[Frontiers in Bioscience 16, 2427-2450, June 1, 2011]
An update on the role of carboxypeptidase U (TAFIa) in fibrinolysis
Evelien Heylen1, Johan Willemse1, Dirk Hendriks1
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TABLE OF CONTENTS
1. ABSTRACT
Since its discovery more than 20 years ago, a lot has been revealed about the biochemistry and physiological behaviour of carboxypeptidase U (CPU). Recent advances in CPU research include the unravelling of the crystal structure of proCPU and revealing the molecular mechanisms for the marked instability of the active enzyme, CPU. The recent development of two highly sensitive assays has cleared the path toward the direct measurement of CPU in circulation or the determination of CPU generation, rather than the measurement of total proCPU concentration in plasma. Finally, since CPU is known to have a prominent bridging function between coagulation and fibrinolysis, the development of CPU inhibitors as profibrinolytic agents is an attractive new concept and has gained a lot of interest from several research groups and from the pharmaceutical industry. These recent advances in CPU research are reviewed in this literature update.
2. INTRODUCTION
The coagulation and fibrinolytic systems safeguard the patency of the vasculature and surrounding tissues. Both cascades have long been considered as separate entities but the discovery of procarboxypeptidase U (proCPU) or thrombin activatable fibrinolysis inhibitor (TAFI) greatly improved our understanding of cross regulation of both systems.
Carboxypeptidase U (CPU) was first described in 1989 by Hendriks et al. who reported the presence of a novel unstable basic carboxypeptidase in fresh human serum (1). This enzyme was named procarboxypeptidase U (proCPU), where the 'U' stands for 'unstable'. This discovery was confirmed by three independent research groups. Campbell et al. named the newly identified carboxypeptidase proCPR, since it showed a preference for arginine (R) containing substrates (2). In 1991, Eaton et al. provided a first clue of the important role of CPU in fibrinolysis. This group was able to purify a new plasminogen-binding protein (3). After isolation of the cDNA and sequence analysis, the protein was named plasma procarboxypeptidase B (plasma proCPB), based on its high sequence similarity to the well-known pancreatic CPB. The binding of the proenzyme to plasminogen, combined with the role of C-terminal lysine residues in the binding and activation of plasminogen on fibrin suggested a role for plasma CPB in fibrinolysis (3). The final link between coagulation and fibrinolysis was disclosed by Bajzar et al. in 1995 (4). They found that the antifibrinolytic effect of thrombin during fibrinolysis was due to the activation of a proenzyme, which they named thrombin activatable fibrinolysis inhibitor (TAFI) (4). Amino-terminal sequencing demonstrated that proCPU, proCPR, plasma proCPB and TAFI are identical (3-6).
Venous and arterial thromboembolism is the largest cause of disease and death in the Western World. Therapy available today includes thrombolytics, anticoagulants and antiplatelet drugs. Because of its prominent bridging function between coagulation and fibrinolysis, the development of CPU inhibitors as profibrinolytic agents is an attractive new concept (7). Furthermore, since the coagulation cascade is unaffected, CPU inhibition may result in fewer bleeding complications than conventional therapy. In recent years several small synthetic and naturally occurring CPU inhibitors have been evaluated in animal thrombosis models and existing in vivo data are intriguing and call for further evaluation in humans (7).
3. BIOCHEMICAL CHARACTERIZATION OF PROCPU
3.1. General considerations
Metallocarboxypeptidases (EC 3.4.17) are a group of enzymes capable of cleaving a single amino acid from the C-terminus of peptide or protein substrates. Their catalytic activity depends on a zinc atom in the active site (8). CPU (EC 3.4.17.20) is a basic metallocarboxypeptidase, meaning that only basic carboxy-terminal amino acids i.e. arginine (Arg) and lysine (Lys) are cleaved off from peptides and proteins (1, 8-11).
ProCPU is a zymogen, circulating in plasma with an apparent molecular mass of 60 kDa on SDS-page (3, 12, 13). ProCPU is synthesized in the liver as a prepropeptide consisting of 423 amino acids (aa), composed of a 22 aa signal peptide, a 92 aa activation peptide and a 309 aa catalytic domain. During secretion, the N-terminal signal peptide is efficiently cleaved off, resulting in the release of proCPU in circulation (3). ProCPU is a glycoprotein with 4 asparagine (Asn) linked glycosylation sites in the activation peptide (Asn22, Asn51, Asn63 and Asn86) (3, 4, 13, 14). Also in the catalytic domain a N-linked glycosylation site (Asn219) has been described, but since it is entirely buried in the protein structure, glycosylation is restricted to the activation peptide (13). After a single cleavage at Arg92, the highly glycosylated activation peptide is cleaved off, releasing the 36 kDa catalytic unit, CPU (3, 4, 13, 14).
