Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI)

Primary structure of bovine TAFI

The amino acid sequence of bovine TAFI was deduced from a cDNA library and published recently [45]. The sequence was 78.6% identical to that of the human protein. The bovine protein consisted of 401 amino acid residues, including a 92-amino acid residue pro-peptide that is released by cleavage at Arg⁹². All potential glycosylation sites were conserved and found glycosylated in both species, with exception of the fifth site (Asn²¹⁹), which remained unglycosylated in bovine TAFI. The location of cysteine residues was identical in both species, with the exception of Cys⁶⁹. This cysteine residue, which is located in the activation peptide, was absent from bovine TAFI. In human TAFI, Cys⁶⁹ does not form a disulfide bridge and therefore, is unlikely to affect tertiary structure. All sites involved in catalysis as well as substrate and zinc binding were identical, suggesting that the two proteins have the same proteolytic properties.

Generation and activity of TAFIa

SDS-PAGE of purified bovine TAFI produced a single sharp band at around 56 kDa, which is slightly lower than the position of human TAFI (Fig 1). TAFIa generated by either trypsin (Fig 1A) or thrombin/solulin complex (Fig 1B) migrated at the same position for both species, suggesting that differences in the migration of the full-length species can be attributed to differences in carbohydrates attached to the pro-peptide. It is obvious from the results of the SDS-PAGE that greater amounts of proteinases were required to generate human TAFIa (Fig 1). Since the thrombin/solulin complex is considered to be responsible for release of the TAFI pro-peptide in vivo [14] and since trypsin seems to inactivate TAFIa more aggressively, only the complex was used to determine the optimal conditions for generation of bovine and human TAFIa. Increasing amounts of the thrombin/solulin were incubated with TAFI, and the enhanced activity was monitored by HPLC based activity assay. As shown in Fig 1C, the amount of proteinase complex required to achieve 100% TAFIa activity was much lower than that needed for the human TAFI. Furthermore, the kinetic constants for both species were determined (Table 1). The intrinsic activity of TAFI is similar between species, while the Vmax and Km for bovine TAFa is somewhat higher in comparison to the human protein.

Identification of proteolysis products generated upon proteinase addition to TAFI

The thrombin/solulin complex produced similar proteolytic fragmentation in both the bovine and human TAFI. The generated products were identified by Edman degradation and are summarized in Table 2. SDS-PAGE of bovine TAFI and thrombin/solulin mixture fashioned a strong band not only at 56 kDa (corresponding to full length TAFI), but also at 36 kDa, which was confirmed to be TAFIa (Fig 2 and Table 2). In contrast to the human TAFI pro-peptide, the released bovine pro-peptide was clearly visible by Coomassie staining, with a mass of around 29 kDa (Fig 2). As expected, large amounts of thrombin/solulin complex truncated human TAFIa (36 kDa) at Arg³⁰², liberating the 11.0 kDa C-terminal peptide to produce a proteolytically inactivated form of TAFIa (24.7 kDa) (Fig 1B). However, no further proteolytic products were observed for the bovine protein in the higher end of the titration using the proteinase complex.

When TAFI was cleaved by trypsin, which is more potent than thrombin/solulin complex, a somewhat dissimilar fragmentation pattern was generated (Fig 2). SDS-PAGE of the human protein yielded a typical pattern consisting of full length TAFI at 58 kDa, mature TAFIa at 36 kDa, C-terminal processed TAFIa (through cleavage at Arg³³⁰) at 28 kDa, and the released C-terminal peptide at 8 kDa (Fig 2). Trypsin cleavage occurred at the same site in both the human and bovine protein, creating a 36 kDa TAFIa through truncation at Arg⁹² (Fig 2). Interestingly, in contrast to human TAFIa, bovine TAFIa was initially proteolytically inactivated by cleavage at the N-terminus, rather than the C-terminus. This processing occurred right after Arg¹⁴⁷, creating a 29.2 kDa fragment, detected around 32 kDa on SDS gel (Fig 2 and Table 2). The pro-peptide was detected at around 29 kDa (Fig 2). However, it is most likely immediately processed at the C-terminus, as a 25 kDa band was detected that also contained the TAFI N-terminal sequence (Fig 2). Trypsin and small trypsin fragments were also detected, along with the released 6.7 kDa TAFIa N-terminal peptide (Fig 2 and Table 2).

