Purification and characterization of a new arginine carboxypeptidase in human serum

Hendriks, Dirk; Wang, Wei; Scharpé, Simon; Lommaert, Marie-Paule; Sande, M. Van

doi:10.1016/0304-4165(90)90157-r

Cited by 120 publications

(86 citation statements)

References 29 publications

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“…It initially appeared as an unstable carboxypeptidase B-like entity in human serum and was described by Hendriks et al (20). Then Eaton et al (21) discovered it as a contaminant in preparations of ␣ 2 -antiplasmin; they cloned the cDNA, deduced the amino acid sequence, described its activation by trypsin, and analyzed its enzymatic properties toward synthetic carboxypeptidase B substrates.…”

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confidence: 99%

TAFI, or Plasma Procarboxypeptidase B, Couples the Coagulation and Fibrinolytic Cascades through the Thrombin-Thrombomodulin Complex

Bajzar

Morser²,

Nesheim

1996

Journal of Biological Chemistry

623

607

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TAFI (thrombin-activatable fibrinolysis inhibitor) is a recently discovered plasma protein that can be activated by thrombin-catalyzed proteolysis to a carboxypeptidase B-like enzyme that inhibits fibrinolysis. This work shows that the thrombin-thrombomodulin complex, rather than free thrombin, is the most likely physiologic activator. Thrombomodulin increases the catalytic efficiency of the reaction by a factor of 1250, an effect expressed almost exclusively through an increase in k cat . The kinetics of the reaction conform to a model whereby thrombin can interact with either TAFI (K m ‫؍‬ 1.0 M) or thrombomodulin (K d ‫؍‬ 8.6 nM), and either binary complex so formed can then interact with the third component to form the ternary thrombin-thrombomodulin-TAFI complex from which activated TAFI is produced with kcat ‫؍‬ 1.2 s ؊1. This work also shows that activated TAFI down-regulates tPA-induced fibrinolysis half-maximally at a concentration of 1.0 nM in a system of purified components. This concentration of TAFI is about 2% of the level of the zymogen in plasma, which indicates that ample activated TAFI could be generated to very significantly modulate fibrinolysis in vivo. Therefore, TAFI in vitro and possibly in vivo defines an explicit molecular connection between the coagulation and fibrinolytic cascades, such that expression of activity in the former down-regulates the activity of the latter.The coagulation and fibrinolytic cascades comprise a series of zymogen to enzyme conversions which terminate in the proteolytic enzymes thrombin and plasmin, respectively (1-3). These enzymes catalyze the deposition and removal of fibrin. A proper balance between the activities of the two cascades is required both to protect the organism from excessive blood loss upon injury and to maintain blood fluidity within the vascular system. Imbalances are characterized by either bleeding or thrombotic tendencies, the latter of which are manifested as heart attacks and strokes.Thrombomodulin is a component of the blood vessel wall which binds thrombin and changes its specificity from fibrinogen to protein C, yielding anticoagulant rather procoagulant activity (4). The thrombin-thrombomodulin complex catalyzes cleavage of protein C to activated protein C, which then downregulates the coagulation cascade by proteolytically inactivating the essential cofactors Factor Va and Factor VIIIa (5). These events are essential in the regulation of the coagulation cascade (4).Early studies suggested that activated protein C is not only anticoagulant but also profibrinolytic, both in vitro and in vivo (6 -9). Subsequent work from our laboratory showed that activated protein C only appears profibrinolytic because it prevents the thrombin-catalyzed activation of a previously unknown fibrinolysis inhibitor (10). The precursor of this inhibitor was isolated from plasma (11) and was designated TAFI (thrombinactivatable fibrinolysis inhibitor). The zymogen is activated by thrombin to an enzyme with carboxypeptidase B-like activity. This enzyme, des...

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mentioning

confidence: 99%

TAFI, or Plasma Procarboxypeptidase B, Couples the Coagulation and Fibrinolytic Cascades through the Thrombin-Thrombomodulin Complex

Bajzar

Morser²,

Nesheim

1996

Journal of Biological Chemistry

623

607

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“…It is a procarboxypeptidase synthesized by the liver and named thrombin activatable fibrinolysis inhibitor (TAFI) or procarboxypeptidase B or U. [7][8][9] Upon activation by thrombin or plasmin, it is converted to an enzyme (TAFIa) with carboxypeptidase B-like activity that inhibits fibrinolysis through the removal of C-terminal lysines from partially degraded fibrin. 10,11 In so doing, TAFIa reduces the cofactor function of fibrin in the plasminogen activation catalyzed by t-PA, thereby decreasing plasmin generation.…”

