Immunological Assay for the Determination of Procarboxypeptidase U Antigen Levels in Human Plasma

Strömqvist, Mats; Schatteman, Katinka; Leurs, Judith; Verkerk, Robert; Andersson, Jan-Olof; Johansson, Tomas; Scharpé, Simon; Hendriks, Dirk

doi:10.1055/s-0037-1612656

Cited by 50 publications

(51 citation statements)

References 18 publications

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“…Some research groups showed a good correlation between ELISAs and activity assays (21)(22)24 ). The most important advantage of activity assays compared with antigen assays is that the 2 described polymorphisms in the coding region of proCPU (Thr147Ala and Thr325Ile) have similar activation kinetics and the activated enzymes have similar carboxypeptidase B-like activity toward small synthetic substrates (26,28 ).…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

“…proCPU concentrations in pooled plasma varied from 73 nmol/L (4.4 g/mL) to 250 nmol/L (15 g/mL) (20 -22, 25 ). Strö mqvist et al (22 ) measured the plasma proCPU concentration in 479 apparently healthy volunteers and found a mean (SD) of 13.4 (2.5) g/mL, or 223 nmol/L.…”

Section: Drawbacksmentioning

confidence: 99%

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

Willemse

Hendriks²

2006

Clinical Chemistry

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“…6); as such, a change in concentration of TAFI in plasma would result in a corresponding change in the rate of TAFI activation by thrombin or thrombin-thrombomodulin. Indeed, a strong correlation between plasma TAFI concentrations and in vitro clot lysis times has been observed (35,37).…”

Section: Fig 5 Mutagenesis Of the Gre In The Human Tafi Promotermentioning

confidence: 99%

Acute Phase Mediators Modulate Thrombin-activable Fibrinolysis Inhibitor (TAFI) Gene Expression in HepG2 Cells

Boffa¹,

Hamill²,

Maret³

et al. 2003

Journal of Biological Chemistry

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Thrombin-activable fibrinolysis inhibitor (TAFI) has recently been identified as a positive acute phase protein in mice, an observation that may have important implications for the interaction of the coagulation, fibrinolytic, and inflammatory systems. Activated TAFI (TAFIa) inhibits fibrinolysis by removing the carboxylterminal lysines from partially degraded fibrin that are important for maximally efficient plasminogen activation. In addition, TAFIa has been shown to be capable of removing the carboxyl-terminal arginine residues from the anaphylatoxins and bradykinin, thus implying a role for the TAFI pathway in the vascular responses to inflammation. In the current study, we investigated the ability of acute phase mediators to modulate human TAFI gene expression in cultured human hepatoma (HepG2) cells. Surprisingly, we found that treatment of HepG2 cells with a combination of interleukin (IL)-1␤ and IL-6 suppressed endogenous TAFI mRNA abundance in HepG2 cells (ϳ60% decrease), while treatment with IL-1␤ or IL-6 alone had no effect. Treatment with IL-1␤ and/or IL-6 had no effect on TAFI promoter activity as measured using a luciferase reporter plasmid containing the human TAFI 5 -flanking region, whereas treatment with IL-1␤ and IL-6 in combination, but not alone, decreased the stability of the endogenous TAFI mRNA. Treatment with the synthetic glucocorticoid dexamethasone resulted in a 2-fold increase of both TAFI mRNA levels and promoter activity. We identified a functional glucocorticoid response element (GRE) in the human TAFI promoter between nucleotides ؊92 and ؊78. The GRE was capable of binding the glucocorticoid receptor, as assessed by gel mobility shift assays, and mutation of this element markedly decreased the ability of the TAFI promoter to be activated by dexamethasone.Thrombin activable fibrinolysis inhibitor (TAFI) 1 was first identified in 1989 by two independent groups as a basic carboxypeptidase present in fresh serum that was distinct from the constitutive basic carboxypeptidase N (1, 2). By virtue of the intrinsic instability of this enzyme, whose activity disappeared within 2 h upon incubation at 37°C, Hendriks et al.(1) designated the novel activity "unstable" carboxypeptidase or carboxypeptidase U (1). Campbell and Okada (2) determined that the enzyme removed arginine residues from substrates more efficiently than lysines and therefore designated it carboxypeptidase R (2). In 1991, Eaton et al. (3) isolated a cDNA encoding the zymogen form of the enzyme and found that it was highly homologous to pancreatic procarboxypeptidase B. Bajzar et al. (4) independently isolated a protein on the basis of its ability to inhibit fibrinolysis in the setting of sustained activation of the coagulation cascade; on the basis of this property, they named the protein TAFI. Amino acid sequence analysis of TAFI revealed it to be identical to plasma procarboxypeptidase B and procarboxypeptidases U and R. TAFI can be activated by thrombin (4), plasmin (5), and thrombin in complex with thrombomodulin (6), wi...

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“…In vitro human plasma models have measured a half-maximal antifibrinolytic effect at roughly 1 nM of the enzyme. Even though plasma TAFI concentrations have been reported as ranging between 75 and 250 nM (11)(12)(13), a maximum antifibrinolytic effect is reached at about 20 nM TAFIa. Therefore, only a small fraction of the potential plasma TAFI pool needs to be activated to mediate a pronounced antifibrinolytic effect.…”

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confidence: 99%

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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Immunological Assay for the Determination of Procarboxypeptidase U Antigen Levels in Human Plasma

Cited by 50 publications

References 18 publications

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

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

Acute Phase Mediators Modulate Thrombin-activable Fibrinolysis Inhibitor (TAFI) Gene Expression in HepG2 Cells

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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