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Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:423-428
Published online before print December 8, 2005, doi: 10.1161/01.ATV.0000199246.08616.98
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:423.)
© 2006 American Heart Association, Inc.

Thrombosis

Development of ELISAs Measuring the Extent of TAFI Activation

Erik Ceresa; Els Brouwers; Miet Peeters; Christina Jern; Paul J. Declerck; Ann Gils

From the Laboratory for Pharmaceutical Biology and Phytopharmacology (E.C., E.B., M.P., P.J.D., A.G.), Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium; and The Institute of Clinical Neuroscience (C.J.), Sahlgrenska Academy, Göteborg University, Göteborg, Sweden.

Correspondence to Ann Gils, Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, O&N2-PB824, Herestraat 49, B-3000 Leuven, Belgium. E-mail Ann.Gils{at}pharm.kuleuven.be


*    Abstract
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Objectives— To date, quantitation of TAFI antigen levels has been mainly focused on "total" antigen levels and has been shown to yield ambiguous results because of the existence of different isoforms and various degrees of activation. Our objective was to develop assays that allow measuring the extent of TAFI activation.

Methods and Results— A variety of enzyme-linked immunosorbent assays (ELISAs) were evaluated for their preferential reactivity toward TAFI before and after activation, and toward the recombinantly expressed activation peptide. Three ELISAs with distinct reactivities were selected: recognizing either exclusively nonactivated TAFI, the released activation peptide, or exclusively TAFIa (activated TAFI). Evaluation of TAFI activation during clot lysis revealed that decreases of TAFI levels are associated with increases of the released activation peptide and TAFIa levels. In addition, antigenic measurement of TAFIa parallels activity measured by chromogenic assay. Analyzing plasma samples revealed that subjects with hyperlipidemia had significantly higher plasma levels of both the activation peptide (109.2 versus 95.5; P<0.001) and TAFIa (112.1 versus 103.3; P=0.03), and not of TAFI antigen (92.5 versus 87.9; P=0.07) (results in % of plasma pooled from normolipidemic subjects).

Conclusion— ELISAs that allow to measure the extent of TAFI activation were developed. These ELISAs constitute more sensitive markers in studies on the relationship between TAFI and cardiovascular diseases.

We developed immunologic assays that allow to measure the extent of TAFI activation. These ELISAs constitute more sensitive markers in studies on the relationship between TAFI and cardiovascular related diseases.


Key Words: fibrinolysis • monoclonal antibodies • TAFI • ELISA • extent of TAFI activation


*    Introduction
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The proenzyme thrombin activatable fibrinolysis inhibitor (TAFI; procarboxypeptidase U [proCPU]) can be activated to the active enzyme (TAFIa; carboxypeptidase U [CPU]) by trypsin-like enzymes such as thrombin, plasmin, or the thrombin/thrombomodulin complex (T/TM).1–3 On activation of TAFI (Phe1-Val401; 56 kDa), the activation peptide (AP) (Phe1-Arg92; 20 kDa, 4 N-linked glycosylations4) is released from the catalytic domain (TAFIa, Ala93-Val401; 36 kDa). Subsequently, TAFIa is inactivated through a conformational change to an inactive form (TAFIai), followed by proteolytic cleavage resulting in fragments of 25 and 11 kDa.5 TAFIa exerts an antifibrinolytic effect by removing C-terminal lysine residues from fibrin, resulting in a decreased plasmin formation and a retardation of clot lysis.6 TAFIa is highly unstable in vitro (half-life at 37°C between 8 to 15 minutes).7–10

