ProCPU Is Expressed by (Primary) Human Monocytes and Macrophages and Expression Differs between States of Differentiation and Activation.

Carboxypeptidase U (CPU, TAFIa, CPB2) is a potent attenuator of fibrinolysis that is mainly synthesized by the liver as its inactive precursor proCPU. Aside from its antifibrinolytic properties, evidence exists that CPU can modulate inflammation, thereby regulating communication between coagulation and inflammation. Monocytes and macrophages play a central role in inflammation and interact with coagulation mechanisms resulting in thrombus formation. The involvement of CPU and monocytes/macrophages in inflammation and thrombus formation, and a recent hypothesis that proCPU is expressed in monocytes/macrophages, prompted us to investigate human monocytes and macrophages as a potential source of proCPU. CPB2 mRNA expression and the presence of proCPU/CPU protein were studied in THP-1, PMA-stimulated THP-1 cells and primary human monocytes, M-CSF-, IFN-γ/LPS-, and IL-4-stimulated-macrophages by RT-qPCR, Western blotting, enzyme activity measurements, and immunocytochemistry. CPB2 mRNA and proCPU protein were detected in THP-1 and PMA-stimulated THP-1 cells as well as in primary monocytes and macrophages. Moreover, CPU was detected in the cell medium of all investigated cell types and it was demonstrated that proCPU can be activated into functionally active CPU in the in vitro cell culture environment. Comparison of CPB2 mRNA expression and proCPU concentrations in the cell medium between the different cell types provided evidence that CPB2 mRNA expression and proCPU secretion in monocytes and macrophages is related to the degree to which these cells are differentiated. Our results indicate that primary monocytes and macrophages express proCPU. This sheds new light on monocytes and macrophages as local proCPU sources.

Proline-specific peptidase activities (DPP4, PRCP, FAP and PREP) in plasma of hospitalized COVID-19 patients.

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.

Acute Ischemic Stroke Severity, Progression, and Outcome Relate to Changes in Dipeptidyl Peptidase IV and Fibroblast Activation Protein Activity.

Dipeptidyl peptidase IV (DPPIV) inhibition may be a promising therapeutic strategy for acute stroke treatment, given its potential to prolong the biological half-life of neuroprotective substrates. A related protease, fibroblast activation protein (FAP), was recently shown to inactivate the same substrates. Therefore, it should also be investigated as a potential target in stroke. The study aimed to investigate whether stroke severity and outcome correlate with DPPIV and FAP activities and their kinetics shortly after acute ischemic stroke. DPPIV and FAP activities were analyzed in the serum of 50 hyperacute stroke patients at admission, 1 day, 3 days, and 7 days after stroke onset and in 50 age-matched healthy controls. This was done as part of the Middelheim's Interdisciplinary Stroke Study. DPPIV activity tended to increase shortly after stroke compared to the control population. DPPIV and FAP activities steadily decreased in the first week after stroke onset. Higher infarct volumes (≥5 ml) and a more severe stroke (NIHSS >7) at admission were correlated with a stronger decrease in the activities of both enzymes. Moreover, these patients more often developed a progressive stroke, were more often institutionalized. Patients with a stronger increase in DPPIV activity at admission and decrease in the activity of both DPPIV and FAP during the first week after stroke onset had a more severe stroke and worse short-term outcomes.

Mapping of carboxypeptidase m in normal human kidney and renal cell carcinoma: expression in tumor-associated neovasculature and macrophages.

Although the kidney generally has been regarded as an excellent source of carboxypeptidase M (CPM), little is known about its renal-specific expression level and distribution. This study provides a detailed localization of CPM in healthy and diseased human kidneys. The results indicate a broad distribution of CPM along the renal tubular structures in the healthy kidney. CPM was identified at the parietal epithelium beneath the Bowman's basement membrane and in glomerular mesangial cells. Capillaries, podocytes, and most interstitial cells were CPM negative. Tumor cells of renal cell carcinoma subtypes lose CPM expression upon dedifferentiation. Tissue microarray analysis demonstrated a correlation between low CPM expression and tumor cell type. CPM staining was intense on phagocytotic tumor-associated macrophages. Immunoreactive CPM was also detected in the tumor-associated vasculature. The absence of CPM in normal renal blood vessels points toward a role for CPM in angiogenesis. Coexistence of CPM and the epidermal growth factor receptor (EGFR) was detected in papillary renal cell carcinoma. However, the different subcellular localization of CPM and EGFR argues against an interaction between these h proteins. The description of the distribution of CPM in human kidney forms the foundation for further study of the (patho)physiological activities of CPM in the kidney.

Carboxypeptidase M in apoptosis, adipogenesis and cancer.

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.

Development of a fast kinetic method for the determination of carboxypeptidase U (TAFIa) using C-terminal arginine containing peptides as substrate.

Carboxypeptidase U (CPU, TAFIa) is a novel determinant of the fibrinolytic rate. It circulates in blood as an inactive zymogen, procarboxypeptidase U, which is activated during the process of coagulation and fibrinolysis. CPU has a very short half-life at 37 degrees C. Its intrinsic instability complicates the determination of kinetic parameters of different substrates using an endpoint method. We developed a fast kinetic assay for measuring continuously the release of the C-terminal arginine by CPU independent of the nature of the substrate peptide used, allowing us to perform substrate specificity studies of CPU. This method uses arginine kinase, pyruvate kinase, and lactate dehydrogenase as auxiliary enzymes. The CPU activities measured using this kinetic assay were in the range of 97-103% of those determined with our HPLC-assisted reference assay, and the obtained K(m) and k(cat) values for hippuryl-l-arginine and bradykinin were in good accordance with those described in the literature. As expected, no arginine cleaving was seen using dipeptides and peptide substrates with a proline in the penultimate position. The presented kinetic assay enables the fast screening of substrates with a C-terminal arginine and is a valuable new tool for the kinetic evaluation of both synthetic and physiological substrates of CPU.

Raloxifene reduces procarboxypeptidase U, an antifibrinolytic marker. A 2-year randomized, placebo-controlled study in healthy early postmenopausal women.

OBJECTIVE: The aim of this study was to compare the long-term effects of two dosages of raloxifene with oral hormone therapy (HT; conjugated equine estrogens combined with medroxyprogesterone acetate) on procarboxypeptidase U. DESIGN: In a randomized, double-blind, placebo-controlled, 2-year study, 95 healthy, nonhysterectomized, early postmenopausal women received either daily raloxifene 60 mg (n = 24), raloxifene 150 mg (n = 23), HT (conjugated equine estrogens 0.625 mg + medroxyprogesterone acetate 2.5 mg, n = 24), or placebo (n = 24). At baseline and after 6, 12, and 24 months, fasting plasma procarboxypeptidase U concentrations were measured. RESULTS: Six months of treatment with raloxifene 60 mg and raloxifene 150 mg were associated with significant decreases in plasma procarboxypeptidase U concentrations, which were sustained after 12 and 24 months. Raloxifene 60 mg: t = 0, 619 +/- 89 U/L (mean +/- SD); t = 6, 574 +/- 87 U/L; t = 12, 571 +/- 96 U/L; t = 24, 568 +/- 92 U/L; ANCOVA versus placebo, P = 0.026. Raloxifene 150 mg: t = 0, 608 +/- 67 U/L; t = 6, 580 +/- 73 U/L; t = 12, 578 +/- 70 U/L; t = 24, 562 +/- 61 U/L; ANCOVA versus placebo, P = 0.039. No significant changes were found in the HT group. CONCLUSION: Long-term treatment with raloxifene reduced procarboxypeptidase U plasma concentrations.