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confidence: 99%

Deficiency of Thrombin Activatable Fibrinolysis Inhibitor in Cirrhosis Is Associated With Increased Plasma Fibrinolysis

et al. 2003

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Hyperfibrinolysis is thought to contribute to bleeding associated with advanced cirrhosis. Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma precursor of a carboxypeptidase (TAFIa) with antifibrinolytic activity and was recently shown to be reduced in cirrhosis. In this study, we evaluated the influence of TAFI deficiency on in vitro fibrinolysis in cirrhotic patients. Fifty-three patients with cirrhosis and 43 healthy controls were studied. TAFI antigen was measured by enzyme-linked immunosorbent assay and TAFI activity by chromogenic assay. Fibrinolysis was evaluated as tissue plasminogen activator-induced plasma clot lysis time in the absence and in the presence of a specific inhibitor of TAFIa. TAFI antigen and activity levels were markedly reduced in cirrhotic patients (P < .0001). In these patients, the lysis time of plasma clots was shorter than in controls (median, interquartile range: 25 minutes, 21-36 minutes vs. 48 minutes, 40-60 minutes, respectively; P < .0001) and was poorly influenced by the TAFIa inhibitor. Accordingly, TAFIa and thrombin activity, generated in cirrhotic samples during clot lysis, were significantly lower than in control samples. Addition of purified TAFI to cirrhotic plasma prolonged the lysis time and enhanced the response to TAFIa inhibitor in a dose-dependent manner. In conclusion, our results indicate that in vitro plasma hyperfibrinolysis in cirrhosis is largely due to a defective TAFIa generation resulting from low TAFI levels and probably from impaired thrombin generation. Impairment of the antifibrinolytic TAFI pathway might contribute to bleeding associated with this disease. (HEPATOLOGY 2003;38:230-237.) H yperfibrinolysis is a common finding in cirrhosis and is thought to contribute to the bleeding tendency associated with this disease by causing a premature removal of the hemostatic plug at sites of vascular injury. The abnormalities of the fibrinolytic system encountered in cirrhosis are complex and result from both impaired synthesis and altered clearance of the fibrinolytic factors. 1 Of particular relevance to hyperfibrinolysis are the imbalance between tissue plasminogen activator (t-PA) and its specific inhibitor (PAI-1), which results in an enhancement of free t-PA in the circulation and the reduction of ␣ 2 -plasmin inhibitor (␣ 2 -PI). [2][3][4][5][6] In recent years, a new plasma protein has been identified that may play an important regulatory role in fibrinolysis. It is a procarboxypeptidase synthesized by the liver and named thrombin activatable fibrinolysis inhibitor (TAFI) or procarboxypeptidase B or U. 7-9 Upon activation by thrombin or plasmin, it is converted to an enzyme (TAFIa) with carboxypeptidase B-like activity that inhibits fibrinolysis through the removal of C-terminal lysines from partially degraded fibrin. 10,11 In so doing, TAFIa reduces the cofactor function of fibrin in the plasminogen activation catalyzed by t-PA, thereby decreasing plasmin generation. The most likely physiologic activator of TAFI is thrombin, 12,13 whi...

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“…The zymogen of PCB, pro-PCB, is activated by limited proteolysis to expose carboxypeptidase activity directed at C-terminal Lys and Arg residues (3,4). Partially degraded fibrin is decorated with C-terminal basic residues that serve to accelerate fibrinolysis by providing binding sites for tissue-type plasminogen activator (t-PA), plasminogen (Plg), and plasmin (5).…”

mentioning

confidence: 99%

Characterization of Plasmin-mediated Activation of Plasma Procarboxypeptidase B

Mao

Cooper

Wood

et al. 1999

Journal of Biological Chemistry

123

109

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Plasma carboxypeptidase B (PCB) is an exopeptidase that exerts an antifibrinolytic effect by releasing C-terminal Lys and Arg residues from partially degraded fibrin. PCB is produced in plasma via limited proteolysis of the zymogen, pro-PCB. In this report, we show that the K m (55 nM) for plasmin-catalyzed activation of pro-PCB is similar to the plasma concentration of pro-PCB (50 -70 nM), whereas the K m for the thrombin-or thrombin:thrombomodulin-catalyzed reaction is 10 -40-fold higher than the pro-PCB level in plasma. Additionally, tissue-type plasminogen activator triggers activation of pro-PCB in blood plasma in a reaction that is stimulated by a neutralizing antibody versus ␣ 2 -antiplasmin. Together, these results show that plasmin-mediated activation of pro-PCB can occur in blood plasma. Heparin (UH) and other anionic glycosaminoglycans stimulate pro-PCB activation by plasmin but not by thrombin or thrombin:thrombomodulin. Pro-PCB is a more favorable substrate for plasmin in the presence of UH (16-fold increase in k cat /K m ). UH also stabilizes PCB against spontaneous inactivation. The presence of UH in clots prepared with prothrombin-deficient plasma delays tissue-type plasminogen activator-triggered lysis; this effect of UH on clot lysis is blocked by a PCB inhibitor from potato tubers. These results show that UH accelerates plasmin-catalyzed activation of pro-PCB in plasma and PCB, in turn, stabilizes fibrin against fibrinolysis. We propose that glycosaminoglycans in the subendothelial extracellular matrix serve to augment the levels of PCB activity thereby stabilizing blood clots at sites where there is a breach in the integrity of the vasculature.

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Purification and characterization of a new arginine carboxypeptidase in human serum

Cited by 120 publications

References 29 publications

TAFI, or Plasma Procarboxypeptidase B, Couples the Coagulation and Fibrinolytic Cascades through the Thrombin-Thrombomodulin Complex

TAFI, or Plasma Procarboxypeptidase B, Couples the Coagulation and Fibrinolytic Cascades through the Thrombin-Thrombomodulin Complex

Deficiency of Thrombin Activatable Fibrinolysis Inhibitor in Cirrhosis Is Associated With Increased Plasma Fibrinolysis

Characterization of Plasmin-mediated Activation of Plasma Procarboxypeptidase B

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