Different clinical studies have investigated the possible relationship between TAFI and cardiovascular events. A positive correlation was found between TAFI levels and the risk for coronary artery disease,11 venous thrombosis,12 angina pectoris,13 and ischemic stroke.14 However, another study revealed that elevated TAFI may be protective against myocardial infarction.15 A possible explanation for these contradictory results was that the different studies used different methods for TAFI determination.16 Silveira et al used quantitative activation of the zymogen followed by determination of the total enzymatic activity with a chromogenic assay. Van Tilburg et al measured TAFI levels with an electroimmunoassay. A commercially available kit from Milan Analytica, based on affinity-purified sheep anti-TAFI IgG, was used in the studies of Morange et al and Juhan-Vague et al. Finally, Montaner et al obtained their results with a commercially available Zymutest TAFI antigen kit from Hyphen BioMed.14 Malyszko et al found that elevated TAFI (Actichrome TAFIa; American Diagnostica) might be a link in the pathogenesis of impaired fibrinolysis in diabetic nephropathy and thus atherosclerosis progression.17 Saibeni et al found that increased TAFI levels (enzyme-linked immunosorbent assay [ELISA]; Affinity Biologicals) might contribute to the prothrombotic state observed in inflammatory bowel syndrome through the induction of hypofibrinolysis.18 Eichinger et al showed that patients with higher TAFI levels (ELISA; American Diagnostica) have a higher risk for recurrent venous thromboembolism.19 To date, quantitation of TAFI antigen levels, as a putative risk marker for cardiovascular events, has been mainly focused on "total" antigen levels (either by ELISA or by activity assays after full activation of TAFI), reporting levels of 4 to 15 µg/mL.16,20–22 However, it should be stressed that in most of these studies the used immunologic assays were not validated with respect to putative differential reactivities toward different isoforms and fragments of TAFI. Therefore, it cannot be excluded that apparently increased or decreased TAFI levels associated with particular disease states is not caused by differences in total TAFI protein levels but rather to differences in isoforms or differences in extent of activation. In this respect, it is important to note that recent studies,23,24 revealing a threshold phenomenon, have suggested that not the total amount of TAFI protein, but the amount of activated TAFI may play a critical role in the interference with fibrinolysis. Therefore, and because of the complications in the use of activity assays, we hypothesized that immunologic measurement of the extent of TAFI activation (ie, either by measuring the released activation peptide or by measuring TAFIa) could represent a more relevant parameter.

In this study, we screened a variety (>1500) of monoclonal antibody (MA)-based ELISAs for their preferential reactivity toward TAFI before and after activation and toward the recombinantly expressed AP. This allowed us to select 3 ELISAs with distinct properties, recognizing either exclusively nonactivated TAFI, the released AP, or exclusively TAFIa.


*    Methods
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Please see http://atvb.ahajournals.org for detailed Methods.


*    Results
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 *Results
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Construction, Selection, and Characterization of Sandwich-Type ELISAs for Detection of Different TAFI Fragments
Forty monoclonal antibodies were pair-wise tested for their reactivity toward pTAFI before and after activation. Two groups of ELISAs could be distinguished: one group reacts only with intact, nonactivated TAFI and a second group reacts only with TAFI after it has been activated. Within this second group, 2 subgroups were identified: one subgroup reacted with the recombinant AP, whereas the other subgroup did not. Based on these preferential reactivities toward different TAFI fragments, 3 ELISA combinations were selected for further characterization: MA-T12D11/MA-T30E5-HRP, reacting with nonactivated TAFI, MA-T12D11/MA-T18A8-HRP, reacting with TAFI after activation as well as with the recombinant AP, and MA-T30E5/MA-T17D7-HRP, reacting with TAFI after activation but not with the recombinant AP.

Preliminary experiments, using Western blotting (data not shown), indicated that the epitopes of MA-T12D11 and MA-T18A8 reside in the region of the AP, whereas for MA-T17D7 the epitope resides in the 36-kDa fragment. Because MA-T30E5 does not work in blotting experiments, no information on the binding region is available.

For calibration of the selected ELISAs, 2 standards were developed, ie, S0 and S10 (cfr methods). The response of S10 in the MA-T12D11/MA-T30E5-HRP ELISA was 11±3.2% compared with S0, whereas the response of S0 in the MA-T12D11/MA-T18A8-HRP and MA-T30E5/MA-T17D7-HRP ELISA was 0.7±0.6% and 4.0±2.1% compared with S10, respectively. Only MA-T12D11/MA-T18A8-HRP reacted with the recombinant AP: 100 µg/mL recombinant AP gave a response of 101±11% compared with S10. Typical standard curves of the three ELISAs are shown in Figure 1.


Figure 1
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  		Figure 1. Typical standard curves of the 2 developed standards S0 (•) and S10 ({circ}). In MA-T12D11/MA-T30E5-HRP (A), in MA-T12D11/MA-T18A8-HRP (B), and in MA-T30E5/MA-T17D7-HRP (C). Y-axis represents the OD at 430 nm and X-axis represents the different plasma dilutions ranging from 1:2560 to 1:40 (of which 1:320, 1:160, 1:80, and 1:40 dilutions are indicated). ({square}) represents the reactivity of recombinant AP applied at a concentration equimolar to that in plasma (ie, 10 µg/mL TAFI contains 3.57 µg/mL AP). The 1/40 dilution of the standards corresponds with 90 ng/mL rAP.

