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.

Procarboxypeptidase U (TAFI) is a recently discovered plasma procarboxypeptidase that upon activation by thrombin or thrombin-thrombomodulin turns into a potent antifibrinolytic enzyme. Its prominent bridging function between coagulation and fibrinolysis raised the interest of many research groups and of the pharmaceutical industry. The development of CPU inhibitors as profibrinolytic agents is an attractive concept and possibilities for rational drug design will become more available in the near future due to the recently published crystal structure. Numerous studies have been performed and many of them show beneficial effects of CPU inhibitors for the improvement of endogenous fibrinolysis in different animal sepsis and thrombosis models. CPU inhibitors combined with t-PA seem to increase the efficiency of pharmacological thrombolysis allowing lower dosing of t-PA and subsequently fewer bleeding complications. This review will focus on recently obtained in vivo data and the benefits/risks of targeting CPU for the treatment of thrombotic disorders.

Procarboxypeptidase U [proCPU, thrombin‐activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down‐regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPU's activity. We have shown that single as well as a few 2–4 mutations in human CPU can prolong the half‐life of CPU's activity at 37 °C from 0.2 h of wild‐type CPU to 0.5–5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis.

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The maintenance of the equilibrium between coagulation and fibrinolysis is crucial for normal haemostasis. In contrast, pathologic consequences of imbalance manifest tendencies of bleeding or thrombosis. Procarboxypeptidase U (proCPU, TAFI) is recognized as an important link between the coagulation system and fibrinolysis. Following activation by thrombin (IIa), carboxypeptidase U (CPU) exerts an antifibrinolytic effect by abolishing the cofactor function of partially degraded fibrin in plasminogen (Pg) activation. This review article focuses on the role of the proCPU/CPU system in the balance between fibrin deposition and removal. How a disturbed system can lead to a higher thrombotic tendency is discussed, while CPU inhibition as a new drug target for fibrinolytic therapy is extensively reviewed.