BACKGROUND
human thrombin-activatable fibrinolysis inhibitor  , also known as plasma pro-carboxypeptidase b, r, and u, is a plasma metallocarboxypeptidase that attenuates fibrinolysis  <cit> .
tafi circulates in plasma as a  <dig> kda protein with significant intrinsic activity  <cit> .
the majority of the sites that undergo transglutaminase-mediated cross-linking to fibrin are primarily located on the heavily glycosylated pro-peptide, suggesting that tafi becomes incorporated into the fibrin clot during later stages of the coagulation cascade  <cit> .
a variety of trypsin-like proteinases have been shown to remove this peptide, generating the mature protein, tafia  <cit> .
the isoelectric point  of this proteolytically cleaved protein is around ph  <dig> , which is significantly more basic than that of tafi   <cit> .
tafia remains in circulation by forming complexes with α2-macroglobulin and pregnancy zone protein  <cit>  but is highly unstable, a feature initially attributed to proteolytic cleavage.
however, this instability is now thought to result from a temperature-dependent conformational change that occurs within minutes of activation  <cit> .
tafi has been implicated not only in fibrinolysis, but also in inflammation, wound healing, and a variety of other deficiencies and diseases, such as diabetes, kidney failure, lung cancer, and liver illnesses  <cit> .
interestingly, individuals with the more stable ile <dig> variant are apparently more susceptible to meningococcal sepsis  <cit> .
tafi has been studied in multiple animal models, including dog, rabbit, mouse, and rat  <cit> .
intriguingly, the absence of the protein in knock out mice is compatible with murine life  <cit> .
mouse and rat tafi have been characterized, and both show similarity to the human protein  <cit> .
until very recently, the only available structural model for the study of tafi was human pancreatic pro carboxypeptidase b   <cit> .
the protein sequence of pro-cpb is about 40% identical to tafi.
however, in contrast to tafi, pro-cpb lacks intrinsic activity and its active form, carboxypeptidase b , is stable upon activation  <cit> .
efforts to crystallize authentic human tafi have been unsuccessful, most likely due to its sugar heterogeneity when purified from pooled plasma  <cit> .
however, using recombinant human tafi and authentic protein purified from a single cow enabled the zymogen structure to be solved  <cit> .
although bovine tafi is similar to pro-cpb, it also has differences.
significantly, the position of the pro-peptide is rotated 12° away from the active site, exposing access to the catalytic residues.
another significant distinction is the lack of the corresponding salt bridge between asp <dig> and arg <dig> in tafi  <cit> .
these distinctions might explain the intrinsic activity of tafi  <cit> .
furthermore, the structure of bovine tafia in complex with tick carboxypeptidase inhibitor  was determined and found to exhibit a high degree of identify with the cpb-tci structure  <cit> .
interestingly, the bovine tafia structure contains two undefined regions, both of which are part of exposed loops present in the lβ2β <dig> and lα2β <dig> regions and in a heparin affinity region  <cit> .
the domains including arg <dig> and arg <dig>  which are predicted to cause instability in human tafi, were fully ordered in the bovine molecule.
these recent advances prompted us to perform a thorough biochemical characterization of the bovine protein, purified from bovine plasma.
this biochemical characterization included analysis of stability, n-linked glycosylation, generation of tafia by removal of the pro-peptide by trypsin and thrombin/solulin, the antifibrinolytic effects of tafia, as well as analysis of the intrinsic activity of the full length protein and its potential to become crosslinked to fibrin by transglutaminases.