TAFI fragments formed on activation of pTAFI with T/TM are shown in the inset of Figure 2A. At 5 minutes, the 56-kDa fragment of TAFI is completely converted to the 36-kDa fragment. Subsequently, at 10 minutes, the degradation products of 25 and 11 kDa are formed and eventually, and after 30 minutes, the 36 kDa fragment is completely degraded. The activation peptide of TAFI has a theoretical Mr of 19.4 kDa (with carbohydrate). In agreement with the literature, no distinct activation peptide after activation of TAFI was detected using SDS-PAGE and silver staining.10,25,26 However, Guimaraes et al showed, using SDS-PAGE and Western blotting, that the released activation peptide co-migrates as a 33-kDa broadband with the 36 kDa catalytic domain of TAFI.27


Figure 2
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  		Figure 2. Response of TAFI on activation in the 3 selected ELISAs. TAFIa activity ({circ}) generated on activation of pTAFI (A) and plasma (B) between 0 and 120 minutes determined with a chromogenic assay. Response on activation between 0 and 120 minutes in MA-T12D11/MA-T30E5-HRP ({blacksquare}), MA-T12D11/MA-T18A8-HRP ({blacktriangleup}), and MA-T30E5/MA-T17D7 ({blacktriangledown}) (mean±SD, n≥3). Inset, SDS-PAGE followed by silver staining of the reaction products formed on activation of TAFI with thrombin/thrombomodulin during a 120-minutes time course. TAFI (56 kDa) is activated to TAFIa (36 kDa). TAFIa is inactivated through conformational change to TAFIai (36 kDa), followed by proteolytic cleavage resulting in fragments of 25 and 11 kDa.

TAFIa activity, generated on activation of pTAFI with T/TM (Figure 2A), reaches a maximum at 5 minutes and then decreases, reaching a minimum at 20 minutes, in agreement with the appearance and the disappearance of the 36-kDa fragment. The response of pTAFI (Figure 2A) in MA-T12D11/MA-T30E5-HRP decreases on activation, reaching a minimum after 5 minutes (<2%, 47±15-fold decrease, P<0.0001 versus t=0 minutes). In contrast, the response in MA-T12D11/MA-T18A8-HRP increases in function of time reaching a maximum at 5 minutes, which is maintained to 20 minutes (74±26-fold increase, P<0.0001 versus t=0 minutes), followed by a slow decrease. The response in MA-T30E5/MA-T17D7-HRP increases on activation reaching a maximum at 5 minutes (70±10-fold increase, P<0.0001 versus t=0 minutes) and then decreases reaching a minimum after 60 minutes (<2%). Similar results were obtained using plasmin to activate TAFI (data not shown) and using 2 recombinant TAFI isoforms (ie, TAFI-Ala147Thr325 and TAFI-Ala147Ile325) (see http://atvb.ahajournals.org). The response in MA-T30E5/MA-T17D7-HRP decreased after reaching a maximum with a half-life of 14.0±0.2 minutes for TAFI-Ala147Thr325 versus 15.9±0.3 minutes for TAFI-Ala147Ile325 (P<0.05), respectively, in line with the increased stability of TAFIa of the Ile325 isoform.8

TAFIa activity in plasma (Figure 2B) reaches a maximum after 10 minutes and then decreases, reaching a minimum after 30 minutes. The response of pooled human plasma (Figure 2B) in MA-T12D11/MA-T30E5-HRP decreases on activation, reaching a minimum after 30 minutes (<15%, 7.6±2.6-fold decrease, P<0.0001 versus t=0 minutes). In contrast, the response in MA-T12D11/MA-T18A8-HRP increases on activation reaching a maximum plateau after 30 minutes (60±18-fold increase, P<0.0001 versus t=0 minutes). The response in MA-T30E5/MA-T17D7-HRP increases on activation reaching a maximum after 10 minutes (24±14-fold increase, P=0.002 versus t=0 minutes), immediately followed by a decrease, reaching a minimum after 60 to 120 minutes (<25%).

Taken together, all aforementioned results demonstrate that MA-T12D11/MA-T30E5-HRP preferentially reacts with intact nonactivated TAFI, MA-T12D11/MA-T18A8-HRP reacts with the released AP and MA-T30E5-T17D7-HRP reacts with TAFIa and/or TAFIai.

Quantitation of TAFI in Plasma
A linear dose-response curve was observed in all 3 ELISAs when plasma (either nonactivated for the MA-T12D11/MA-T30E5-HRP ELISA or activated for the MA-T12D11/MA-T18A8-HRP and MA-T30E5/MA-T17D7-HRP ELISA) was diluted between 1/40-fold and 1/2560-fold. Within the linear portion of these curves, correlation coefficients exceeded 0.98.

For recoveries, intra-assay, inter-assay, and interdilution coefficients of variation and detection limits of the 3 ELISAs (please see http://atvb.ahajournals.org).

Application of the Developed Assays In Vitro and In Vivo
During clot formation and dissolution, TAFIa activity was measured using the chromogenic assay and antigen levels of TAFI and TAFI fragments were followed using the 3 described ELISAs (Figure 3).


