI activation, it is unclear whether the activation peptide is released or remains attached to the catalytic domain, and the role of the activation peptide in the activity and stability of TAFIa remains controversial [4,9,10]. It has been reported that TAFI zymogen itself exerts intrinsic carboxypeptidase activity, ranging from 5.6% [11] to less than 1% [9] as compared with TAFIa. Whether the intrinsic activity of TAFI plays a physiologic role in the regulation of fibrinolysis remains controversial [11–13]. A TAFI variant, TAFI-S305C-T325I-T329I-H333YH335Q (TAFI-CIIYQ), containing stabilizing mutations in the dynamic flap region, has been reported previously [14]. Its activated form, TAFIa-CIIYQ, has been reported to be a stable TAFIa variant with a 180-fold increased half-life (1140 66 min, 37 °C). In the current study, a deletion mutant of TAFI-CIIYQ, TAFI-CIIYQ-Δ1–73, in which the first 73 amino acids (which form the globular structure) of the activation peptide are lacking, is described. The activity, functional stability and activation of this deletion mutant by thrombin and thrombin– thrombomodulin were determined and compared with those of TAFI-CIIYQ. Materials and methods Construction, expression and purification of TAFI-CIIYQ-Δ1–73 and N-His-TAFI-CIIYQ-Δ1–73
The TAFI-CIIYQ-Δ1–73-coding sequence was generated by overlap extension PCR, based on the full-length cDNA of TAFI-CIIYQ in pcDNA5/FRT, yielding pcDNA/FRT-TAFI-CIIYQ-Δ1–73. The construct was transiently transfected into HEK293T cells. For efficient purification of TAFI-CIIYQ-Δ1–73 from the conditioned medium, an N-terminal hexahistidine tag was inserted in frame in the pcDNA/FRT-TAFI-CIIYQ-Δ1–73 construct. pcDNA/FRT-N-His-TAFI-CIIYQ-Δ1–73 was transiently transfected into HEK293T cells, and N-His-TAFI-CIIYQ-Δ1–73 was purified by standard immobilized metal affinity chromatography.
© 2015 International Society on Thrombosis and Haemostasis
Evaluation of TAFI-CIIYQ-Δ1–73 and N-His-TAFI-CIIYQ-Δ1–73 antigen levels
Expression and secretion of TAFI-CIIYQ-Δ1–73 in the conditioned medium were evaluated by western blot (WB) analysis, with MA-TCK27A4 [15] as the primary antibody, and goat anti-mouse–horseradish peroxidase (HRP) as secondary antibody, followed by electrochemiluminescence (ECL) staining. Antigen levels of TAFICIIYQ-Δ1–73 in the conditioned medium or of purified N-His-TAFI-CIIYQ-Δ1–73 were quantified with in-house ELISA (MA-TCK27A4 for capture and MA-TCK28F8– HRP for detection). Analysis of the intrinsic activities of TAFI-CIIYQ and N-HisTAFI-CIIYQ-Δ1–73
The intrinsic activities of TAFI-CIIYQ and N-HisTAFI-CIIYQ-Δ1–73 were determined with a chromogenic assay, as described previously, with minor modifications [9,16]. Briefly, TAFI-CIIYQ (5.4 nM during substrate conversion) or N-His-TAFI-CIIYQ-Δ1–73 (3.0 nM during substrate conversion) was incubated with the synthetic substrate Hip-Arg (11.1 mM during substrate conversion). Substrate conversion was allowed to proceed for 1 h at 22 °C. Reactions were stopped, 6% cyanuric chloride (dissolved in 1,4-dioxane) was added, and the mixtures were vortexed and centrifuged (16 100 9 g, 2 min). The absorbance of the supernatant was measured and the intrinsic activity was calculated as described previously [9]. One unit (U) of intrinsic activity or carboxypeptidase activity is defined as the amount of enzyme converting 1 lmol min–1 at 22 °C. Evaluation of TAFI-CIIYQ and N-His-TAFI-CIIYQ-Δ1–73 fragmentation patterns generated upon treatment with thrombin or thrombin–thrombomodulin
Fragments of TAFI-CIIYQ or N-His-TAFI-CIIYQ-Δ1–73 upon treatment with thrombin or thrombin–thrombomodulin were evaluated with WB analysis, as described previously [9], with the following modifications. TAFI-CIIYQ (8.9 nM during activation) or N-His-TAFI-CIIYQΔ1–73 (5.0 nM) was treated with thrombin (100 nM) or thrombin–thrombomodulin (thrombin, 100 nM; thrombomodulin, 100 nM) and CaCl2 (5 mM) in 60 lL of HEPES buffer (25 mM HEPES, 137 mM NaCl, 3.5 mM KCl, pH 7.4, supplemented with 0.1% bovine serum albumin) at 37 °C for 5 min, 10 min, or 30 min. The reactions were stopped at the indicated time points by addition of SDS (1% final concentration). The samples were heated at 100 °C for 30 s, and subjected to SDS-PAGE (10–15% gradient), WB analysis with MA-T3D8 [17] as primary antibody, and goat anti-mouse–HRP as secondary antibody, and ECL staining.
