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Background: COVID-19 patients experience several features of dysregulated immune system observed in sepsis. We previously showed a dysregulation of several proline-selective peptidases such as dipeptidyl peptidase 4 (DPP4), fibroblast activation protein alpha (FAP), prolyl oligopeptidase (PREP) and prolylcarboxypeptidase (PRCP) in sepsis. In this study, we investigated whether these peptidases are similarly dysregulated in hospitalized COVID-19 patients., Methods: Fifty-six hospitalized COVID-19 patients and 32 healthy controls were included. Enzymatic activities of DPP4, FAP, PREP and PRCP were measured in samples collected shortly after hospital admission and in longitudinal follow-up samples., Results: Compared to healthy controls, both DPP4 and FAP activities were significantly lower in COVID-19 patients at hospital admission and FAP activity further decreased significantly in the first week of hospitalization. While PRCP activity remained unchanged, PREP activity was significantly increased in COVID-19 patients at hospitalization and further increased during hospital stay and stayed elevated until the day of discharge., Conclusion: The changes in activities of proline-selective peptidases in plasma are very similar in COVID-19 and septic shock patients. The pronounced decrease in FAP activity deserves further investigation, both from a pathophysiological viewpoint and as its utility as a part of a biomarker panel., (Copyright © 2022 Elsevier B.V. All rights reserved.)

This review covers carboxypeptidase M (CPM) research that appeared in the literature since 2009. The focus is on aspects that are new or interesting from a clinical perspective. Available research tools are discussed as well as their pitfalls and limitations. Evidence is provided to suggest the potential involvement of CPM in apoptosis, adipogenesis and cancer. This evidence derives from the expression pattern of CPM and its putative substrates in cells and tissues. In recent years CPM emerged as a potential cancer biomarker, in well differentiated liposarcoma where the CPM gene is co-amplified with the oncogene MDM2; and in lung adenocarcinoma where coexpression with EGFR correlates with poor prognosis. The available data call for extended investigation of the function of CPM in tumor cells, tumor-associated macrophages, stromal cells and tumor neovascularisation. Such experiments could be instrumental to validate CPM as a therapeutic target., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Patients with hereditary mucocutaneous bleeding are difficult to diagnose and many of them fulfill the category of bleeders of unknown cause (BUC). The pathogenic role of hyperfibrinolysis has received little attention, despite the successful use of antifibrinolytic drugs in treating many of these patients. Theoretically, decreased plasma procarboxypeptidase U (proCPU) levels or lower carboxypeptidase U (CPU) stability would result in higher fibrinolytic activity and bleeding tendency., Methods: We analyzed plasma proCPU and the distribution of the Thr325Ile proCPU polymorphism in 193 patients with mucocutaneous bleeding of whom 116 were bleeders of unknown cause (BUC), and in 143 healthy, age and sex-matched controls., Results: ProCPU concentration was higher in women than in men, increased with age, and was significantly correlated with clot lysis time, platelet count, APTT, and PT. However, proCPU levels were unexpectedly higher in patients than in controls (968+/-134 vs. 923+/-147 U/L, p=0.004). The allele distribution of the Thr325Ile proCPU polymorphism was similar in both groups, with a low percentage of homozygous Ile/Ile., Conclusions: Our results indicate that the proCPU system is not of major importance in the bleeding pathogenesis of these patients. The higher proCPU levels in the patients may even modestly counteract the bleeding tendency.

Carboxypeptidase M (EC 3.4.17.12) belongs to the family of the carboxypeptidases. These enzymes remove C-terminal amino acids from peptides and proteins and exert roles in the physiological processes of blood coagulation/fibrinolysis, inflammation, food digestion and pro-hormone and neuropeptide processing. Among the carboxypeptidases CPM is of particular importance because of its constitutive expression in an active form at the surface of specialized cells and tissues in the human body. Despite the fact that the function(s) of this enzyme is not fully understood several suggestions have been made since its discovery more than two decades ago. Based on potential substrates and its presence, often on the boundary between the host and environment, a role in inflammation was proposed. This review describes how recent discoveries affected the insights in the cellular and physiological functions of CPM. A critical analysis of the potential endogenous peptide and protein substrates is provided. The distribution of CPM on different cell types and tissues and its expression in states of disease are discussed. There is evidence that CPM functions not only as a protease but also as a binding partner in cell-surface protein-protein interactions.