Figure 3
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  		Figure 3. Time-dependent evaluation of TAFI and TAFI fragments during clot lysis. Measurement of TAFI, the released activation peptide, and the catalytic domain of TAFI during clot lysis in the presence of 0.5 nM TM or 5 nM TM is shown (A and B, respectively). Clot formation and dissolution (···) and TAFIa activity ({circ}) are shown in the top figure (mean±SD, n≥3), the response in the MA-T12D11/MA-T30E5-HRP ({blacksquare}), MA-T12D11/MA-T18A8-HRP ({blacktriangleup}), and MA-T30E5/MA-T17D7-HRP ELISA ({blacktriangledown}) in the figure below (mean±SD, n≥3). Clot formation and dissolution were followed by monitoring the turbidity at 405 nm. Coagulation of plasma is indicated by an increase, dissolution by a decrease in turbidity. The TAFIa activity during clot lysis was determined every 10 minutes with a chromogenic assay.

As reported before,28 a biphasic pattern of TAFIa activity generation during in vitro clot lysis was observed. A first TAFIa activity peak was found immediately after clot formation, and a second peak during clot dissolution. Our data show that in the presence of 0.5 nM TM (Figure 3A), TAFI antigen levels (measured with MA-T12D11/MA-T30E5-HRP) decreased, whereas released AP antigen levels (measured with MA-T12D11/MA-T18A8-HRP) increased concomitantly, both in a similar biphasic pattern. Antigen levels of TAFIa/ai (measured with MA-T30E5/MA-T17D7-HRP) increased during clot formation, followed by a decrease and increased slightly during clot dissolution, followed by a decrease. Under these conditions, TAFI antigen levels decreased, in a biphasic pattern, to 83±16% after clot formation and to 49±8.6% after clot dissolution; the released AP levels increased, in a biphasic pattern, to 25±1.7% after clot formation and to 47±5.3% after clot dissolution. TAFIa/ai antigen levels increased to 27±3.5% during clot formation and to 7.3±0.3% during dissolution. Strikingly, the biphasic changes as observed in the ELISAs coincide with the biphasic pattern observed in the TAFIa activity assay.

As reported before,28 higher TM concentrations (Figure 3A versus Figure 3B) increased the height and width of the first activity peak. With 5 nM TM (Figure 3B), the biphasic pattern was not observed. TAFI antigen levels immediately decreased to 3.2±1.2% after clot formation, released activation peptide antigen levels immediately increased to 108±15.2% after clot formation. TAFIa/ai antigen levels increased transiently to 79±3.7% during clot formation. Again, these data demonstrate the differential reactivities of the ELISAs and their respective association with the activity assay. A decrease of intact TAFI antigen levels is associated with an increase in released AP antigen levels and with a transient increase of TAFIa/ai antigen levels.

The in vivo clearance of the released AP and TAFIa/ai was determined in female Balb/c mice using MA-T12D11/MA-T18A8-HRP and MA-T30E5/MA-T17D7-HRP, respectively. The biological half-lives of the released AP and TAFIa/ai were 27±5.4 and 11±3.9 minutes, respectively.

Plasma samples of 13 healthy individuals were analyzed in the different ELISAs, before and after 10 minutes of activation with T/TM (cfr chromogenic assay) (http://atvb.ahajournals.org).

Quantitation of TAFI and TAFI Fragments in a Swedish Study Population
Characteristics of the study population (n=300) are given in Methods and Materials (http://atvb.ahajournals.org). The plasma levels of TAFI and the released AP and TAFIa were 92.1% (median; interquartiles=78.8% to 103.2%), 106.5% (median; interquartiles=93.0% to 118.2%), and 110.9% (median; interquartiles=96.9% to 132.1%), respectively. TAFI and TAFI fragments (TAFI variables) did not show a significant correlation to age and there were no significant gender differences except a lower plasma level of the activation peptide in men compared with women (103.8% versus 110.7%, P=0.01). A significant correlation was observed between the activation peptide and intact TAFI (r=0.39, P<0.001), between TAFIa and the activation peptide (r=0.19, P=0.001), but not between TAFIa and intact TAFI. Strikingly, intact TAFI, TAFIa, and the activation peptide showed significant correlations to cholesterol (ie, r=0.15, P<0.05; r=0.12, P<0.05 and r=0.27, P<0.001, respectively) and triglycerides (ie, r=0.14, P<0.05; r=0.14, P<0.05; and r=0.23, P<0.001, respectively), whereas no significant associations were detected for high-density lipoprotein, insulin, blood pressure, or body mass index, with the exception for a correlation between the activation peptide and body mass index (r=0.13, P=0.03). Subjects with hyperlipidemia had significantly higher plasma levels of both the activation peptide (109.2 versus 95.5; P<0.001) and TAFIa (112.1 versus 103.3; P=0.03), whereas the difference for intact TAFI antigen did not reach the conventional level of statistical significance (92.5 versus 87.9; P=0.07). Twenty-eight subjects were on lipid-lowering therapy. If they were excluded from the analysis, similar correlations between the 3 TAFI variables and cholesterol as well as triglycerides were observed, and differences between subjects with hyperlipidemia and normolipidemia remained (activation peptide 109.9 versus 95.5, P<0.001; TAFIa 115.0 versus 103.3, P=0.01; intact TAFI 92.6 versus 87.9, P=0.03). There were no significant differences for any TAFI variable with regard to hypertension, diabetes, or smoking status (P>0.2 throughout). The activation peptide showed significant correlations to tissue-type plasminogen activator antigen (r=0.17, P<0.01), plasminogen activator inhibitor-1 (r=0.19, P<0.01), and fibrinogen (r=0.18, P<0.01), and an inverse correlation to tissue-type plasminogen activator activity (r=–0.17, P<0.01), whereas intact TAFI and TAFIa revealed no significant correlation.


