Enzymatic Assays for the Diagnosis of Bradykinin-Dependent Angioedema

Abstract

BackgroundThe kinins (primarily bradykinin, BK) represent the mediators responsible for local increase of vascular permeability in hereditary angioedema (HAE), HAE I-II associated with alterations of the SERPING1 gene and HAE with normal C1-Inhibitor function (HAE-nC1INH). Besides C1-Inhibitor function and concentration, no biological assay of kinin metabolism is actually available to help physicians for the diagnosis of angioedema (AE). We describe enzymatic tests on the plasma for diagnosis of BK-dependent AE.MethodsThe plasma amidase assays are performed using the Pro-Phe-Arg-p-nitroanilide peptide substrate to evaluate the spontaneous amidase activity and the proenzyme activation. We analyzed data of 872 patients presenting with BK-dependent AE or BK-unrelated diseases, compared to 303 controls. Anti-high MW kininogen (HK) immunoblot was achieved to confirm HK cleavage in exemplary samples. Reproducibility, repeatability, limit of blank, limit of detection, precision, linearity and receiver operating characteristics (ROC) were used to calculate the diagnostic performance of the assays.ResultsSpontaneous amidase activity was significantly increased in all BK-dependent AE, associated with the acute phase of disease in HAE-nC1INH, but preserved in BK-unrelated disorders. The increase of the amidase activity was associated to HK proteolysis, indicating its relevance to identify kininogenase activity. The oestrogens, known for precipitating AE episodes, were found as triggers of enzymatic activity. Calculations from ROC curves gave the optimum diagnostic cut-off for women (9.3 nmol⋅min−1⋅mL−1, area under curve [AUC] 92.1%, sensitivity 80.0%, and specificity 90.1%) and for men (6.6 nmol·min−1⋅mL−1, AUC 91.0%, sensitivity 87.0% and specificity 81.2%).ConclusionThe amidase assay represents a diagnostic tool to help physicians in the decision to distinguish between BK-related and –unrelated AE.

Distinct Conditions Support a Novel Classification for Bradykinin-Mediated Angio-Oedema

“…However, the existence of both acquired (idiopathic non-histaminergic AAO - InH-AAO) and hereditary (HAO of unknown origin - U-HAO) has been acknowledged in the most recent classification of AO under the patronage of the European Academy of Allergy and Clinical Immunology [10]. From our side, we demonstrated that some of these AOs could be related to an increased kinin formation and/or to a decreased BK catabolism associated with a deficiency in one or several kininase(s) [15,22,23,24]. …”

“…C1-INH analysis included quantitative and functional measurements [30]. Kinin formation was evaluated by measurement of the plasma spontaneous amidase activity using the HD-Pro-Phe-Arg- p NA substrate as recently described [24]. This assay evaluates the activity of the enzymes responsible for BK production after cleaving high-molecular weight kininogen, the serine proteases of contact phase (kallikrein, FXII).…”

Hereditary and acquired C1-inhibitor-dependent angioedema: from pathophysiology to treatment

“…Conceptually, given the fact that these mutations affect the prolin-rich domain of FXII, which is considered to play a role in the interaction with negatively charged surfaces, one might speculate that these forms of FXII are more prone to activation upon contact with negatively charged surfaces (58). Spontaneous amidase activity in plasma as a surrogate marker for serine protease activity of the CP and fibrinolytic system has been shown to be increased in patients with FXII-HAE regardless the clinical severity (60). Interestingly, ACE and CPN activity levels inversely correlated with disease severity pointing to the important role of BKdegrading enzymes in the pathogenesis of HAE type III, but most probably in other forms as well (61).…”

“…Clotting factor-based assays may help to identify increased activity of FXIIa in plasma (57). In a collective of 100 symptomatic patients suffering from FXII-HAE spontaneous amidase activity (see above) has been shown to correlate with disease severity (60). However, specific genetic testing for the known FXII mutations or sequencing of the FXII gene is the most appropriate diagnostic tools to confirm or exclude HAE type III (9).…”

Presentation, diagnosis and treatment of angioedema without wheals: a retrospective analysis of a cohort of 1058 patients

“…The therapeutic response to specific antagonists characterizes IH-AAE. Bradykinin mediates ACEI-AAE and C1-INH-HAE, and possibly also FXII-HAE, U-HAE and InH-AAE [12,13]. Leukotrienes, and other yet undefined factors, are likely to be involved in some cases of drug-related angioedema [1].…”