RESULTS
primary structure of bovine tafi
the amino acid sequence of bovine tafi was deduced from a cdna library and published recently  <cit> .
the sequence was  <dig> % identical to that of the human protein.
the bovine protein consisted of  <dig> amino acid residues, including a 92-amino acid residue pro-peptide that is released by cleavage at arg <dig>  all potential glycosylation sites were conserved and found glycosylated in both species, with exception of the fifth site , which remained unglycosylated in bovine tafi.
the location of cysteine residues was identical in both species, with the exception of cys <dig>  this cysteine residue, which is located in the activation peptide, was absent from bovine tafi.
in human tafi, cys <dig> 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  <dig> kda, which is slightly lower than the position of human tafi .
tafia generated by either trypsin  or thrombin/solulin complex  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 .
since the thrombin/solulin complex is considered to be responsible for release of the tafi pro-peptide in vivo  <cit>  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 .
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.
*the values for km and vmax were determined by the direct fit of the michaelis-menten equation employing four graphical methods.
the data represent the enzyme-catalysed reaction for  <dig>  μm tafi and  <dig>  μm tafia.
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  <dig>  sds-page of bovine tafi and thrombin/solulin mixture fashioned a strong band not only at  <dig> kda , but also at  <dig> kda, which was confirmed to be tafia .
in contrast to the human tafi pro-peptide, the released bovine pro-peptide was clearly visible by coomassie staining, with a mass of around  <dig> kda .
as expected, large amounts of thrombin/solulin complex truncated human tafia  at arg <dig>  liberating the  <dig>  kda c-terminal peptide to produce a proteolytically inactivated form of tafia  .
however, no further proteolytic products were observed for the bovine protein in the higher end of the titration using the proteinase complex.
bovine tafia
cut at the
bovine tafi
bovine tafi
bovine tafia
*determined by edman degradation.
**upon cleavage of bovine tafi by either trypsin or solulin/thrombin complex, fragments were separated by sds-page, transferred to pvdf membrane and subjected to edman –degradation as described in the method section.
the obtained n-terminal sequences of fragments and their molecular weight is listed.
when tafi was cleaved by trypsin, which is more potent than thrombin/solulin complex, a somewhat dissimilar fragmentation pattern was generated .
sds-page of the human protein yielded a typical pattern consisting of full length tafi at  <dig> kda, mature tafia at  <dig> kda, c-terminal processed tafia  at  <dig> kda, and the released c-terminal peptide at  <dig> kda .
trypsin cleavage occurred at the same site in both the human and bovine protein, creating a  <dig> kda tafia through truncation at arg <dig> .
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 arg <dig>  creating a  <dig>  kda fragment, detected around  <dig> kda on sds gel .
the pro-peptide was detected at around  <dig> kda .
however, it is most likely immediately processed at the c-terminus, as a  <dig> kda band was detected that also contained the tafi n-terminal sequence .
trypsin and small trypsin fragments were also detected, along with the released  <dig>  kda tafia n-terminal peptide .
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 .
the half-life of bovine tafia was longer than that of human tafia.
human tafia activity decreased by 50% after  <dig> min, while this decrease in bovine tafia activity occurred at  <dig> min .
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 .
moreover, hippuric acid release was blocked when tafi was incubated with the substrate in the presence of  <dig> mm  <dig>  10-phenantroline, a chelating agent and known carboxypeptidase inhibitor .
this confirms that bovine tafi has genuine intrinsic activity.
this activity was also inhibited by  <dig>  μm tci, a potentially physiologically relevant inhibitor of tafi .
isoelectric point variation between tafi and tafia
isoelectric focusing revealed that the isoelectric point of tafi and tafia varied greatly in both species .
the full length bovine protein migrated in the lower end of the ph gradient and appeared as multiple bands between a pi of  <dig>  and  <dig> , likely due to glycosylation heterogeneity.
this migration position was slightly higher than that of the human tafi, which migrated to a pi of  <dig>  to  <dig>  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  <dig> .
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 .
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  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  tafi to effect clot lysis .
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  incorporated dansylcadaverine .
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  tof ms/ms .
thirty-five n-glycans were observed from the four n-glycosylation sites present within the n-terminal activation region  .