*    Discussion
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 *Discussion
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TAFIa is formed on activation of TAFI with T/TM or plasmin by release of the activation peptide and exerts an antifibrinolytic effect. Two polymorphisms (ie, Ala147Thr and Thr325Ile), resulting in the existence of 4 different isoforms, have been described.29,30 It has been suggested that increased TAFI plasma levels or increased activation is associated with cardiovascular events such as thrombosis, angina pectoris, and myocardial infarction.11–15 Therefore, different clinical studies have aimed to investigate the relationship between TAFI and cardiovascular events, based on the determination of TAFI levels in plasma. TAFI levels are determined either by immunologic assays, presumed to measure "total" TAFI antigen but not characterized in detail, or by activation of the zymogen followed by determination of total enzymatic activity reflecting total activatable TAFI levels.11–15 Alternatively, clot lysis experiments31 or the use of fluorescently labeled plasminogen32 may also be used to quantitate TAFI levels or TAFIa activity, respectively. Apart from a previous study16 demonstrating that the reactivity of a majority of available assays is partially dependent on the Thr325 polymorphism, it should be realized that interpretation of TAFI antigen levels as determined with noncharacterized immunologic assays is also quite ambiguous because TAFI can occur in different forms (TAFI, TAFIa, TAFIai, fragmentation products of TAFIai, released AP). Therefore, there is currently a strong need for well-characterized immunoassays for the evaluation of the levels of different TAFI forms. In addition, immunologic measurement of the extent of TAFI activation, either by measurement of the released AP or by measurement of TAFIa (ie, TAFIa and/or TAFIai), could be a more relevant parameter. Therefore, our objective was to develop well-characterized assays that allow measurement of the extent of TAFI activation.

In this study, a panel of sandwich-type ELISAs was screened for their reactivity toward TAFI before and after activation. Three types of ELISAs could be distinguished. From the group of ELISAs, exclusively reacting with intact nonactivated TAFI, MA-T12D11/MA-T30E5-HRP was selected for further characterization. The response of purified TAFI or human plasma in MA-T12D11/MA-T30E5-HRP immediately decreases on activation, reaching a minimum at a time point corresponding with the time of exhaustion of activatable TAFI (Figure 2B). Taking all evidence together, including Western blotting experiments (data not shown), it can be concluded that this particular ELISA specifically reacts with intact, nonactivated TAFI.

From the group of ELISAs reacting with TAFIa, 2 ELISAs were selected, ie, MA-T12D11/MA-T18A8-HRP and MA-T30E5/MA-T17D7-HRP. These ELISAs reacted differentially on activation of TAFI (in plasma or purified). The response in MA-T12D11/MA-T18A8-HRP increases reaching a maximum plateau whereas the increased response on activation in the MA-T30E5/MA-T17D7-HRP is immediately followed by a strong decrease (Figure 2). This reveals that these 2 ELISAs reacted with different fragments from TAFI. To explore this ambiguity, we cloned, expressed, and purified the AP of human TAFI. This recombinant AP reacted only in MA-T12D11/MA-T18A8-HRP, although with a much lower reactivity compared with activated TAFI. The response of purified TAFI or human plasma in MA-T12D11/MA-T18A8-HRP increases on activation, reaching a maximum plateau value at a time-point corresponding with the time of exhaustion of activatable TAFI. All these data demonstrate that MA-T12D11/MA-T18A8-HRP specifically reacts with the released AP from TAFI. It should be mentioned that in contrast to purified TAFI, the response observed for plasma on activation remained stable after reaching the maximum. This is most likely caused by stabilization of the released AP in a plasma environment. This was confirmed by the observation that the response of activated pTAFI added to TAFI-depleted plasma remained stable (data not shown).