Bovine TAFIa stability at 37°C

The thermal stability of TAFIa was investigated with HPLC based kinetic assays using Hip-Arg as a substrate and the thrombin/solulin complex as an activator (Fig 3). The half-life of bovine TAFIa was longer than that of human TAFIa. Human TAFIa activity decreased by 50% after 5 min, while this decrease in bovine TAFIa activity occurred at 10 min (Fig 3).

Intrinsic activity of bovine TAFI

The intrinsic activity of the bovine protein was determined by activity assays using the Hip-Arg substrate and compared to that of human TAFI, by measuring the released hippuric acid by HPLC. TAFI activity was undoubtedly detected using this method (Fig 4A). Moreover, hippuric acid release was blocked when TAFI was incubated with the substrate in the presence of 5 mM 1, 10-phenantroline, a chelating agent and known carboxypeptidase inhibitor (Fig 4B). This confirms that bovine TAFI has genuine intrinsic activity. This activity was also inhibited by 2.3 μM TCI, a potentially physiologically relevant inhibitor of TAFI (Fig 4C).

Isoelectric point variation between TAFI and TAFIa

Isoelectric focusing revealed that the isoelectric point of TAFI and TAFIa varied greatly in both species (data not shown). The full length bovine protein migrated in the lower end of the pH gradient and appeared as multiple bands between a pI of 6.0 and 6.5, likely due to glycosylation heterogeneity. This migration position was slightly higher than that of the human TAFI, which migrated to a pI of 5.1 to 6.0 and also appeared as multiple isoforms. Upon release of the heavily glycosylated activation peptide, both human and bovine TAFIa appeared as a single band at a much higher pI of around 8.5. Thus, the difference in migration between TAFI in the two species is due to differences in the carbohydrate modifications to their respective pro-peptides.

Bovine TAFI attenuates clot lysis in vitro

The effect of bovine TAFI on fibrinolysis was examined by conducting a fibrinolyses assay in a purified system (Fig 5). The clot was generated in microtiter wells by addition of thrombin to fibrinogen and the clot lysis initiated by further addition of plasminogen and tPA simultaneously. Purified bovine or human TAFI (1 μg) added to the wells, in the presence and absence of solulin, was able to delay clot lysis. Moreover, this effect was reversed by the carboxypeptidase inhibitors, PCI or TCI, confirming capability of bovine (and human) TAFI to effect clot lysis (data not shown).

Bovine TAFI is a substrate for tissue transglutaminase

To test whether bovine TAFI has the potential to become cross-linked to a fibrin meshwork in the same manner as the human protein, we monitored the incorporation of a fluorescent donor, dansylcadaverine, into the protein by tissue transglutaminase. Visualization of SDS-polyacrylamide gels with UV light revealed that both human TAFI and α-2AP (another known tissue transglutaminase substrate) incorporated dansylcadaverine (Fig 6). Importantly, dansylcadaverine was also successfully incorporated into bovine TAFI under these conditions, showing that bovine TAFI can serve as a transglutaminase substrate.

Bovine TAFI contains four N-linked carbohydrate structures, which are located solely on the activation peptide

To characterize the glycans of bovine TAFI, we performed tryptic digests with and without subsequent PNGase F treatment, and separated the resulting fragments using RP-HPLC. Fractions containing glycopeptides were purified and analyzed using MALDI-TOF MS and subsequently verified based on their fragmentation using MALDI quadrupole (Q) TOF MS/MS (data not shown). Thirty-five N-glycans were observed from the four N-glycosylation sites present within the N-terminal activation region (i.e., N22, N51, N63, and N86) (Fig 7 and Table 3). Biantennary structures without core fucosylations were the sole structures identified by the glycoanalysis. Hence, the substantial microheterogeneity observed was limited to variations in the contents of the two types of sialic acids, N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Up to four sialic acid residues were observed on the N22 glycans, whereas the rest of the occupied sequons contained a maximum of three sialic acid residues (Fig 7). Peptide mass fingerprinting of tryptic bovine TAFI treated with and without PNGase F revealed that neither of the two potential sites located in the middle of the protein (N219 and N226) was occupied by N-glycans. In contrast, N-linked glycosylation has been detected in human TAFIa. The human protein also exhibits a much greater heterogeneity in the N-linked sugars [18]. This may help account for difficulties in determining the three-dimensional structure of human TAFI zymogen.