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  and n-acetylneuraminic acid .
up to four sialic acid residues were observed on the n <dig> glycans, whereas the rest of the occupied sequons contained a maximum of three sialic acid residues .
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  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  <cit> .
this may help account for difficulties in determining the three-dimensional structure of human tafi zymogen.
*theoretical glycopeptide mass values are based on the transformation of the n-terminal gln <dig> to pyroglutamic acid.
**percent occupancy of n-glycans was determined based on ms experiments in which peptide mass fingerprints of tryptic bovine tafi treated ± pngase f were compared to establish the presence of -glycosylated peptides .
***glcnac, n-acetylglucosamine; man, mannose; gal, galactose; neu5ac, 5-n-acetylneuraminic acid; neu5gc, 5-n-glycolylneuraminic acid.
discussion
the structure of tafi and tafia/tci complex has recently been solved using authentic bovine tafi  <cit> .
here, we present a full biochemical characterization of the bovine protein purified from bovine plasma.
the amino acid sequence idenity between the two spieces is  <dig> % and all the important sites, such as the catalytic domain, substrate-binding domain, and zinc-binding domain, are fully conserved  <cit> .
only four of the potential n-linked carbohydrate sites are occupied, all located on the activation peptide.
the fifth site, asn <dig>  which is partially glycosylated in the human protein  <cit> , remained unglycosylated in the bovine protein .
accordingly, this site was found to be buried within the protein structure and has previously been suggested to be unglycosylated  <cit> .
recently, the biochemical importance of human tafi glycosylation has been studied using tafi mutants  <cit> .
interestingly, in some mutants, the absence of carbohydrates increases the activity of full length tafi, but decreases tafia activity.
the increase in intrinsic activity is most apparent in the mutants tafi-n22q and tafi-n22q-n51q-n63q.
these observations corroborate the finding that, in human tafi, access to the active site exists  <cit>  and this access site potentially expands upon carbohydrate removal, possibly imparting a catalytic function to sugars  <cit> .
interestingly, the pronounced microheterogeneity of the tafi glycans was exclusively generated by the variation in the number and type of sialic acid residues located in the termini of the biantennary complex glycans.
neu5ac and neu5gc were found in the tafi glycans and both are known to be abundant sialic acids in bovine glycoconjugates.
in contrast, humans cannot synthesize neu5gc, highlighting a difference between the authentic human and authentic bovine tafi structure.
purified bovine tafi successfully attenuated fibrinolysis of tpa-induced clots in a purified system.
also similar to human tafi, the bovine protein displays considerable stable intrinsic activity, which can be abolished by the same inhibitors used to inhibit tafia.
furthermore, it is most likely crosslinked to the fibrin meshwork during the early stages of fibrinolysis, as the protein seems to act as a substrate for transglutaminases.
bovine tafi contains potential amine acceptor sites, as evidenced by the successful incorporation of dansylcadavarine into the protein by tissue transglutaminase.
bovine tafi, like the human protein, can also be cleaved through proteolysis at arg <dig>  generating the mature form, tafia.
in contrast to the human tafi, bovine tafi is processed into not only the  <dig> kda active enzyme, but also a  <dig> kda tafia fragment following incubation with trypsin.
this n-terminally processed tafia is missing a  <dig> kda n-terminal peptide and is formed through proteolysis of the arg147-ala <dig> bond.
this cleavage takes place prior to the usual inactivation that occurs at the c-terminus.
human tafi contains either a thr or ala at position  <dig>  depending on the variant.
therefore, an identical n-terminal truncation at this position is not possible  <cit> .
similar fragmentation has been observed following activation of rat tafi by plasmin  <cit> .
this may also explain the disordered lβ2β <dig> segment observed in the three-dimensional structure of the tafia/tci complex  <cit> .
in human tafi, substituting his <dig> with tyr or gln increases the half-life of tafia for up to  <dig>  h, while preserving all characteristics of wild type tafi  <cit> .