The response of human plasma (Figure 2B) in MA-T30E5/MA-T17D7-HRP increases transiently on activation, reaching a maximum after 10 minutes. Because the response decreases after reaching the maximum, we can conclude that this ELISA reacts with the 36-kDa fragment of TAFI and not with the 25-kDA and 11-kDa products. Strikingly, the increase in MA-T30E5/MA-T17D7-HRP occurs parallel with the increase in TAFIa enzymatic activity. The delay of the decreasing response in the ELISA compared with the decrease in enzymatic activity strongly indicates that MA-T30E5/MA-T17D7-HRP reacts with both TAFIa and TAFIai. This is also confirmed by the analysis of the fragmentation products by SDS-PAGE illustrating that the reactivity in the ELISA is associated with the presence of the 36-kDa fragment and decreases concomitantly with the appearance of the 25-kDa and 11-kDa products. Analysis of 2 recombinant isoforms (ie, TAFI-Ala147Thr325 and TAFI-Ala147Ile325) revealed similar responses in the 3 ELISAs. However, the response of the Ile325 isoform decreased significantly slower in the MA-T30E5/MA-T17D7-HRP ELISA. This is in agreement with the increased stability of Ile325 isoforms at 37°C.8

When using plasmin to activate TAFI, a similar response was observed except that the response in the MA-T30E5/MA-T17D7-HRP ELISA paralleled the time course of the generation and loss of TAFIa activity (data not shown). This is in agreement with Marx et al5 describing that on plasmin-mediated activation of TAFI, the appearance and disappearance of the 36-kDa fragment (ie, TAFIa/ai, followed by SDS-PAGE) matched the time course of generation and loss of TAFIa activity. This provides further evidence that the MA-T30E5/MA-T17D7-HRP ELISA reacts with both TAFIa and TAFIai.

The 3 characterized ELISAs were also used to follow the antigen levels of TAFI and TAFI fragments during clot formation and clot dissolution. When using 5 nM TM, TAFI is completely activated during clot formation, whereas in the presence of 0.5 nM TM, there is partial activation during clotting followed by subsequent activation during fibrinolysis (Figure 3). This is also reflected by the antigen levels of the released AP and TAFIa. With 5 nM TM, released AP levels immediately increase to 100% and TAFIa levels transiently to 80%, whereas with 0.5 nM TM the released AP increases partially during clot formation and subsequently during clot lysis. In the presence of 0.5 nM TM, the antigen levels of TAFIa follow the pattern of TAFIa activity with a first peak during clot formation and a second lower peak during clot lysis. These results are in agreement with the biphasic pattern of TAFIa generation during in vitro clot lysis in human plasma as described by Leurs et al.28 Leurs et al also found a thrombomodulin-dependent increase of the first activity peak during clot formation, which is also confirmed by our data (Figure 3). 23

Analysis of 300 plasma samples revealed correlations between cholesterol and triglycerides for TAFI as well as TAFIa and AP. These findings are in agreement with the data of Silveira et al, who reported statistical correlations between TAFI and cholesterol contents of plasma and all lipoprotein fractions but high-density lipoprotein and a tendency of TAFI to correlate with the triglyceride contents of all lipoprotein fractions in healthy subjects.11 In the current study, higher plasma levels of both the activation peptide and TAFIa were observed for subjects with hyperlipidemia, compared with subjects with normolipidemia. In contrast, no association between total TAFI antigen levels and hyperlipidemia could be observed. This suggests that there is an increased activation state in hyperlipidemia. These findings provide evidence that indeed measurement of the extent of activation is a more relevant parameter in the search for an association between TAFI levels and risk markers for cardiovascular diseases. Moreover, evidence that measuring the extent of TAFI activation is more relevant is supported by the threshold phenomenon stating that it is not the total amount of TAFI protein but the amount of TAFIa that plays a critical role in fibrinolysis.23 The use of this kind of ELISA in large epidemiological studies might refute the contradictory results observed previously.11–15 Measuring the released activation peptide (MA-T12D11/MA-T18A8-HRP ELISA), independent of intrinsic stability of TAFIa and also exhibiting the strongest correlation with hyperlipidemia, is the preferred assay.

In conclusion, we described the development of 3 different MA-based ELISAs. The MA-T12D11/MA-T30E5-HRP specifically reacts with nonactivated TAFI, the MA-T12D11/MA-T18A8-HRP specifically reacts with the released activation peptide of TAFI and the MA-T30E5/MA-T17D7-HRP specifically reacts with TAFIa and TAFIai. Application in various settings (clot lysis, activation of plasma, clinical samples) has demonstrated that the 2 latter assays reliably provide a measure for the extent of TAFI activation and that the released activation peptide is strongly associated with hyperlipidemia.