Materials

Bovine trypsin, 1, 10-phenantroline, phenylmethylsulfonyl fluoride (PMSF), polyethylene glycol 8000 (PEG), and the chromogenic carboxypeptidase substrate, hippuryl-Arg (Hip-Arg), were obtained from Sigma. Ortho-methylhippuric acid and Pefablock SC were from Aldrich. ECH-Lysine Sepharose was from Amersham Biosciences, GE Healthcare (Uppslala, Sweden). Dansylcadaverine was from Molecular Probes (Eugene, OR).

Proteins

Human TAFI and human α₂-antiplasmin (α-₂AP) were purified from normal human plasma (Statens Serum, Institute, Copenhagen, Denmark using plasminogen-depleted plasma and plasminogen-Sepharose affinity chromatography as described previously [4, 18]. Guinea pig liver (tissue) transglutaminase (EC 2.3.2.13), human fibrinogen and human thrombin (EC 3.4.21.5) were purchased from Sigma. Recombinanat tPA (EC 3.4.21.68) was purchased from ProSpec-Tany TechnoGene LTD., Rehovot, Israel. Recombinant soluble thrombomodulin (solulin) was a generous gift of Dr. Achim Schuettler (PAION GmbH, Aachen, Germany) and Factor XIIIa from Sanofi-Aventis. Potato carboxypeptidase inhibitor (PCI) and TCI were kind gifts from Prof. Francesc. X. Aviles, Dr. Joan Lopez Arolas, and Dr. Laura Sanglas. TAFI-antiserum was raised commercially (Pel-Freez, Rogers, AR). Human plasminogen was purified by affinity chromatography using ECH-Lysine Sepharose as described previously [53].

Purification of bovine TAFI

Bovine TAFI was purified essentially as already described [45]. In short, bovine blood (10 L) was collected at the local slaughterhouse and supplemented with 5 mM EDTA to prevent coagulation. The plasma was separated from erythrocytes by centrifugation at 600 × g for 15 min at 22°C. Plasma was incubated with 6% (w/v) PEG, and after 1 h, the precipitated proteins were removed by centrifugation at 10,000 × g for 40 min at 4°C. Plasminogen was removed from the supernatant by affinity chromatography using 1 L of ECH-Lysine Sepharose equilibrated in binding buffer (50 mM NaH₂PO₄, pH 7.5 and 100 mM NaCl). Plasminogen-depleted plasma was applied to a 500-ml plasminogen Sepharose column equilibrated in binding buffer, and bovine TAFI was eluted using 50 mM γ-amino-caproic acid. After buffer exchange into 20 mM Tris-Cl (pH 7.5), bovine TAFI was separated from other contaminants by ion-exchange chromatography on a 5-ml HiTrapQ column connected to an AKTA Prime system (Amersham Biosciences, GE Healthcare). The column was eluted, at a flow rate of 1 ml/min, using a 0.5%/min linear gradient of Buffer A (20 mM Tris-Cl, pH 7.5) and Buffer B (20 mM Tris-Cl, pH 7.5 containing 1 M NaCl).

Polyacrylamide gel electrophoresis

Proteins were separated by SDS-PAGE in 5 – 15% polyacrylamide gels [54]. Samples were boiled for 5 min in the presence of 30 mM dithiothreitol (DTT) and 1% SDS prior to electrophoresis.

Generation of human and bovine TAFIa

Human and bovine TAFI (1 μg) were incubated with increasing amounts of the thrombin/solulin complex, (0 μg/0 μg to 0.01 μg/0.25 μg) for 30 min at 22°C in 20 mM Tris-HCl and 100 mM NaCl, pH 7.5. For trypsin induced proteolysis, 1 μg TAFI (human and bovine) was incubated with increasing amounts of trypsin (0–0.5 μg) for 20 min at 37°C in 20 mM Tris-HCl and 100 mM NaCl, pH 7.5. All reactions were terminated by addition of Pefablock or PMSF to a final concentration of 5 mM. Optimal conditions to generate TAFIa with peak activity were determined through kinetic assays (using 0.2 μg of TAFI) and SDS-PAGE (using 1.0 μg TAFI) with the physiologically relevant thrombin/solulin complex as an activator only. To activate 1 μg of human TAFI, the optimal trombin/solulin complex ratio (w/w) was 0.06 μg/1.5 μg. Generation of bovine TAFIa was optimal using thrombin/solulin complex ratio of 0.004 μg/0.1 μg to 1 μg TAFI.