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are expressed relative to the initial activity observed at day 0 prior to incubation at 37 °C.
Analysis of TAFIa activity upon thrombin treatment
Upon full cleavage by thrombin (100 nM, 37 °C for 4 h), the TAFIa activities of TAFIa-CIIYQ (8.9 nM during activation) and N-His-TAFIa-CIIYQ-Δ1–73 (5.0 nM during activation) were determined as described previously, with minor changes [9]. Substrate conversion was allowed to proceed for 1 h at 22 °C.
Results and conclusions The construct containing the TAFI-CIIYQ-Δ1–73-coding sequence was transiently transfected into HEK293T cells. Conditioned medium was collected 96 h post-transfection. WB analysis revealed a clear band at 40 kDa, representing TAFI-CIIYQ-Δ1–73 in the conditioned medium. According to ELISA, TAFI-CIIYQ-Δ1–73 was present at a concentration of 180 30 ng mL 1 (mean standard deviation [SD]; n = 3) in the conditioned medium. These levels are 10-fold lower than those found for TAFI-CIIYQ when the corresponding DNA construct was used. For efficient purification of TAFI-CIIYQ-Δ1–73 from the conditioned medium, an N-terminal hexahistidine tag was inserted in frame in the pcDNA/FRT-TAFI-CIIYQ-Δ1–73 construct. TAFI antigen levels of purified N-His-TAFI-CIIYQ-Δ1–73 were 400 ng mL 1 as determined with ELISA. Purified N-His-TAFI-CIIYQ-Δ1–73 showed high intrinsic activity; that is, at 22 °C, the intrinsic activity of N-HisTAFI-CIIYQ-Δ1–73 (54 2 U mg 1, mean SD, n = 4) was ~ 90-fold higher than that of purified TAFI-CIIYQ (0.6 0.2 U mg 1, mean SD, n = 4). The intrinsic activ-
Determination of the functional stability of TAFIa-CIIYQ and N-His-TAFIa-CIIYQ-Δ1–73
The functional stability of N-His-TAFI-CIIYQ-Δ1–73 and TAFI-CIIYQ, before and after thrombin treatment (100 nM, 37 °C for 4 h), was measured as described previously, with some modifications [9]. N-His-TAFI-CIIYQΔ1–73 and TAFI-CIIYQ, either as such or after thrombin treatment (4 h at 37 °C, followed by incubation with Dchloromethylketone), phenylalanyl-L-prolyl-L-arginine were incubated at 37 °C for 7 days. At each time point indicated in Fig. 2B, aliquots were placed on ice until measurement of residual intrinsic activity (i.e. without thrombin pretreatment; substrate conversion, 1 h at 22 °C) or residual TAFIa activity (i.e. with thrombin pretreatment; substrate conversion, 1 h at 22 °C). All values
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Fig. 1. Western blot (WB) analysis of fragmentation patterns of TAFI-CIIYQ and N-His-TAFI-CIIYQ-Δ1–73 upon thrombin and thrombin–thrombomodulin treatment. Upon cleavage for 5 min, 10 min, or 30 min, samples were subjected to SDS-PAGE, followed by WB analysis with MA-T3D8 as primary antibody. (A) TAFI-CIIYQ (8.9 nM, lane 1) was fully activated to TAFIa after thrombin–thrombomodulin treatment for 5 min (lane 3). The cleavage was not complete after thrombin treatment for 30 min (lane 6). (B) N-His-TAFI-CIIYQ-Δ1–73 (5 nM, lane 1) was cleaved to a similar extent, after both thrombin–thrombomodulin and thrombin treatment for 5 min (lanes 2 and 3), 10 min (lanes 4 and 5), or 30 min (lanes 6 and 7). Intact TAFI-CIIYQ (56 kDa) is indicated by the arrow, N-His-TAFI-CIIYQ-Δ1–73 (40 kDa) by the arrowhead, and activated TAFI-CIIYQ (TAFIa-CIIYQ) by the dashed arrow. TAFI, thrombin-activatable fibrinolysis inhibitor; TAFIa, activated thrombin-activatable fibrinolysis inhibitor; T, thrombin; T/M, thrombin–thrombomodulin. © 2015 International Society on Thrombosis and Haemostasis
