Alpha-1-antitrypsin deficiency (AATD) is a genetic disorder that mainly manifests in adults with lung emphysema and liver cirrhosis. Pathogenesis of the liver disease arises from misfolding of alpha-1-antitrypsin mutants such as Z-AAT (E342K) that results in accumulation of Z-AAT polymers in the endoplasmic reticulum of hepatocytes. On the other hand, emphysema derives from AAT deficiency in the bloodstream leading to reduced inhibition of AAT target proteinases such as neutrophil elastase (NE) in the lungs. In general, clinical assays implicitly equate circulating AAT levels to anti-protease activity. However, the activity of circulating Z-AAT variant is further reduced by conformational modifications that impair the inhibitory mechanism and by the presence of inactive polymers released by hepatocytes. Standard assays developed in the past to measure AAT inhibitory capacity in plasma require an experimental set-up that may limit their routine implementation in most clinical laboratories. To overcome this, we developed a new sandwich ELISA to determine NE inhibition by plasma AAT. The inhibitory activity determined by this assay on both purified AAT or control plasma samples were consistent with those determined by classical methods and was not affected by alpha-2-macroglobulin, another abundant inhibitor of serine proteinases in plasma. Application of the new method to plasma samples from patients with the ZZ genotype showed that 20 to 50% of circulating Z-AAT is inactive toward NE. This is partially due to the intrinsic dysfunction of the Z-AAT molecule as well as to the presence of inactive circulating polymers, which we previously showed to range from 8 to 33% of total plasma AAT content in a cohort of ZZ patients. Measuring the anti-elastase protective activity provides an additional level of detail to explain the different severity of lung manifestations observed among patients, and could thereby improve the current diagnostic pipeline of AATD.
Development of a novel assay to measure alpha-1-antitrypsin inhibitory activity toward neutrophil elastase
Bignotti, M;Gambelli, J;Denardo, A;Irving, JA;Fra, A
2024-01-01
Abstract
Alpha-1-antitrypsin deficiency (AATD) is a genetic disorder that mainly manifests in adults with lung emphysema and liver cirrhosis. Pathogenesis of the liver disease arises from misfolding of alpha-1-antitrypsin mutants such as Z-AAT (E342K) that results in accumulation of Z-AAT polymers in the endoplasmic reticulum of hepatocytes. On the other hand, emphysema derives from AAT deficiency in the bloodstream leading to reduced inhibition of AAT target proteinases such as neutrophil elastase (NE) in the lungs. In general, clinical assays implicitly equate circulating AAT levels to anti-protease activity. However, the activity of circulating Z-AAT variant is further reduced by conformational modifications that impair the inhibitory mechanism and by the presence of inactive polymers released by hepatocytes. Standard assays developed in the past to measure AAT inhibitory capacity in plasma require an experimental set-up that may limit their routine implementation in most clinical laboratories. To overcome this, we developed a new sandwich ELISA to determine NE inhibition by plasma AAT. The inhibitory activity determined by this assay on both purified AAT or control plasma samples were consistent with those determined by classical methods and was not affected by alpha-2-macroglobulin, another abundant inhibitor of serine proteinases in plasma. Application of the new method to plasma samples from patients with the ZZ genotype showed that 20 to 50% of circulating Z-AAT is inactive toward NE. This is partially due to the intrinsic dysfunction of the Z-AAT molecule as well as to the presence of inactive circulating polymers, which we previously showed to range from 8 to 33% of total plasma AAT content in a cohort of ZZ patients. Measuring the anti-elastase protective activity provides an additional level of detail to explain the different severity of lung manifestations observed among patients, and could thereby improve the current diagnostic pipeline of AATD.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