Synthesis of proCPU is not entirely restricted to the hepatocytes. Also in megakaryocytes proCPU synthesis has been described during the process of megakaryocytopoiesis (15). This proCPU is present in the α-granules of circulating platelets in a concentration of 50 ng/1 x 109 platelets and is released upon platelet activation (15). Recently, it has been demonstrated that platelet derived proCPU is capable of attenuating platelet-rich thrombus lysis in vitro independently of plasma proCPU, and thus could be significant in regions of vascular damage or pathological thrombosis, where activated platelets are known to accumulate (16).
3.2. Genetic features
The proCPU gene, denoted the CPB2 gene, is located on chromosome 13q14.11 (5, 17). The 11 encoding exons stretch over approximately 48 kb of genomic DNA (5, 17). Transcription is initiated from multiple sites in the 5'-flanking region, which does not seem to carry a conserved TATA box (17). Transcription seems to be under control of liver-specific transcription factors, such as C/EBP (18), and also hepatocyte nuclear factor-1α (HNF-1α) is involved in the liver specific expression of CPB2 (19). However, proCPU synthesis is not restricted to the liver, since it is also reported in megakaryocyt cell lines (15). Several single nucleotide polymorphisms (SNPs) have been identified in the proCPU gene (20-23). Polymorphisms in the 5'-flanking region could influence the binding of specific transcription factors and thus promoter activity, whereas mRNA abundance could be altered by SNPs in the 3'-untranslated region (24). Of the polymorphisms in the coding regions, two result in an amino acid substitution, i.e. Thr147Ala and Thr325Ile (21, 23, 25), where only the latter has an influence on the enzyme characteristics (25). These polymorphisms were thought to be the major cause of proCPU variability seen in a normal population (plasma proCPU concentration ranging between 75 - 275 nM) . However, using antigen assays insensitive to genotype dependent artefacts, Frère et al. reported that only approximately 25 % of the variation can be explained by the proCPU polymorphisms (26). This implicates that non-genetic factors are more important for the explanation of variation in plasma proCPU concentration, such as glucocorticoids, interleukins, sex hormones, etc. (27-32).
3.3. Intrinsic enzymatic activity
A single-site cleavage of proCPU leads to the release of the activation peptide, generating the active enzyme CPU. Although being an inactive zymogen, it has been shown that proCPU can exert some intrinsic enzymatic activity toward small synthetic substrates (33). This feature is shared by proCPA, but is completely absent in proCPB. Comparison of the structure of proCPU with that of proCPA2 and proCPB revealed that, in contrast to the latter, the catalytic sites of proCPU and proCPA2 are in the active conformation (34). This will result in the reported intrinsic enzymatic activity of the zymogen (33).
Further comparison of the three crystal structures revealed a striking difference in the rotation of the activation peptide in proCPU compared to proCPA2 and proCPB (35). In proCPU, the activation peptide is rotated further away from the catalytic cavity as compared to proCPA2 and proCPB, suggesting that the active site in proCPU is more accessible to substrates compared to the other CPs (35). This observation was in line with a recent publication which reported that proCPU does not only show intrinsic activity toward small synthetic substrates, but that the catalytic centre is also accessible for high molecular substrates, thus implying that the zymogen proCPU should also be considered as being an antifibrinolytic enzyme (36). However, in two publications from independent research groups, this finding was contradicted. These papers demonstrated that the zymogen proCPU is not able to down-regulate fibrinolysis and suggested that the previously published results are compromised by in vitro activation of proCPU (37, 38). Additional structural analysis has demonstrated that although slightly larger, the access tunnel to the catalytic site in proCPU is not accessible for high molecular substrates, due to the larger side chains of the activation peptide (35). Further structural and functional research will be necessary to definitely conclude whether the zymogen proCPU has antifibrinolytic properties.
4. CHARACTERISTICS OF CPU
4.1. Generation of CPU
A single-site proteolytic cleavage at Arg92 of proCPU is necessary to generate the active enzyme CPU. There are several enzymes able to activate proCPU in vitro, including thrombin, plasmin, trypsin and neutrophil-derived elastase. However, there remains some debate on which of these proCPU activators are physiologically relevant. Leurs et al. demonstrated that during in vitro clot lysis CPU generation follows a biphasic pattern, where a first peak of CPU activity appeared after initiation of coagulation (through the action of thrombin) and a second rise in CPU activity was observed during fibrinolysis with plasmin as activator (39). Since thrombin generation will usually precede plasmin formation, the importance of plasmin-mediated proCPU activation may be limited (39). With the construction of mutant variants of proCPU resistant to activation by either thrombin or plasmin, Miah et al. were able to confirm these observations (40). In an in vitro clot lysis assay in the absence of thrombomodulin, they demonstrated that thrombin was the only relevant activator of proCPU, under the conditions described, resulting in an attenuation of fibrinolysis (40). The authors suggest that plasmin-mediated proCPU activation becomes more important in situations where there is a reduced fibrinolytic activity and hence a prolonged lysis time (40). Recently, monoclonal antibodies (41, 42), as well as nanobodies (43), against proCPU were developed, which could discriminate between the various modes of proCPU activation. In this respect, these could be regarded as excellent research tools to identify the in vivo relevant activators of proCPU in several pathophysiological conditions (41-43).