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ity of this deletion mutant could be fully inhibited by potato tuber carboxypeptidase inhibitor (PTCI) (data not shown). WB analysis of the fragmentation patterns of N-HisTAFI-CIIYQ-Δ1–73 after thrombin or thrombin–thrombomodulin treatment revealed that the deletion mutant could be cleaved by either thrombin or thrombin–thrombomodulin (Fig. 1B; the primary antibody, MA-T3D8, reacted specifically with the catalytic moiety of TAFI [17]). Full cleavage by thrombin–thrombomodulin or thrombin was not achieved even after treatment for 30 min at 37 °C (Fig. 1B; lanes 6 and 7). In contrast to the activation of TAFI-CIIYQ by thrombin–thrombomodulin and thrombin (Fig. 1A, lanes 2–7), the presence of thrombomodulin did not accelerate the cleavage of NHis-TAFI-CIIYQ-Δ1–73 by thrombin (Fig. 1B, lanes 2–7). These results are in line with the data reported by Plug
et al. [18] and Wu et al. [19], demonstrating that several positively charged residues (Arg12 [18] and Lys42–Lys44 [19]) within the globular structure of the activation peptide might be important for the activation of TAFI by thrombin–thrombomodulin. Indeed, lack of the globular structure in the deletion mutant prevents efficient interaction with thrombomodulin, thereby abolishing the cofactor function. It is notable that, after cleavage of N-His-TAFI-CIIYQ-Δ1–73, two bands (~ 36 kDa) clearly appeared (Fig. 1B, lanes 2–7) on SDS-PAGE. When reducing reagent was applied prior to gel electrophoresis, this double-band migration pattern disappeared, and activated N-His-TAFI-CIIYQ-Δ1–73 migrated as a single band (data not shown), which may suggest that the deletion mutant also exists with partially oxidized cysteines (i.e. fewer than three disulfide bonds are formed). N-His-TAFICIIYQ-Δ1–73
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Fig. 2. Determination of the functional stability before and after cleavage by thrombin. (A) Evaluation of the cleavage levels of TAFI-CIIYQ and N-His-TAFI-CIIYQ-Δ1–73 upon cleavage by thrombin, prior to incubation at 37 °C. Western blot analysis (with MA-T3D8 as primary antibody) showed that TAFI-CIIYQ and N-His-TAFI-CIIYQ-Δ1–73 (lanes 1 and 3, respectively) were completely cleaved after thrombin treatment (lanes 2 and 4, respectively). Intact TAFI-CIIYQ (56 kDa) is indicated by the arrow, N-His-TAFI-CIIYQ-Δ1–73 (40 kDa) by the arrowhead, and TAFIa-CIIYQ by the dashed arrow. (B) Functional stability (37 °C) of N-His-TAFI-CIIYQ-Δ1–73 (■, □) and of TAFI-CIIYQ (●, ○) before (■, ●) and after (□, ○) thrombin treatment. All values are expressed relative to the intrinsic or TAFIa activity observed at day 0 prior to incubation at 37 °C. At day 0, without thrombin treatment, the intrinsic activity of TAFI-CIIYQ without thrombin treatment was 0.6 0.2 U mg 1, whereas the intrinsic activity of N-His-TAFI-CIIYQ-Δ1–73 was ~ 85-fold higher, i.e. 51 3 U mg 1. After thrombin treatment, the TAFIa activity of TAFI-CIIYQ was 42 2 U mg 1, whereas that of N-His-TAFI-CIIYQ-Δ1–73 was 50 5 U mg 1. Data are presented as mean standard deviation, n = 4. TAFI, thrombin-activatable fibrinolysis inhibitor; TAFIa, activated thrombin-activatable fibrinolysis inhibitor; T, thrombin. © 2015 International Society on Thrombosis and Haemostasis