NH₂-terminal amino acid sequencing

Proteolytic fragments of bovine TAFI generated by trypsin or solulin/thrombin complex were separated by SDS/PAGE. The stacking gel was allowed to polymerize one day prior to electrophoresis, and samples were heated for 3 min at only 80°C prior to separation. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore) in 10 mM CAPS and 10% (v/v) methanol (pH 11) [55]. Alternatively, the TAFI-trypsin or TAFI-solulin/thrombin mixture was applied to an activated ProSorb sample preparation cartridge (Applied Biosystems), according to the manufacturer's instructions. Samples were analyzed by automated Edman degradation using an Applied Biosystems PROCISE™ 494 HT sequencer with on-line HPLC (Applied Biosystems Model 120A) for phenylthiohydantoin analysis.

Isoelectric focusing

Isoelectric focusing of bovine TAFI was performed essentially as described previously [18]. Briefly, 10 μg of salt-free protein was focused under native conditions in a Ready IEF gel using the MiniProtean III Cell (Biorad) according to the manufacturer's instructions. Bands were focused in a pH gradient of 3 – 10 using 20 mM lysine and 20 mM arginine as a cathode buffer and 7% phosphoric acid as anode buffer (all in H₂O). Running conditions consisted of 100 V for 60 min, 250 V for 60 min, and 500 V for 30 min. Bands were visualized using IEF staining solution (27% isopropyl alcohol, 10% acetic acid, 0.04% Coomassie Blue R250, and 0.05% Crocein Scarlet in H₂O).

HPLC based kinetic activity assay using Hip-Arg substrate

The activity of both full length and mature TAFI was determined essentially as described previously [56]. A 10-μl sample containing 1 μg of bovine TAFI or 0.2 μg TAFIa was incubated with 40 μl of 30 mM Hip-Arg for 40 min. Some samples were incubated for 15 min with 5 mM phenanthroline or 1 μg TCI prior to substrate addition. The reactions were stopped by addition of 50 μl 1 M HCl. Ten microliters of 15 mM ortho-methylhippuric acid was included in the reaction mixture as an internal standard. The reaction products, as well as the internal standard, were extracted using 300 μl ethyl acetate. One-hundred microliters of the extracted sample were lyophilized, solubilized in 100 μl mobile phase buffer [10 mM KH₂PO₄, pH 3.4 containing 15% acetonitrile (ACN)], and separated on a reverse phase (RP) HPLC column (PTH C18, 5 μm, 220 × 2.1 mm, Applied Biosystems) using the ÄKTA Ettan system (Amersham Biosciences, GE Healthcare).

Determination of TAFI kinetic constants

Human and bovine TAFI kinetic properties were essentially determined as described previously, with small modifications [46]. Briefly, 1 μg of the zymogen and 0.1 μg of TAFIa, generated by the thrombin/solulin complex, for both human and bovine protein, were incubated with increasing concentration of the Hip-Arg substrate (0–30 mM), in duplicates, for 60 min at 37°C in a final volume of 60 μl. The reaction was terminated by addition of 20 μl 1 M HCl, neutralized by addition of 20 μl of 1 M NaOH and buffered with 25 μl of 1 M NaH₂PO₄, pH 7.4. Upon addition of 60 μl 6% cyanuric chloride dissolved in 1,4-dioxane, the samples were vortexed vigorously and centrifuged at 16000 × g for 5 minutes. The supernatant was subsequently transferred to 96-well microtiter plate and the absorbance was measured at 405 nm in a FLUOStar Omega plate reader (BMG Labtech) using the endpoint mode. The kinetic constants were determined using 4 different graphical methods.

Thermal stability of TAFIa enzymatic activity

Bovine and human TAFI (3 μg) were mixed with solulin/thrombin complex using the optimal conditions for generation of TAFIa for each species. At the time of reaction termination with pefablock (5 mM final concentration), the reaction mixture was placed at 37°C. At various intervals over 120 min, 0.2 μg of TAFI protein was removed and incubated with Hip-Arg substrate. Kinetic measurements were then performed using HPLC method described above.

