A sensitive and specific assay to characterize plasma kallikrein activity in plasma from patients with hereditary angioedema.

Introduction: Plasma kallikrein (PKa) activity is increased in the plasma of patients with hereditary angioedema (HAE) and has been implicated in other kallikrein-kinin system (KKS)-mediated diseases. Exogenous substrates commonly used in PKa assays can be cleaved by multiple plasma proteases, which reduce assay specificity and sensitivity for PKa. We describe a sensitive and specific assay to detect PKa activity in plasma as a candidate biomarker for HAE. Methods: PKa activity was measured in plasma samples from patients with HAE with decreased C1 inhibitor (C1INH) levels or activity who were not receiving prophylactic medications for HAE (HAE-C1INH; n = 25), from individuals with a presumptive diagnosis of HAE with normal C1INH (HAE-nC1INH; n = 3), and from age-matched controls without HAE (n = 57). Samples were analyzed at baseline and after 6 h of cold incubation at 4 °C. Amidolytic activity was measured in the absence and presence of a PKa-specific inhibitor (KV999272). Specific PKa (sPKa) activity was quantified by the subtraction of amidolytic activity not inhibited by KV999272 from the total measured amidolytic activity. Results: In control plasma, sPKa activity was 0.69 ± 0.07 nmol/min/mL at baseline and 0.88 ± 0.11 nmol/min/mL after 6 h of cold incubation (mean ± SEM, p = 0.0062); the 95th percentile of sPKa activity was 1.87 nmol/min/mL at baseline and 3.07 nmol/min/mL after cold incubation. In plasma from patients with HAE-C1INH, sPKa activity was 3.43 ± 0.64 nmol/min/mL at baseline and 24.53 ± 8.92 nmol/min/mL after 6 h of cold incubation (p = 0.023). sPKa activity in HAE-C1INH plasma samples was above the 95th percentile for control plasma with assay sensitivity of 84% and specificity of 95%. The area under the receiver operating characteristic curve was 0.98 (p < 0.0001). sPKa activity in all plasma samples from patients with HAE-nC1INH was above the 95th percentile for control plasma after 6 h of cold incubation. Conclusion: We developed a specific PKa assay that can detect low levels of PKa activity in plasma and can differentiate patients with HAE-C1INH from controls without HAE with high sensitivity and specificity. Using this assay, we demonstrated that sPKa activity is elevated during the intercritical period in patients with HAE-C1INH and in those with HAE-nC1INH compared with controls when measured after 6 h of cold incubation. This sensitive and specific PKa assay could be useful to characterize PKa activity in plasma samples from patients with HAE and could potentially serve as a future candidate biomarker for HAE-nC1INH.