site-directed mutagenesis of arg <dig>  arg <dig>  and arg <dig> produces a molecule much less stable than the wild type protein, suggesting that this instability is concentrated in the  <dig> –  <dig> region  <cit> .
the naturally occurring mutation of thr <dig> to ile <dig> has been shown to make human tafi twice as stable  <cit> .
position  <dig> of the bovine protein is occupied by ile, which might account for the longer half-life of bovine tafia  compared to the human tafia .
similarly, mutation of thr <dig> to ile <dig> increases not only the half-life of the cleaved human protein, but also its fibrinolytic effect  <cit> .
again, this position is occupied by ile in bovine tafi.
substitution of human tafi residues with corresponding residues of cpb, such as tafia-ile182arg-ile183glu, does not significantly increase stability.
on the contrary, it reduces antifibrinolytic potential.
nevertheless, lower amounts of thrombin-thrombomodulin complex are required in order to generate tafia from this mutant  <cit> .
this can explain, at least partly, why lower amounts of proteinases are required to generate bovine tafia, in which lys <dig> and glu <dig> naturally occur in sequence.
indeed,  <dig> times less solulin/thrombin complex is required to generate bovine tafia with activity similar to that of human tafia.
in summary, we deduce that human tafi and bovine tafi have similar properties.
the overall secondary structure is conserved, generation of tafia can be achieved in similar manner, and bovine tafia produces a measurable effect on fibrinolysis.
thus, the available three-dimensional structure of bovine tafi is a reliable model for investigation of human tafi, including its in vivo function and the in vivo effects of its inhibition.
CONCLUSIONS
the bovine and human tafi activation occurs at equivalent sites and both tafia and tafi exhibit caroboxypeptidase activity.
additionally, tafi from both species was found to be substrate for transglutaminases.
minor differences in the enzymatic stability of bovine and human tafia was observed as well as differences in the level of glycosylation, isoelectric point and proteolytic by-products in trypsin activation.
however, overall the findings suggested that the the two orthologous proteins are similar and that conclusions reached using the bovine tafi can safely be extrapolated to the human protein.

 human thrombin-activatable fibrinolysis inhibitor, also known as plasma pro-carboxypeptidase b, r, and u, is a plasma metallocarboxypeptidase that attenuates fibrinolysis. tafi circulates in plasma as a kda protein with significant intrinsic activity. the majority of the sites that undergo transglutaminase-mediated cross-linking to fibrin are primarily located on the heavily glycosylated pro-peptide, suggesting that tafi becomes incorporated into the fibrin clot during later stages of the coagulation cascade. a variety of trypsin-like proteinases have been shown to remove this peptide, generating the mature protein, tafia. the isoelectric point of this proteolytically cleaved protein is around ph, which is significantly more basic than that of tafi. tafia remains in circulation by forming complexes with α2-macroglobulin and pregnancy zone protein but is highly unstable, a feature initially attributed to proteolytic cleavage. however, this instability is now thought to result from a temperature-dependent conformational change that occurs within minutes of activation. tafi has been implicated not only in fibrinolysis, but also in inflammation, wound healing, and a variety of other deficiencies and diseases, such as diabetes, kidney failure, lung cancer, and liver illnesses. interestingly, individuals with the more stable ile variant are apparently more susceptible to meningococcal sepsis. tafi has been studied in multiple animal models, including dog, rabbit, mouse, and rat. intriguingly, the absence of the protein in knock out mice is compatible with murine life. mouse and rat tafi have been characterized, and both show similarity to the human protein. until very recently, the only available structural model for the study of tafi was human pancreatic pro carboxypeptidase b. the protein sequence of pro-cpb is about 40% identical to tafi. however, in contrast to tafi, pro-cpb lacks intrinsic activity and its active form, carboxypeptidase b, is stable upon activation. efforts to crystallize authentic human tafi have been unsuccessful, most likely due to its sugar heterogeneity when purified from pooled plasma. however, using recombinant human tafi and authentic protein purified from a single cow enabled the zymogen structure to be solved. although bovine tafi is similar to pro-cpb, it also has differences. significantly, the position of the pro-peptide is rotated 12° away from the active site, exposing access to the catalytic residues. another significant distinction