Proteolytic activation of purified human procarboxypeptidase U

Schatteman, Katinka; Goossens, Filip; Scharpé, Simon; Hendriks, Dirk

doi:10.1016/s0009-8981(99)00205-3

Cited by 50 publications

(37 citation statements)

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“…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 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.…”

Section: Construction Selection and Characterization Of Sandwich-tymentioning

confidence: 99%

“…6 TAFIa is highly unstable in vitro (half-life at 37°C between 8 to 15 minutes). [7][8][9][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.…”

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confidence: 99%

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Development of ELISAs Measuring the Extent of TAFI Activation

Ceresa

Brouwers

Peeters

et al. 2006

ATVB

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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; PϽ0.001) and TAFIa (112.1 versus 103.3; Pϭ0.03), and not of TAFI antigen (92.5 versus 87.9; Pϭ0.07) (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. ; 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][8][9][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 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 g/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...

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Section: Construction Selection and Characterization Of Sandwich-tymentioning

confidence: 99%

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confidence: 99%

Development of ELISAs Measuring the Extent of TAFI Activation

Ceresa

Brouwers

Peeters

et al. 2006

ATVB

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“…drawbacks proCPU is an inactive zymogen; therefore, accurate measurement of proCPU concentrations by activity-based assays requires quantitative conversion of the zymogen to CPU. Thus, a very well standardized activation protocol is required (18,19 ). Because most of the activity assays use synthetic substrates that are not selective for CPU, the interfering activity of plasma CPN must also be accounted for (33 ).…”

Section: Advantagesmentioning

confidence: 99%

“…Thrombin is believed to be the activator of proCPU in vivo, albeit with low catalytic efficiency in vitro. Thrombomodulin stimulates the rate of proCPU activation by thrombin 1250-fold (18,19 ). proCPU circulates in plasma at a concentration of 73-250 nmol/L (4.4 -15.0 g/mL) (20 -23 ).…”

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Measurement of Procarboxypeptidase U (TAFI) in Human Plasma: A Laboratory Challenge

Willemse

Hendriks²

2006

Clinical Chemistry

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Background:The importance of carboxypeptidase U (CPU) as a novel regulator of the fibrinolytic rate has attracted much interest during recent years. CPU circulates in plasma as a zymogen, proCPU, that can be activated by thrombin, thrombin-thrombomodulin (TTm), or plasmin. Given that the proCPU concentration in plasma is far below its K m for activation by the T-Tm complex, the formation of CPU will be directly proportional to the proCPU concentration. A low or high proCPU plasma concentration might therefore tip the balance between profibrinolytic and antifibrinolytic pathways and thereby cause a predisposition to bleeding or thrombosis. Content: To measure plasma proCPU concentrations, different methods have been developed based on 2 different principles: antigen determination and measurement of CPU activity after quantitative conversion of the proenzyme to its active form by addition of T-Tm. The major drawbacks that should be kept in mind when analyzing clinical samples by both principles are reviewed. Conclusions: proCPU is a potential prothrombotic risk factor. Evaluation of its relationship with thrombosis requires accurate assays. Many assays used in different clinical settings are inadequately validated, forcing reconsideration of conclusions made in these reports.

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“…Even though TAFIa is a powerful antifibrinolytic enzyme, there are no known inhibitors of TAFIa in human plasma. Instead, TAFIa is highly unstable, with reported halflives at 37°C of 8 -9 (7,8) and 15 min (9). Whereas TAFIa can also be cleaved by thrombin at Arg-302, this cleavage is subsequent to the spontaneous structural changes corresponding to inactivation of the enzyme (8,10) and is therefore probably not relevant to the regulation of the enzyme.…”

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Two Naturally Occurring Variants of TAFI (Thr-325 and Ile-325) Differ Substantially with Respect to Thermal Stability and Antifibrinolytic Activity of the Enzyme

Schneider¹,

Boffa²,

Stewart³

et al. 2002

Journal of Biological Chemistry

162

201

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Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like zymogen that is activated toTAFIa by plasmin, thrombin, or the thrombin-thrombomodulin complex. The enzyme TAFIa attenuates clot lysis by removing lysine residues from a fibrin clot. Screening of nine human cDNA libraries indicated a common variation in TAFI at position 325 (Ile-325 or Thr-325). This is in addition to the variation at amino acid position 147 (Ala-147 or Thr-147) characterized previously. Thus, four variants of TAFI having either Ala or Thr at position 147 and either Thr or Ile at position 325 were stably expressed in baby hamster kidney cells and purified to homogeneity. The kinetics of activation of TAFI by thrombin/thrombomodulin were identical for all four variants; however, Ile at position 325 extended the half-life of TAFIa from 8 to 15 min at 37°C, regardless of the residue at position 147. In clot lysis assays with thrombomodulin and the TAFI variants, or with pre-activated TAFI variants, the Ile-325 variants exhibited an antifibrinolytic effect that was 60% greater than the Thr-325 variants. Similarly, in the absence of thrombomodulin, the Ile-325 variants exhibited an antifibrinolytic effect that was 30 -50% greater than the Thr-325 variants. In contrast, the variation at position 147 had little if any effect on the antifibrinolytic potential of TAFIa. The increased antifibrinolytic potential of the Ile-325-containing TAFI variants reflects the fact that these variants have an increased ability to mediate the release of lysine from partially degraded fibrin and suppress plasminogen activation. These findings imply that individuals homozygous for the Ile-325 variant of TAFI would likely have a longer lived and more potent TAFIa enzyme than those homozygous for the Thr-325 variant. Thrombin-activable fibrinolysis inhibitor (TAFI)1 is a zymogen found in human plasma (1), which is also known as plasma procarboxypeptidase B (2) and procarboxypeptidase U (3). It can be activated by thrombin (1), plasmin (4), or the thrombinthrombomodulin complex (5) to the carboxypeptidase B-like enzyme, TAFIa. When exposed to a fibrin clot, TAFIa catalyzes the removal of carboxyl-terminal lysines, thereby diminishing the cofactor activity for plasminogen activation (6). Less efficient plasminogen activation on the fibrin clot corresponds to prolongation of fibrinolysis, and in this way TAFIa can serve as a potent antifibrinolytic enzyme. Studies performed using an in vitro human plasma model have found that clot lysis times can be attenuated up to 3-fold in the presence of TAFIa as compared with clots lysed in the absence of TAFIa (5).Activation of TAFI is catalyzed only slowly by thrombin alone; however, in the presence of thrombin/thrombomodulin, the efficiency of activation is increased 1000-fold. Despite the large thrombomodulin dependence of TAFI activation, in vitro clot lysis assays done in the absence of thrombomodulin still exhibit prolonged clot lysis times as compared with similar assays performed with TAFI-depleted plas...

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Proteolytic activation of purified human procarboxypeptidase U

Cited by 50 publications

References 20 publications

Development of ELISAs Measuring the Extent of TAFI Activation

Development of ELISAs Measuring the Extent of TAFI Activation

Measurement of Procarboxypeptidase U (TAFI) in Human Plasma: A Laboratory Challenge

Two Naturally Occurring Variants of TAFI (Thr-325 and Ile-325) Differ Substantially with Respect to Thermal Stability and Antifibrinolytic Activity of the Enzyme

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