*    Acknowledgments
 
This study is supported by research grants OT/02/55 of the research fund of K. U. Leuven and by 1.5.019.04N of the Fund for Scientific Research-Flanders. A.G. is a postdoctoral fellow of the Fund for Scientific Research-Flanders.

Received July 4, 2005; accepted November 17, 2005.


*    References
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 up arrowAbstract
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 *References
 

  1. Hendriks D, Scharpe S, van-Sande M, Lommaert MP. Characterisation of a carboxypeptidase in human serum distinct from carboxypeptidase N. J Clin Chem Clin Biochem. 1989; 27: 277–285.[Medline] [Order article via Infotrieve]
  2. Eaton DL, Malloy BE, Tsai SP, Henzel W, Drayna D. Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma. J Biol Chem. 1991; 266: 21833–21838.[Abstract/Free Full Text]
  3. Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. J Biol Chem. 1995; 270: 14477–14484.[Abstract/Free Full Text]
  4. Bouma BN, Marx PF, Mosnier LO, Meijers JM. Thrombin-activatable fibrinolysis inhibitor (TAFI, plasma procarboxypeptidase B, procarboxypeptidase R, procarboxypeptidase U). Thrombosis Research. 2001; 101: 329–354.[CrossRef][Medline] [Order article via Infotrieve]
  5. Marx PF, Dawson PE, Bouma BN, Meijers JC. Plasmin-mediated activation and inactivation of thrombin-activatable fibrinolysis inhibitor. Biochemistry. 2002; 41: 6688–6696.[CrossRef][Medline] [Order article via Infotrieve]
  6. Wang W, Boffa MB, Bajzar L, Walker JB, Nesheim ME. A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor. J Biol Chem. 1998; 273: 27176–27181.[Abstract/Free Full Text]
  7. Boffa MB, Wang W, Bajzar L, Nesheim ME. Plasma and recombinant thrombin-activable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/thrombomodulin-dependent activation, thermal stability, and enzymatic properties. J Biol Chem. 1998; 273: 2127–2135.[Abstract/Free Full Text]
  8. Schneider M, Boffa MB, Stewart R, Rahman M, Koschinsky M, Nesheim M. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme. J Biol Chem. 2002; 277: 1021–1030.[Abstract/Free Full Text]
  9. Boffa MB, Bell R, Stevens WK, Nesheim ME. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor. J Biol Chem. 2000; 275: 12868–12878.[Abstract/Free Full Text]
  10. Schatteman KA, Goossens FJ, Scharpe SS, Hendriks DF. Proteolytic activation of purified human procarboxypeptidase U. Clin Chim Acta. 2000; 292: 25–40.[CrossRef][Medline] [Order article via Infotrieve]
  11. Silveira A, Schatteman K, Goossens F, Moor E, Scharpe S, Stromqvist M, Hendriks D, Hamsten A. Plasma procarboxypeptidase U in men with symptomatic coronary artery disease. Thromb Haemost. 2000; 84: 364–368.[Medline] [Order article via Infotrieve]
  12. van-Tilburg NH, Rosendaal FR, Bertina RM. Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis. Blood. 2000; 95: 2855–2859.[Abstract/Free Full Text]
  13. Morange PE, Juhan Vague I, Scarabin PY, Alessi MC, Luc G, Arveiler D, Ferrieres J, Amouyel P, Evans A, Ducimetiere P. Association between TAFI antigen and Ala147Thr polymorphism of the TAFI gene and the angina pectoris incidence. Thromb Haemost. 2003; 89: 554–560.[Medline] [Order article via Infotrieve]
  14. Montaner J, Ribo M, Monasterio J, Molina CA, Alvarez-Sabin J. Thrombin-activable fibrinolysis inhibitor levels in the acute phase of ischemic stroke. Stroke. 2003; 34: 1038–1040.[Abstract/Free Full Text]
  15. Juhan Vague I, Morange PE, Aubert H, Henry M, Aillaud MF, Alessi MC, Samnegard A, Hawe E, Yudkin J, Margaglione M, Di-Minno G, Hamsten A, Humphries SE. Plasma thrombin-activatable fibrinolysis inhibitor antigen concentration and genotype in relation to myocardial infarction in the north and south of Europe. Arterioscler Thromb Vasc Biol. 2002; 22: 867–873.[Abstract/Free Full Text]
  16. Gils A, Alessi MC, Brouwers E, Peeters M, Marx P, Leurs J, Bouma B, Hendriks D, Juhan-Vague I, Declerck PJ. Development of a genotype 325-specific proCPU/TAFI ELISA. Arterioscler Thromb Vasc Biol. 2003; 23: 1122–1127.[Abstract/Free Full Text]