is the lack of the corresponding salt bridge between asp and arg in tafi. these distinctions might explain the intrinsic activity of tafi. furthermore, the structure of bovine tafia in complex with tick carboxypeptidase inhibitor was determined and found to exhibit a high degree of identify with the cpb-tci structure. interestingly, the bovine tafia structure contains two undefined regions, both of which are part of exposed loops present in the lβ2β and lα2β regions and in a heparin affinity region. the domains including arg and arg which are predicted to cause instability in human tafi, were fully ordered in the bovine molecule. these recent advances prompted us to perform a thorough biochemical characterization of the bovine protein, purified from bovine plasma. this biochemical characterization included analysis of stability, n-linked glycosylation, generation of tafia by removal of the pro-peptide by trypsin and thrombin/solulin, the antifibrinolytic effects of tafia, as well as analysis of the intrinsic activity of the full length protein and its potential to become crosslinked to fibrin by transglutaminases.  primary structure of bovine tafi the amino acid sequence of bovine tafi was deduced from a cdna library and published recently. the sequence was % identical to that of the human protein. the bovine protein consisted of amino acid residues, including a 92-amino acid residue pro-peptide that is released by cleavage at arg all potential glycosylation sites were conserved and found glycosylated in both species, with exception of the fifth site, which remained unglycosylated in bovine tafi. the location of cysteine residues was identical in both species, with the exception of cys this cysteine residue, which is located in the activation peptide, was absent from bovine tafi. in human tafi, cys 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 kda, which is slightly lower than the position of human tafi. tafia generated by either trypsin or thrombin/solulin complex 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. since the thrombin/solulin complex is considered to be responsible for release of the tafi pro-peptide in vivo 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. 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. *the values for km and vmax were determined by the direct fit of the michaelis-menten equation employing four graphical methods. the data represent the enzyme-catalysed reaction for μm tafi and μm tafia. 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 sds-page of bovine tafi and thrombin/solulin mixture fashioned a strong band not only at kda, but also at kda, which was confirmed to be tafia. in contrast to the human tafi pro-peptide, the released bovine pro-peptide was clearly visible by coomassie staining, with a mass of around kda. as expected, large amounts of thrombin/solulin complex truncated human tafia at arg liberating the kda c-terminal peptide to produce a proteolytically inactivated form of tafia. however, no further proteolytic products were observed for the bovine protein in the higher end of the titration using the proteinase complex. bovine tafia cut at the bovine tafi bovine tafi bovine tafia *determined by edman degradation. **upon cleavage of bovine tafi by either trypsin or solulin/thrombin complex, fragments were separated by sds-page, transferred to pvdf membrane and subjected to edman –degradation as described in the method section. the obtained n-terminal sequences of fragments and their molecular weight is listed. when tafi was cleaved by trypsin, which is more potent than thrombin/solulin complex, a somewhat dissimilar fragmentation pattern was generated. sds-page of the human protein yielded a typical pattern consisting of full length tafi at kda, mature tafia at kda, c-terminal processed tafia at kda, and the released c-terminal peptide at kda. trypsin cleavage occurred at the same site in both the human and bovine protein, creating a kda tafia through truncation at arg. 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 arg creating a kda fragment, detected around kda on sds gel. the pro-peptide was detected at around kda. however, it is most likely immediately processed at the c-terminus, as a kda band was detected that also contained the tafi n-terminal sequence. trypsin and small trypsin fragments were also detected, along with the released kda tafia n-terminal peptide. 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. the half-life of bovine tafia was longer than that of human tafia. human tafia activity decreased by 50% after min, while this decrease in bovine tafia activity occurred at min. 