The catalytic parameters of proCPU activation by thrombin (IIa) are a Km of 2.14 �M and a kcat of 0.0021 s-1, implying that IIa is a relatively weak activator of proCPU (44). Following the initial phase of coagulation - tissue factor (TF)-pathway or extrinsic pathway - small amounts of IIa are generated, leading to fibrin formation. However, these low amounts of IIa are insufficient for proCPU activation (44-49). In the second phase of coagulation - through the intrinsic pathway - IIa is able to boost its own generation through the activation of factor XI. This positive feedback loop results in the release of high amounts of IIa, leading to proCPU activation (44-49). In the presence of an intact intrinsic pathway of coagulation, the CPU concentration that is generated, will be sufficient to prolong clot lysis substantially (45, 50). Bajzar et al. demonstrated that half-maximal inhibition of clot lysis time was achieved at a CPU concentration of 1 nM (44). Since proCPU is present in plasma in a concentration of 75 - 275 nM, it is clear that even a minimal activation of proCPU will lead to a substantial attenuation of fibrinolysis (44). To demonstrate the importance of the intrinsic pathway in proCPU activation by IIa, Minnema et al. demonstrated that CPU dependent retardation of clot lysis could be attenuated in vivo by neutralization of factor XI through the addition of specific antibodies (51).
In the presence of thrombomodulin (TM), activation of proCPU by IIa is enhanced 1250 times, almost entirely through an effect on the kcat (kcat = 0.4 - 1.2 s-1 in the presence of TM) (44). Since the cofactor TM is able to accelerate proCPU activation by IIa so drastically, it has been postulated that the IIa-TM complex is the physiological activator of proCPU (44). A recent study by Wu et al. identified several positively charged residues of proCPU to be important in its activation by IIa-Tm, through their interaction with negatively charged residues on the C-loop of the TM-EGF-like domain 3 (52). Besides the generation of the antifibrinolytic enzyme CPU, the IIa-TM complex is also responsible for the conversion of protein C to the anticoagulant enzyme activated-protein C (APC) (53). By inactivating factor Va and VIIIa, APC inhibits further IIa formation. These two actions of the IIa-TM complex seem to have opposing effects and are demonstrated to be under strict regulation (Figure 1). (54-56).
A second important activator of proCPU is plasmin. This key enzyme of fibrinolysis is a more potent activator of proCPU compared to IIa alone, with a 10-fold increased catalytic efficiency (kcat/Km = 0.008 �M-1s-1 for plasmin vs. kcat/Km = 0.00098 �M-1s-1 for IIa) . In the presence of glycosaminoglycans, such as are found in the subendothelial matrix upon arterial injury, plasmin-mediated proCPU activation becomes 20-fold more efficient. Nevertheless, the catalytic efficiency for plasmin activation remains 10-fold lower compared to IIa-TM, whereby its physiological relevance remains unclear.
To date, there remains some debate on whether the activation peptide is actually released from or stays attached to the catalytic moiety upon activation of proCPU. In attempts to purify CPU, Mao et al. used a Concanavalin A Sepharose column, which interacts with sugar residues in the protein. Since glycosylation of proCPU is restricted to the activation peptide, purification of CPU is only possible if the activation peptide is still attached to the catalytic domain (57). In an attempt to provide additional evidence that the activation peptide remains noncovalently attached to the catalytic residue upon activation, Buelens et al. performed Western blotting experiments on activated CPU, using an activation peptide specific monoclonal antibody (58). This work showed that the activation peptide stays in close proximity of the catalytic moiety upon activation. Moreover, this interaction was suggested to affect CPU activity (58). The latter was challenged in recent work by Marx et al. where it was demonstrated that the activation peptide was not required for CPU activity, nor did it have an effect on CPU stability (59). The exact role of the highly glycosylated activation peptide remains unclear, and is in need for further investigation. Some of its ascribed functions are ensuring structural integrity of the proenzyme, shielding the catalytic site for physiological substrates, ensuring cellular secretion and increasing the solubility of proCPU (59).