In vitro clot lysis assays

Clot lysis assays were performed essentially as described previously [57] using 96-well microtiter plates, with some modifications. Twenty μl of fibrinogen (20 μl/μg), 1 μl of plasminogen (0.5 μg/μl) and 12.5 μl of factor XIIIa (0.8 μg/μl) were mixed in a final volume of 100 μl in 20 mM Hepes and 150 mM NaCl, 5 mM CaCl₂, pH 7.4 (reaction buffer) in a set of wells. In a proximate set of wells, 1 μl of tPA (0.002 μg/μl) and 2 μl of thrombin (20 U/ml) were combined in a final volume of 50 μl using the reaction buffer. Clotting was initiated by addition of 50 μl of the fibrinogen/plasminogen/factor XIIIa mixture to wells containing tPA and thrombin. In some wells, 10 μl of solulin (0.1 μg/μl) was added to the tPA/thrombin mixture prior to clot initiation. Purified human or bovine TAFI (1 μg), was added to certain wells containing tPA, thrombin and (+/-) solulin, moments prior to the start of the clotting generation. Some wells contained additionally 1.27 μM TCI or 4.65 μM PCI. The turbidity of the clot was measured continuously at 405 nm in a plate reader (FLUOstar Omega, BMG LABTECH GmbH) at 37°C. The lysis time was defined as the time required for a 50% reduction in optical density.

Incorporation of dansylcadaverine using tissue transglutaminase

Human TAFI, bovine TAFI, or α-2AP (2 μg of each) were incubated with varying amounts of tissue transglutaminase (0 – 2 μg) for 3 h at 37°C in 20 mM Tris-Cl and 100 mM NaCl (pH 7.5) containing 10 mM Ca²⁺, 0.5 mM DTT, and 0.5 mM dansylcadaverine. The reaction was stopped by addition of 10 mM EDTA, and samples were analyzed by reducing SDS-PAGE. The gel was visualized under UV light.

Amino acid sequence analysis

To determine the accurate concentration of TAFI used in this study, we performed each analysis in triplicate. For each analysis, approximately 2 μg of purified bovine or human TAFI was dried in 500 μl polypropylene vials. The lids were punctured, and the vials were placed in a 25-ml glass vial equipped with a MinInert valve (Pierce Biotechnology, Rockford, IL, USA). Two-hundred microliters of 6 N HCl containing 0.1% phenol was placed in the bottom of the glass and blown with argon before a vacuum was applied. The samples were incubated at 110°C for 18 h. They were subsequently redissolved in 50 μl 0.20 M sodium citrate loading buffer, pH 2.20 (Biochrom, Cambridge, UK), transferred to microvials, and loaded on a BioChrom 30 amino acid analyzer (Biochrom). Data analysis was performed using software developed in house.

Proteolytic digestion of bovine TAFI and purification of glycosylated peptides

Modified trypsin (2 μg, Promega, Madison, WI) was added to approximately 40 μg of purified bovine TAFI in 20 mM Tris and 200 mM NaCl (pH 7.5) and then incubated overnight at 37°C. The resulting peptide mixture was split into two samples. N-glycosidase F (1 U, Roche, Mannheim, Germany) was added to one of the samples and incubated overnight at 37°C. The other sample was stored at -18°C. The two samples were applied separately to a reversed phase HPLC column (Jupiter C18 250 mm × 2 mm, 5 μm, 300 Å, Phenomenex, Torrance, CA) connected to an ÄKTA Basic instrument (Amersham Pharmacia Biotech, GE, Uppsala, Sweden). The sample was applied in buffer A [0.06% trifluoroacetic acid (TFA) in water] and eluted using the following three-step gradient in buffer B (0.05% TFA and 90% ACN in water): 5 to 40% in 30 min, 40 to 60% in 5 min, and 60 to 90% in 3 min. Differences in the corresponding chromatograms revealed the fractions potentially containing glycopeptides. These fractions were dried and redissolved in 5% formic acid for further analysis.

Characterization of glycosylated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)

The fractions containing glycopeptides were concentrated and desalted using hydrophobic microcolumns packed with Poros R2 (20 μm, Applied Biosystems, Framingham, MA) in GelLoader pipette tips (Eppendorf, Hamburg, Germany) as described elsewhere [58]. The samples were eluted directly onto the MS target with 0.5 μl 2,5-dihydroxybenzoic acid (20 g/L) in 70% ACN and 0.1% TFA. Alternatively, fractions were not desalted and analyzed by mixing 0.5 μl sample and 0.5 μl matrix directly on the target. All samples were analyzed in positive polarity mode by MALDI MS using a Bruker Ultraflex (Bruker Daltonics, Bremen, Germany) with TOF-TOF technology or a MALDI Q-TOF Ultima (Waters, Micromass, Manchester, UK). The spectra were internally calibrated, or external calibration was performed by placing a tryptic lactoglobulin digest near the actual target spot.