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. moreover, hippuric acid release was blocked when tafi was incubated with the substrate in the presence of mm 10-phenantroline, a chelating agent and known carboxypeptidase inhibitor. this confirms that bovine tafi has genuine intrinsic activity. this activity was also inhibited by μm tci, a potentially physiologically relevant inhibitor of tafi. isoelectric point variation between tafi and tafia isoelectric focusing revealed that the isoelectric point of tafi and tafia varied greatly in both species. the full length bovine protein migrated in the lower end of the ph gradient and appeared as multiple bands between a pi of and, likely due to glycosylation heterogeneity. this migration position was slightly higher than that of the human tafi, which migrated to a pi of to 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. 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. 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 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 tafi to effect clot lysis. 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 incorporated dansylcadaverine. 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 tof ms/ms. thirty-five n-glycans were observed from the four n-glycosylation sites present within the n-terminal activation region. 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 and n-acetylneuraminic acid. up to four sialic acid residues were observed on the n glycans, whereas the rest of the occupied sequons contained a maximum of three sialic acid residues. 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 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. this may help account for difficulties in determining the three-dimensional structure of human tafi zymogen. *theoretical glycopeptide mass values are based on the transformation of the n-terminal gln to pyroglutamic acid. **percent occupancy of n-glycans was determined based on ms experiments in which peptide mass fingerprints of tryptic bovine tafi treated ± pngase f were compared to establish the presence of -glycosylated peptides. ***glcnac, n-acetylglucosamine; man, mannose; gal, galactose; neu5ac, 5-n-acetylneuraminic acid; neu5gc, 5-n-glycolylneuraminic acid. discussion the structure of tafi and tafia/tci complex has recently been solved using authentic bovine tafi. here, we present a full biochemical characterization of the bovine protein purified from bovine plasma. the amino acid sequence idenity between the two spieces is % and all the important sites, such as the catalytic domain, substrate-binding domain, and zinc-binding domain, are fully conserved. only four of the potential n-linked carbohydrate sites are occupied, all located on the activation peptide. the fifth site, asn which is partially glycosylated in the human protein, remained unglycosylated in the bovine protein. accordingly, this site was found to be buried within the protein structure and has previously been suggested to be unglycosylated. recently, the biochemical importance of human tafi glycosylation has been studied using tafi mutants. interestingly, in some mutants, the absence of carbohydrates increases the activity of full length tafi, but decreases tafia activity. the increase in intrinsic activity is most apparent in the mutants tafi-n22q and tafi-n22q-n51q-n63q. these observations corroborate the finding that, in human tafi, access to the active site exists and this access site potentially expands upon carbohydrate removal, possibly imparting a catalytic function to sugars. interestingly, the pronounced microheterogeneity of the tafi glycans was exclusively generated by the variation in the number and type of sialic acid residues located in the termini of the biantennary complex glycans. neu5ac and neu5gc were found in the tafi glycans and both are known to be abundant sialic acids in bovine glycoconjugates. in contrast, humans cannot synthesize neu5gc, highlighting a difference between the authentic human and authentic bovine tafi structure. purified bovine tafi successfully attenuated fibrinolysis of tpa-induced clots in a purified system. also similar to human tafi, the bovine protein displays considerable stable intrinsic activity, which can be abolished by the same inhibitors used to inhibit tafia. furthermore, it is most likely crosslinked to the fibrin meshwork during the early stages of fibrinolysis, as the protein seems to act as a substrate for transglutaminases. bovine tafi contains potential amine acceptor sites, as evidenced by the successful incorporation of dansylcadavarine into the protein by tissue transglutaminase. bovine tafi, like the human protein, can also be cleaved through proteolysis at arg generating the mature form, tafia. in contrast to the human tafi, bovine tafi is processed into not only the kda active enzyme, but also a kda tafia fragment following incubation with trypsin. this n-terminally processed tafia is missing a kda n-terminal peptide and is formed through proteolysis of the arg147-ala bond. this cleavage takes place prior to