4.2. Instability of CPU
As indicated by its name, CPU is characterized by a profound thermal instability (1). At body temperature, the half-life of CPU is approximately 10 minutes, whereas at room temperature the instability is much less distinct with a half-life of 2 hours. At 0 �C, CPU is highly stable (1, 60-62). Not only a decrease in temperature could result in a significant increase in CPU stability, also the presence of competitive inhibitors (62) and an excess of substrate has been shown to improve CPU stability (63).
It is generally accepted that the marked thermal instability of CPU is the result of conformational changes within the enzyme, rather than a proteolytic cleavage (60, 61). This hypothesis was endorsed by the observation that there are no known physiologic inhibitors of CPU, in contrast to most coagulation and fibrinolytic enzymes. Thrombin is able to proteolytically cleave CPU at Arg302, however this cleavage seems to appear after a conformational change within the enzyme leading to its inactivation (60, 61). This was confirmed with an Arg302Gln mutant showing marked instability at 37 �C, without proteolytic cleavage by thrombin on SDS-page (60, 61). In contrast, proteolytic degradation of proCPU and CPU by plasmin can occur prior to the conformational change (64). However, this represents only a minor pathway for the regulation of CPU activity.
Comparison of CPU to the stable pancreatic CPB - a highly homologous enzyme with 48% sequence similarity - has revealed that the 300-330 amino acid region shows the least sequence similarity (65). In this region, a naturally occurring variation in human proCPU was discovered at position 325 (21). Threonine (Thr) at this position results in a CPU half-life of 7 min, whereas substitution to isoleucine (Ile) at position 325 leads to a 2-fold increase in half-life, i.e. 15 min (25). Extensive mutagenesis studies have been conducted to unravel the mechanism of CPU instability, where all mutations with a stabilizing effect were located in the above mentioned 300-330 amino acid region (60, 61, 65-70).
It wasn't until 2008 that a more in-depth explanation for CPU instability was revealed by Marx et al., with the resolution of the crystal structure (35). The crystal structure of proCPU shows that CPU stability is directly related to a highly dynamic region between residues 296-350. This region includes the cryptic thrombin cleavage site Arg302 and interacts with the activation peptide through hydrophobic interactions between Tyr341 and residues Val35 and Leu39 (35). Through these interactions, the dynamic region is stabilized by reducing its mobility. Upon activation of proCPU, these stabilizing interactions are lost, leading to increased mobility of the dynamic region. This will result in irreversible unfolding of the protein, resulting in disruption of the catalytic site and thus in loss of activity (35). This conformational change also leads to exposure of the cryptic thrombin cleavage site Arg302, making further proteolytic cleavage of inactivated CPU possible. Moreover, the dynamics of this region are markedly reduced by substrates as well as by reversible inhibitors, such as guanidinoethylmercaptosuccinic acid GEMSA, known stabilizers of the active enzyme CPU (35, 62). The structural data of Marx et al. were confirmed by two publications of another group (71, 72).
4.3. CPU at the interface between coagulation and fibrinolysis
After damage of the vasculature, coagulation is initiated through the tissue-factor pathway, leading to the formation of thrombin. This key enzyme of the coagulation will activate soluble fibrinogen generating fibrin monomers, which will ultimately form a stable thrombus after cross-linking through factor XIIIa. In order to prevent excessive clot formation and safeguard the fluidity of the blood, the fibrinolytic cascade will be activated. The key enzyme of the fibrinolysis is plasmin, which will break down the thrombus, leading to the generation of soluble fibrin degradation products (73-75). Plasmin is formed by the action of tissue-type plasminogen activator (t-PA) on its inactive precursor plasminogen and cleaves fibrin specifically after arginine or lysine residues. This initial phase of fibrinolysis results in the generation of partially degraded fibrin containing C-terminal arginine and lysine residues (73-75). These residues participate in a multifaceted positive feedback loop, since they (i) increase the binding-affinity for plasminogen on the fibrin surface and therefore increase plasmin formation (76-79), (ii) convert Glu-plasminogen to Lys-plasminogen which is a much better substrate for t-PA (75) and (iii) protect plasmin from inactivation by alpha2-antiplasmin (80, 81). This feedback loop results in the acceleration phase of fibrinolysis.
High amounts of thrombin - as generated upon the intrinsic pathway of the coagulation - are able to activate proCPU. By continuously removing the C-terminal lysine residues generated by the action of plasmin on fibrin, CPU prevents the fibrinolysis from proceeding into the acceleration phase (77, 78). Through this system, CPU has been shown to push the fibrinolytic system toward the antifibrinolytic state by (i) abrogating the enhanced cofactor activity of partially degraded fibrin on Glu-plasminogen activation, (ii) inhibiting the conversion of Glu-plasminogen to Lys-plasminogen and (iii) promoting the inhibition of plasmin by α2-antiplasmin (77, 78, 80, 81). It was shown independently by two research groups that CPU attenuates fibrinolysis through a threshold dependent mechanism (82, 83). As long as the plasma CPU activity remains above a certain key threshold value - which depends on the t-PA concentration - fibrinolysis stays in its initial phase, only to accelerate once the CPU activity decays to a level below this threshold value (82, 83). The time interval over which the CPU level will stay above the threshold is determined by the plasma proCPU concentration, the extent of proCPU activation by the coagulation cascade and most importantly by the stability of CPU (66, 82, 83). The process of fibrinolysis and the action of CPU are presented schematically in figure 2.