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The functional stability of N-His-TAFI-CIIYQ-Δ1–73 before and after thrombin treatment was measured and compared with that of TAFI-CIIYQ before and after thrombin treatment. Full cleavage of N-His-TAFI-CIIYQΔ1–73 and TAFI-CIIYQ to TAFIa after thrombin treatment was confirmed by WB analysis (Fig. 2A). At day 0, the intrinsic activity (without thrombin treatment) of N-HisTAFI-CIIYQ-Δ1–73 was comparable to the TAFIa activity (after thrombin treatment) of N-His-TAFIa-CIIYQ-Δ1–73 (51 3 U mg 1; 50 5 U mg 1; P > 0.05). At day 0, the intrinsic activity of TAFI-CIIYQ (without thrombin treatment) was 0.6 0.2 U mg 1, whereas the TAFIa activity of TAFIa-CIIYQ was 42 2 U mg 1. As illustrated in Fig. 2B, without thrombin treatment, N-HisTAFI-CIIYQ-Δ1–73 showed similar stability to that of TAFI-CIIYQ, with 75% residual intrinsic activity after incubation for 7 days at 37 °C. After thrombin treatment, N-His-TAFI-CIIYQ-Δ1–73 and TAFI-CIIYQ were activated to N-His-TAFIa-CIIYQ-Δ1–73 and TAFIa-CIIYQ, respectively. As expected, the activity of TAFIa-CIIYQ resulting from the activation of TAFI-CIIYQ decreased to 35% at day 2 and to almost 0% after 7 days, owing to heat inactivation at 37 °C (half-life, 33 1 h; mean SD, n = 4). NHis-TAFIa-CIIYQ-Δ1–73 resulting from the activation of NHis-TAFI-CIIYQ-Δ1–73 showed a similar time-dependent stability pattern; that is, the activity of thrombin-treated NHis-TAFI-CIIYQ-Δ1–73 dropped after incubation at 37 °C. However, thrombin-treated N-His-TAFI-CIIYQ-Δ1–73 showed slightly higher TAFIa stability (half-life, 40 2 h; mean SD, n = 4) and an apparent residual TAFIa activity of ~ 30% after incubation for 7 days. Also, this residual activity could be inhibited by PTCI. Overall, these data indicate that thrombin treatment of N-His-TAFI-CIIYQ-Δ1–73 resulted in cleavage of the Arg92–Ala93 peptide bond, leading to the formation of an ‘activated’ form that was less stable than the uncleaved deletion mutant. Thus, removal of the short linker region Ala74–Arg92 by thrombin results in a concomitant loss of stability. Therefore, these results suggest that the segment Ala74–Arg92 may also contribute to the role of the activation peptide in stabilization of the catalytic moiety in TAFI zymogen, apart from the hydrophobic interactions between Tyr341 and Leu35 and Val39, as hypothesized by Marx et al. [3]. The stability of TAFI zymogen may be attributed to a combined effect of the stability of the dynamic flap mediated by Leu35, Val39, and Tyr341, and the interactions between segment Ala74–Arg92 and the catalytic moiety. In conclusion, we generated a stable deletion mutant of TAFI exerting full TAFIa activity prior to cleavage of the Arg92–Ala93 bond. This deletion mutant can be cleaved by thrombin. However, the cleavage of N-His-TAFI-CIIYQΔ1–73 by thrombin was not accelerated by thrombomodulin. Strikingly, this deletion mutant does not behave as a zymogen, as the intrinsic activity is not further increased by cleavage. The availability of this stable, active TAFI deletion mutant may facilitate further elucidation of the TAFIa
structure and the mechanisms of TAFIa destabilization. Most importantly, it can be used as a potent biochemical tool to screen TAFIa inhibitors, and may pave the way for the development of new profibrinolytic drugs that target TAFI or TAFIa to prevent thrombosis. Addendum X. Zhou performed the research, analyzed and interpreted the data, performed statistical analysis, and wrote the manuscript. P. J. Declerck conceived and designed the study, coordinated the experiments, and reviewed the manuscript. Both authors read and approved the final manuscript.
Disclosure of Conflict of Interests The authors state that they have no conflict of interest.
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