the usual inactivation that occurs at the c-terminus. human tafi contains either a thr or ala at position depending on the variant. therefore, an identical n-terminal truncation at this position is not possible. similar fragmentation has been observed following activation of rat tafi by plasmin. this may also explain the disordered lβ2β segment observed in the three-dimensional structure of the tafia/tci complex. in human tafi, substituting his with tyr or gln increases the half-life of tafia for up to h, while preserving all characteristics of wild type tafi. site-directed mutagenesis of arg arg and arg produces a molecule much less stable than the wild type protein, suggesting that this instability is concentrated in the – region. the naturally occurring mutation of thr to ile has been shown to make human tafi twice as stable. position of the bovine protein is occupied by ile, which might account for the longer half-life of bovine tafia compared to the human tafia. similarly, mutation of thr to ile increases not only the half-life of the cleaved human protein, but also its fibrinolytic effect. again, this position is occupied by ile in bovine tafi. substitution of human tafi residues with corresponding residues of cpb, such as tafia-ile182arg-ile183glu, does not significantly increase stability. on the contrary, it reduces antifibrinolytic potential. nevertheless, lower amounts of thrombin-thrombomodulin complex are required in order to generate tafia from this mutant. this can explain, at least partly, why lower amounts of proteinases are required to generate bovine tafia, in which lys and glu naturally occur in sequence. indeed, times less solulin/thrombin complex is required to generate bovine tafia with activity similar to that of human tafia. in summary, we deduce that human tafi and bovine tafi have similar properties. the overall secondary structure is conserved, generation of tafia can be achieved in similar manner, and bovine tafia produces a measurable effect on fibrinolysis. thus, the available three-dimensional structure of bovine tafi is a reliable model for investigation of human tafi, including its in vivo function and the in vivo effects of its inhibition.  the bovine and human tafi activation occurs at equivalent sites and both tafia and tafi exhibit caroboxypeptidase activity. additionally, tafi from both species was found to be substrate for transglutaminases. minor differences in the enzymatic stability of bovine and human tafia was observed as well as differences in the level of glycosylation, isoelectric point and proteolytic by-products in trypsin activation. however, overall the findings suggested that the the two orthologous proteins are similar and that conclusions reached using the bovine tafi can safely be extrapolated to the human protein. 
the enzymatic stability and the kinetic constants of tafia differed somewhat between the two proteins, as did the isoelectric point of tafi, but not tafia. the three-dimensional crystal structures of authentic mature bovine tafi in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor, and recombinant human tafi have recently been solved. in light of these recent advances, we have characterized authentic bovine tafi biochemically and compared it to human tafi. the four n-linked glycosylation sequons within the activation peptide were all occupied in bovine tafi, similar to human tafi, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. equivalent to human tafi, bovine tafi was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. furthermore, bovine tafi exhibited intrinsic activity and tafia attenuated tpa-mediated fibrinolysis similar to the human protein. the findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine tafi may be extrapolated to the human protein.
 tafi is a plasma protein assumed to be an important link between coagulation and fibrinolysis. the three-dimensional crystal structures of authentic mature bovine tafi in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor, and recombinant human tafi have recently been solved. in light of these recent advances, we have characterized authentic bovine tafi biochemically and compared it to human tafi.  the four n-linked glycosylation sequons within the activation peptide were all occupied in bovine tafi, similar to human tafi, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. the enzymatic stability and the kinetic constants of tafia differed somewhat between the two proteins, as did the isoelectric point of tafi, but not tafia. equivalent to human tafi, bovine tafi was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. furthermore, bovine tafi exhibited intrinsic activity and tafia attenuated tpa-mediated fibrinolysis similar to the human protein.  the findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine tafi may be extrapolated to the human protein. 