5. MEASUREMENT OF PROCPU IN PLASMA
For the measurement of proCPU in plasma several assays exist, including immunoassays (ELISAs) as well as activity-based assays. All of these methods have their own inherent advantages and disadvantages and none benefits from the existence of an internationally recognized reference standard for proCPU. The complicated matter of proCPU measurement is extensively reviewed in (84) and here discussed briefly.
Immunoassays for the measurement of proCPU are widespread and have the major advantage of being easy to perform (84). However, one has to take into account that proCPU and CPU are present in plasma in different forms. Antibodies constructed to target proCPU could also have a nonnegligible reactivity toward the active moiety CPU, the activation peptide, inactivated CPU, proCPU bound to plasminogen, etc. (85-87). A second important disadvantage of immunoassays is the genotype dependent reactivity. Several antibodies have been described to show an altered reactivity toward a naturally occurring polymorphism at position 325 of the proCPU protein (85). It is therefore of utmost importance that these assays are well characterized with respect to genotype dependent reactivity. Although ELISAs are very attractive and easy to use, care should be taken with the interpretation of the generated results.
Most available activity-based assays are based on the principle that CPU is able to cleave off C-terminal lysine (Lys) or arginine (Arg) residues from small synthetic substrates. The released Lys or Arg on the one hand or the des-Arg or des-Lys product on the other hand can be quantified (84). A major advantage of activity-based assays is that only the enzymatically active CPU is measured, without the interference of the profragment or inactivated CPU. However, this approach requires quantitative activation of the zymogen proCPU (44, 88). Moreover, activity-based assays are compromised by the interference of carboxypeptidase N (CPN). This plasmatic enzyme is constitutively active and can interfere with CPU measurement due to its similar basic carboxypeptidase activity (89). Finally, in activity-based assays one has to take into account the pronounced thermal instability of CPU, which is genotype dependent (25). Stabilization of the active enzyme can be obtained by placing the samples on ice, by incubating with an excess of substrate or by using a short incubation interval during which linear substrate conversion can be guaranteed (63, 90). It is of utmost importance to validate activity-based assays with respect to CPU stability and linear substrate conversion.
Due to the lack of well characterized assays for the measurement of proCPU, results of several studies evaluating the possible role for the plasma proCPU concentration as risk factor for the occurrence of thrombotic disorders have to be interpreted with caution (84). This concern is shared by the Scientific Standardisation Committee (SSC) on fibrinolysis of the International Society of Thrombosis and Haemostasis (ISTH), which expressed the high need for a thorough characterization of proCPU assays (84). Recently, we have evaluated various commercially available proCPU assays, especially with regard to genotype dependent reactivity and were able to show that several assays that are on the market today still display these genotype dependent artefacts. As a result, great care should be taken when choosing an assay for proCPU measurement in a clinical setting, as well as with the interpretation and comparison of reported results (data in submission).
6. MEASUREMENT OF CPU IN CIRCULATION
In normal physiological conditions, CPU circulates in plasma as an inactive precursor, proCPU. Upon activation of the coagulation cascade, CPU is generated which will exert its antifibrinolytic activity and thus helps to stabilize the clot. The measurement of the proenzyme in plasma is well established (see above (84)). With the discovery of the threshold phenomenon more evidence was revealed that the direct measurement of CPU in circulation or the extent of CPU generation could be more relevant than the measurement of the proenzyme itself. However, the direct measurement of the active enzyme CPU is not straightforward. A first critical step in CPU measurement is the sample collection. Since CPU concentrations in venous circulation are expected to be very low (low pM range, see infra), ex vivo activation of proCPU must be avoided (91). Blood must be collected on citrate anticoagulant with the addition of D-phenylalanyl-L-prolyl-arginyl chloromethyl ketone (PPACK) and aprotinin, inhibitors of thrombin and plasmin, respectively. Recently, we demonstrated the importance of correct sample collection (91). A second obstacle is the intrinsic instability of CPU (25). To prevent rapid decay of CPU, samples must be placed on ice immediately after collection and subsequent centrifugation must be performed at 4 �C. Despite its thermal instability, CPU activity does not seem to be affected by freeze-thaw cycles, provided that the samples are thawed on ice (92). Apart from the preanalytical problems, there are also several analytical challenges that must be addressed. First, there is a need to measure a CPU concentration in circulation that is expected to be very low. The half-maximal effect of CPU on clot lysis occurs at 1 nM, whereas the maximal effect occurs at 20 nM. Since the circulating concentration of proCPU is 75 - 275 nM, only a small portion needs to be activated to have a significant effect on clot lysis (44). At the site of the thrombus, local CPU concentrations are most likely to be much higher than the CPU concentration in venous circulation. ProCPU is present in both plasma and in platelets and is secreted when platelets are activated by thrombin (15), causing a boost in the local proCPU concentration at the site of blood clotting. Moreover, proCPU can be cross-linked to fibrin by factor XIIIa and this interaction can possibly lead to a stabilization of this carboxypeptidase increasing its antifibrinolytic potential dramatically (93). Finally, the assay procedure must be specific enough to measure low amounts of CPU when set against a rather high background activity of CPN circulating in plasma at a concentration of 30 �g/mL or 100 nM (94).