  17. Malyszko J, Malyszko JS, Hryszko T, Mysliwiec M. Thrombin activatable fibrinolysis inhibitor (TAFI) and markers of endothelial cell injury in dialyzed patients with diabetic nephropathy. Thromb Haemost. 2004; 91: 480–486.[Medline] [Order article via Infotrieve]
  18. Saibeni S, Bottasso B, Spina L, Bajetta M, Danese S, Gasbarrini A, de Franchis R, Vecchi M. Assessment of thrombin-activatable fibrinolysis inhibitor (TAFI) plasma levels in inflammatory bowel diseases. Am J Gastroenterol. 2004; 99: 1966–1970.[CrossRef][Medline] [Order article via Infotrieve]
  19. Eichinger S, Schonauer V, Weltermann A, Minar E, Bialonczyk C, Hirschl M, Schneider B, Quehenberger P, Kyrle PA. Thrombin-activatable fibrinolysis inhibitor and the risk for recurrent venous thromboembolism. Blood. 2004; 103: 3773–3776.[Abstract/Free Full Text]
  20. Bajzar L, Nesheim ME, Tracy PB. The profibrinolytic effect of activated protein C in clots formed from plasma is TAFI-dependent. Blood. 1996; 88: 2093–2100.[Abstract/Free Full Text]
  21. 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: 829–835.[Medline] [Order article via Infotrieve]
  22. Stromqvist M, Schatteman K, Leurs J, Verkerk R, Andersson JO, Johansson T, Scharpe S, Hendriks D. Immunological assay for the determination of procarboxypeptidase U antigen levels in human plasma. Thromb Haemost. 2001; 85: 12–17.[Medline] [Order article via Infotrieve]
  23. Leurs J, Nerme V, Sim Y, Hendriks D. Carboxypeptidase U (TAFIa) prevents lysis from proceeding into the propagation phase through a threshold-dependent mechanism. J Thromb Haemost. 2004; 2: 416–423.[CrossRef][Medline] [Order article via Infotrieve]
  24. Walker JB, Bajzar L. The intrinsic threshold of the fibrinolytic system is modulated by basic carboxypeptidases, but the magnitude of the antifibrinolytic effect of activated thrombin-activable fibrinolysis inhibitor is masked by its instability. J Biol Chem. 2004; 279: 27896–27904.[Abstract/Free Full Text]
  25. Valnickova Z, Thogersen IB, Christensen S, Chu CT, Pizzo SV, Enghild JJ. Activated human plasma carboxypeptidase B is retained in the blood by binding to alpha2-macroglobulin and pregnancy zone protein. J Biol Chem. 1996; 271: 12937–12943.[Abstract/Free Full Text]
  26. Broze G-JJ, Higuchi DA. Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma. Blood. 1996; 88: 3815–3823.[Abstract/Free Full Text]
  27. 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: 780–784.[CrossRef][Medline] [Order article via Infotrieve]
  28. Leurs J, Wissing BM, Nerme V, Schatteman K, Bjorquist P, Hendriks D. Different mechanisms contribute to the biphasic pattern of carboxypeptidase U (TAFIa) generation during in vitro clot lysis in human plasma. Thromb Haemost. 2003; 89: 264–271.[Medline] [Order article via Infotrieve]
  29. Zhao L, Morser J, Bajzar L, Nesheim M, Nagashima M. Identification and characterization of two thrombin-activatable fibrinolysis inhibitor isoforms. Thromb Haemost. 1998; 80: 949–955.[Medline] [Order article via Infotrieve]
  30. Brouwers GJ, Vos HL, Leebeek FW, Bulk S, Schneider M, Boffa M, Koschinsky M, van-Tilburg NH, Nesheim ME, Bertina RM, Gomez-Garcia EB. A novel, possibly functional, single nucleotide polymorphism in the coding region of the thrombin-activatable fibrinolysis inhibitor (TAFI) gene is also associated with TAFI levels. Blood. 2001; 98: 1992–1993.[Free Full Text]
  31. Guimaraes AH, Bertina RM, Rijken DC. A new functional assay of thrombin activatable fibrinolysis inhibitor. J Thromb Haemost. 2005; 3: 1284–1292.[CrossRef][Medline] [Order article via Infotrieve]
  32. Neill EK, Stewart RJ, Schneider M, Nesheim ME. A functional assay for measuring activated thrombin-activatable fibrinolysis inhibitor in plasma. Anal Biochem. 2004; 330: 332–341.[CrossRef][Medline] [Order article via Infotrieve]



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