Background: Measurement of procarboxypeptidase U (TAFI) in plasma by activity-based assays is complicated by the presence of plasma carboxypeptidase N (CPN). Accurate blank measurements, correcting for this interfering CPN activity, should therefore be performed. A selective CPU substrate will make proCPU determination much less time-consuming., Methods: We searched for selective and sensitive CPU substrates by kinetic screening of different Bz-Xaa-Arg (Xaa=a naturally occurring amino acid) substrates using a novel kinetic assay., Results: The presence of an aromatic amino acid (Phe, Tyr, Trp) resulted in a fairly high selectivity for CPU which was most pronounced with Bz-Trp-Arg showing a 56-fold higher k(cat)/K(m) value for CPU compared to CPN. Next we performed chemical modifications on the structure of those aromatic amino acids. This approach resulted in a fully selective CPU substrate with a 2.5-fold increase in k(cat) value compared to the commonly used Hip-Arg (Bz-Gly-Arg)., Discussion: We demonstrated significant differences in substrate specificity between CPU and CPN that were previously not fully appreciated. The selective CPU substrate presented in this paper will allow straightforward determination of proCPU in plasma in the future.

Background: Carboxypeptidase N is a plasma zinc metallocarboxypeptidase which is constitutively expressed in the liver and was identified as the enzyme responsible for inactivating bradykinin and kallidin by removing the C-terminal arginine. Because CPN can cleave the C-terminal arginine of C3a, C4a and C5a it is often referred to as anaphylatoxin inactivator. Markedly reduced levels of circulating CPN are associated with recurrent angioedema and abnormal cutaneous polymorphonuclear cell infiltration., Methods: In this paper we describe a fast kinetic coupled enzymatic assay for the sensitive measurement of carboxypeptidase N activities in serum samples. The assay makes use of the excellent CPN substrate Benzoyl-L-Alanyl-L-Arginine., Results: This novel assay is very fast, easy to perform and combines good reliability and reproducibility with excellent correlation with the HPLC-assisted assay (r=0.927; n=140)., Conclusion: The presented assay can be used for high throughput screening of this important regulator of inflammation in clinical plasma or serum samples.

Background: Carboxypeptidase U (EC 3.4.17.20, TAFIa) is a new member of the metallocarboxypeptidase family circulating in human plasma as a zymogen. It is activated during coagulation and is considered as an important player in the regulation of fibrinolysis., Methods: Heterologous expression of human plasma procarboxypeptidase U (proCPU, TAFI) was obtained in mammalian cells (C127 and DON) and in insect cells (Sf21 and H5 cells). Conditioned media were purified by cation-exchange chromatography and plasminogen affinity chromatography to yield an essentially pure protein., Results: All systems gave high expression levels (6-20 mg/l). Due to differences in glycosylation of the activation peptide, the recombinant variants of proCPU migrated differently on SDS-PAGE (52-65 kDa). However, after activation, all active recombinant enzymes migrated at 35 kDa, similar to native CPU and no evidence for post-translational modification of the catalytic domains could be detected. For the mammalian cell produced variants, activation was more efficient after desialylation. After activation, CPU showed low solubility (0.2 mg/ml) but was inhibited similarly as native CPU., Conclusions: Mammalian cell systems were the most efficient for the production of human plasma recombinant proCPU. The obtained zymogen differs with respect to the extent and the heterogeneity of glycosylation but, after activation, the experiments did not reveal any alteration between the recombinant and native protein.