In 2004, Neill and co-workers developed a functional assay for the measurement of CPU in circulation (92). This assay is based on the fact that CPU decreases the cofactor activity of high-molecular-weight fibrin degradation products in the stimulation of plasminogen activation in a concentration-dependent manner (95-97). In a later publication of the same group, Kim and co-workers modified this functional assay to overcome its shortcomings (98). The starting point of this modified assay is similar; it is based on the ability of CPU to remove C-terminal lysine residues that are exposed on plasmin modified fibrin, thereby releasing fluorescently labelled plasminogen that was bound to these lysines by its kringle domains. The present assay directly measures the release of plasminogen rather than measuring the extent of plasminogen activation (98). The functional assay as described by Kim et al. was shown to be sensitive for CPU at a concentration as low as 12 pM. Moreover, it was not confounded by the naturally occurring proCPU Thr325Ile polymorphism or by endogenous plasminogen in the plasma. (98). With this assay, Kim et al. were able to detect basal CPU levels in the plasma of healthy individuals at a concentration of 20.3 � 9.1 pomp (n=5) (98).
Another approach for the direct measurement of CPU in circulation is the use of an activity-based assay. In a study on ischemic stroke patients, an HPLC-assisted activity-based assay was used to demonstrate CPU generation during thrombolytic treatment (91). This assay uses hippuryl-L-arginine (HipArg) as a substrate. However, since this substrate is not selective for CPU, this assay will suffer from rather high background activities from the CPN present in the plasma sample. To overcome this analytical challenge of CPN interference, a specialized procedure was used where a set of two CP-inhibitors were combined, resulting in a limit of detection (LOD) of 1 U/L or 200 pM (91, 99). A more straightforward approach would be the development of a substrate with improved selectivity toward CPU combined with minimal residual activity toward CPN. Recent screening of Bz-Xaa-Arg peptides with an aromatic amino acid at the P1 position and further modifications in this position, lead to the discovery of a selective CPU substrate, benzoyl-ortho-cyano-phenylalanyl-arginine (Bz-o-cyano-Phe-Arg) (100). Very recently, our group published a novel activity-based assay for the measurement of CPU in plasma using the selective substrate Bz-o-cyano-Phe-Arg, thereby limiting the interference of CPN as well as excluding the intrinsic activity of proCPU. The novel assay is easy to perform and its high specificity is translated into a LOD as low as 0.05 U/L or 10 pM. With this assay, basal CPU levels in healthy individuals were found to be below 10 pM (n=15).
In 2005, Ceresa and co-workers reported on the development of ELISAs measuring the extent of proCPU activation (101). A variety of monoclonal antibody (MA) based ELISAs were evaluated for their preferential reactivity toward proCPU before and after activation, identifying immunologic assays that measure the amount of CPU (active as well as inactivated form CPUi) or that are directed toward the released activation peptide (101). In a large study on 300 patients with hyperlipidemia, higher levels of both the activation peptide and CPU/CPUi were observed compared to normolipidemic controls, using the newly identified ELISAs. In contrast, no association was found between total proCPU antigen and hyperlipidemia, suggesting that the extent of activation is a more relevant parameter in CPU research (101).
7. PROCPU/CPU SYSTEM IN THROMBOSIS
In a review article by Leurs et al., published in 2005, an extensive overview of the role of proCPU in thrombotic and haemorrhagic conditions was given (102). Since then, the proCPU/CPU system gained more interest by the scientific community, as indicated by the exponential increase in publications on its pathophysiological role. In Table 1, we focus on recent findings in the field (> 2005). Results of some studies may be compromised by the use of a proCPU assay that lacks thorough validation, as was discussed before.
