Carboxypeptidase U (CPU, TAFIa, CPB2) in Thromboembolic Disease: What Do We Know Three Decades after Its Discovery?

Abstract

Procarboxypeptidase U (proCPU, TAFI, proCPB2) is a basic carboxypeptidase zymogen that is converted by thrombin(-thrombomodulin) or plasmin into the active carboxypeptidase U (CPU, TAFIa, CPB2), a potent attenuator of fibrinolysis. As CPU forms a molecular link between coagulation and fibrinolysis, the development of CPU inhibitors as profibrinolytic agents constitutes an attractive new concept to improve endogenous fibrinolysis or to increase the efficacy of thrombolytic therapy in thromboembolic diseases. Furthermore, extensive research has been conducted on the in vivo role of CPU in (the acute phase of) thromboembolic disease, as well as on the hypothesis that high proCPU levels and the Thr/Ile325 polymorphism may cause a thrombotic predisposition. In this paper, an overview is given of the methods available for measuring proCPU, CPU, and inactivated CPU (CPUi), together with a summary of the clinical data generated so far, ranging from the current knowledge on proCPU concentrations and polymorphisms as potential thromboembolic risk factors to the positioning of different CPU forms (proCPU, CPU, and CPUi) as diagnostic markers for thromboembolic disease, and the potential benefit of pharmacological inhibition of the CPU pathway.

Keywords: arterial thrombosis; carboxypeptidase B2; carboxypeptidase U; fibrinolysis; thrombin-activatable fibrinolysis inhibitor; thromboembolic diseases; venous thrombosis.

Figure 1

Measurement of different forms of the CPB2 gene product. The zymogen procarboxypeptidase U (proCPU) (1) can be measured with antigen- or activity-based assays. Carboxypeptidase U (CPU) activation can also be determined. Upon activation with thrombin, the thrombin-thrombomodulin (TM) complex, or plasmin, the activation peptide (AP; 2) is released and can be measured with an antigen-based assay. The active enzyme CPU can be directly measured by enzymatic assays (3). The active enzyme CPU is thermally inactivated into CPUi (4) which can be measured with antigen-based assays that detect both CPU and CPUi simultaneously (5).

Figure 2

Functional assessment of carboxypeptidase U (CPU). The contribution of CPU to the total clot lysis time (ΔCLT; d) can be visualized by means of in vitro clot lysis experiments in the absence (a) or presence (b) of potato tuber carboxypeptidase inhibitor (PTCI) or a selective CPU inhibitor. During in vitro clot lysis, CPU is generated shortly after initiation of the coagulation—when proCPU is activated by thrombin(-thrombomodulin)—resulting in a first CPU activity peak (c) and halting fibrinolysis as long as the CPU level remains above a certain threshold value (e). This threshold concentration is dictated by steady-state plasmin concentrations, and thus dependent on tissue-type plasminogen activator (tPA) and plasmin inhibitor concentrations. Once CPU activity falls below this critical threshold value, fibrinolysis accelerates and the generated plasmin gives rise to the formation of a second CPU activity peak (c,e). The time that the CPU stays above the threshold (f) is defined by (1) the procarboxypeptidase U (proCPU) concentration, (2) the extent of proCPU activation—and thus the concentrations of thrombin(-thrombomodulin)—and (3) the CPU half-life, defined by the Thr/Ile325 polymorphism.

Figure 3

General structure of a low molecular weight (LMW) carboxypeptidase inhibitor (a; based on Bunnage et al. [146]) and structures of selective LMW carboxypeptidase U inhibitors disclosed to date (b–j). Inhibitors containing an imidazole—(b–d), thiol—(e,f), dialkyl phosphinic acid—(g–h), sulfamide—(i) or selenium-group (j) as the functional group involved in the Zn²⁺-coordination.