8. CPU: A NEW DRUG TARGET ?
The prominent bridging function of CPU between coagulation and fibrinolysis raised the interest of several research groups and of the pharmaceutical industry. The development of CPU inhibitors as profibrinolytic agents is an attractive new concept, as it will leave the coagulation cascade unaffected (7). Possibilities for rational drug design become more readily available as a result of the recently published crystal structure of CPU (35, 71, 72). An overview of CPU inhibitors and their potential role as profibrinolytic drugs is given in a recently published review by Willemse et al. (7).
The organic inhibitors 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MERGETPA) and guanidinoethylmercaptosuccinic acid (GEMSA) are two of the most widely used CPU inhibitors for both in vitro and in vivo studies (10, 12, 14, 62). Both compounds have two major drawbacks from a drug discovery point of view, i.e. they lack selectivity and also inhibit CPN, which is believed to have an important function in plasma as an inactivator of anaphylatoxins and in the processing of peptide hormones (94, 196); moreover, they have a low oral availability, due to their polar nature (102). Major efforts have been made to obtain more potent and selective drugs with favourable pharmacokinetic properties.
Apart from the synthetic inhibitors, protein inhibitors have also been described. The most familiar one is potato tuber carboxypeptidase inhibitor (PTCI), a 39 amino acid protein that competitively inhibits CPU with a Ki in the nanomolar range; which is commonly used in in vitro settings as well as in in vivo animal models (197-200). Other protein inhibitors are the 66 amino acid residue leech carboxypeptidase inhibitor (LCI) found in Hirudo medicinalis (201) and the recently isolated tick carboxypeptidase inhibitor (TCI) from the soft tick Rhipicephalus bursa (202). A major advantage of this kind of inhibitors is the high selectivity toward CPU.
It has been observed that several competitive inhibitors exert a biphasic effect in in vitro experiments, where clot lysis is stimulated at high concentrations - as expected - but prolonged at low concentrations (62, 203). A possible explanation for this could be the equilibrium between free and (inhibitor) bound CPU. Free CPU is highly unstable and will inactivate irreversibly, due to increased dynamic segment mobility. The bound form on the other hand is protected from loss of activity through interaction of the inhibitor with the dynamic segment of the enzyme moiety. This stable bound form will be released to replenish the free pool, so as to maintain the equilibrium (62, 203). As long as the free CPU concentration stays above the t-PA dependent threshold value, fibrinolysis will stay in its initial phase (82, 83). Although this stabilizing effect of CPU inhibitors has raised some concerns about their use as profibrinolytic drugs, this observed paradoxal behaviour has not been substantiated in in vivo models. Moreover, recent insights reveal that this phenomenon is most probably compound-related.
Besides direct CPU inhibition, another profibrinolytic strategy is to prevent the activation of the proenzyme proCPU. In this regard, several monoclonal antibodies (MA) as well as nanobodies have been described, inhibiting proCPU activation by plasmin or by the thrombin-thrombomodulin complex (41-43). As already mentioned above, activation of proCPU by plasmin as well as by the thrombin-thrombomodulin complex occurs through a single-site proteolytic cleavage at Arg92. However, Hillmayer et al. were able to generate MA that could discriminate between plasmin and thrombin-thrombomodulin mediated activation, implying that these MA do not bind to the cleavage site. Moreover, different binding sites in the enzyme moiety have been identified for thrombin or plasmin. MA that inhibit exclusively the activation of proCPU by thrombin-thrombomodulin bind to Gly66, whereas MA that inhibit activation by both plasmin and thrombin-thrombomodulin bind to Val41. These MA are of particular interest for revealing the physiological activator of proCPU in in vivo models (204) as well as for therapeutic use, since they do not cross-react with CPN (42).
Surprisingly, initial in vivo data from proCPU knockout mice did not reveal an important function of the proCPU/CPU system, raising questions about its in vivo significance (205-207). A recent update on proCPU knockout mice is given by Morser et al. (207), concluding that proCPU deficient mice without being challenged displayed no overt phenotype, suggesting that proCPU can regulate fibrinolysis under defined conditions whereby its deficiency is only observable when normal fibrinolysis is compromised (207). For instance, in proCPU deficient mice with a heterozygous plasminogen-deficient background, a role of proCPU was demonstrated in models of pulmonary embolism (PE) and peritoneal inflammation (208). These results led to the conclusion that proCPU is able to modulate the in vivo functions of plasminogen in fibrinolysis and cell migration (208). Moreover, when fibrinogen was depleted in glomerulonephritis (209) and lung fibrosis models (210), the protective effects of proCPU deficiency were abrogated, demonstrating enhanced fibrinolysis to be at the basis for the protective phenotype in proCPU deficient mice. More recent data on proCPU knockout mice with a normal plasminogen status have been published describing a protective role of proCPU deficiency in a 3.5 % ferric chloride induced vena cava thrombosis model (211) and linking proCPU deficiency to enhanced endogenous fibrinolysis (212).
The search for clinical utility of CPU inhibitors has been focussed on improvement of endogenous fibrinolysis on the one hand and adjuvants for thrombolytic therapy on the other hand. Several studies have been conducted to evaluate whether a CPU inhibitor alone improves endogenous thrombolysis, albeit with contradictory results (reviewed in (7)). The in vivo profibrinolytic efficiency of a CPU inhibitor alone depends on the type of thrombosis model and the studied animal species, the type of inhibitor and whether this inhibitor is administered before or after thrombus induction (51, 200, 213-216). Recently, a phase II, single-blind, multicentre study was presented which investigated the effect of the novel CPU inhibitor AZD9684 in PE (217). Fifty-eight patients with confirmed PE were randomized to receive AZD9684 or placebo, on top of once-daily dalteparin for 5-7 days. In the patient group receiving AZD9684, fibrinolysis biomarkers in plasma were higher and sustained for a longer period of time, implying that inhibition of CPU by AZD9684 stimulates endogenous fibrinolysis. Moreover, lung deficiency scintigraphy scores improved over the treatment period. In addition, no difference in the occurrence of adverse effects was seen between both treatment groups (217).
Also the use of CPU inhibitors as adjuvants of thrombolytic therapy is an attractive concept. Since thrombolytic therapy is still characterized by major shortcomings - large therapeutic doses, limited fibrin specificity, significant bleeding tendency and reocclusion - major benefit can be expected from adjunctive therapy that potentiates the t-PA mediated thrombolytic effect, enabling dose reduction and thus limiting unfavourable side effects of plasminogen activators (218, 219). In recent years, consistent data have been obtained in animal studies on the utility of CPU inhibitors as adjuvant therapy of thrombolytics (reviewed in (7)). Administration of a CPU inhibitor along with low-dose t-PA leads to a significant enhancement of thrombolytic efficiency, with a 3-fold reduction in time to reperfusion and similarly improved vessel patency compared to low-dose t-PA alone (199, 213, 215, 216).
An important concern about the use of CPU inhibitors as profibrinolytic drugs is the question whether inhibition of CPU leads to an increased bleeding risk. The use of a CPU inhibitor in many in vivo models however was not associated with an increased bleeding risk, either when used alone or in combination with t-PA (206).
9. CONCLUSIONS
By providing an important link between coagulation and fibrinolysis, the proCPU system is considered to be a potential target for the treatment of thrombotic disorders. Several selective CPU inhibitors have been designed and tested in animal thrombosis models, showing improved endogenous fibrinolysis and an increased efficiency of t-PA mediated thrombolysis upon inhibition of the (pro)CPU system. Recently, a phase II, single-blinded, multicentre study in patients with PE also showed enhancement of endogenous fibrinolysis upon administration of a selective CPU inhibitor. With the availability of the crystal structure, a major step forward has been made in CPU research, boosting the development of potent and selective CPU inhibitors in the near future.
10. ACKNOWLEDGEMENTS
E. Heylen is a research assistant of the Fund for Scientific Research Flanders (FWO-Vlaanderen).
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Abbreviations: proCPU: procarboxypeptidase U; CPU: carboxypeptidase U; TAFI: thrombin activatable fibrinolysis inhibitor; TAFIa: activated thrombin activatable fibrinolysis inhibitor; CPB: carboxypeptidase B; CPN: carboxypeptidase N; SNP: single nucleotide polymorphism; IIa: thrombin; TM: thrombomodulin; TF: tissue factor; EGF: epidermal growth factor; GEMSA: guanidinoethylmercaptosuccinic acid; MERGETPA: 2-mercapto-methyl-3-guanidinoethylthiopropanoic acid; PTCI: potato tuber carboxypeptidase inhibitor; LCI: leech carboxypeptidase inhibitor; TCI: tick carboxypeptidase inhibitor; t-PA: tissue plasminogen activator; MA: monoclonal antibody; ELISA: enzyme linked immunosorbent assay; HPLC: high pressure liquid chromatography; PPACK: D-phenylalanyl-L-prolyl-arginyl chloromethyl ketone; LOD: limit of detection;
Key Words: Procarboxypeptidase U, ProCPU, Carboxypeptidase U, CPU, Thrombin Activatable Fibrinolysis Inhibitor, TAFI, Activated Thrombin Activatable Fibrinolysis Inhibitor, Tafia, Fibrinolysis, Thrombosis, Review
Send correspondence to: Dirk Hendriks, Laboratory of Medical Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium,Tel: 3232652727, Fax: 3232652745, E-mail:dirk.hendriks@ua.ac.be