Abstract
Neprilysin (NEP) is an endogenous protease that degrades a wide range of peptides including amyloid beta (Aβ), the main pathological component of Alzheimer’s disease (AD). We have engineered NEP as a potential therapeutic for AD but found in pre-clinical safety testing that this variant increased prothrombin time (PT) and activated partial thromboplastin time (APTT). The objective of the current study was to investigate the effect of wild type NEP and the engineered variant on coagulation and define the mechanism by which this effect is mediated. PT and APTT were measured in cynomolgus monkeys and rats dosed with a human serum albumin fusion with an engineered variant of NEP (HSA-NEPv) as well as in control plasma spiked with wild type or variant enzyme. The coagulation factor targeted by NEP was determined using in vitro prothrombinase, calibrated automated thrombogram (CAT) and fibrin formation assays as well as N-terminal sequencing of fibrinogen treated with the enzyme. We demonstrate that HSA-NEP wild type and HSA-NEPv unexpectedly impaired coagulation, increasing PT and APTT in plasma samples and abolishing fibrin formation from fibrinogen. This effect was mediated through cleavage of the N-termini of the Aα- and Bβ-chains of fibrinogen thereby significantly impairing initiation of fibrin formation by thrombin. Fibrinogen has therefore been identified for the first time as a substrate for NEP wild type suggesting that the enzyme may have a role in regulating fibrin formation. Reductions in NEP levels observed in AD and cerebral amyloid angiopathy may contribute to neurovascular degeneration observed in these conditions.
Highlights
Neprilysin (NEP) is an integral type II membrane-bound zinc-dependent peptidase that degrades a number of physiological peptides that are involved in processes such as blood pressure regulation and nociception [1]
We have engineered NEP to have increased proteolytic activity on Aβ peptide and tested our improved variant for efficacy in rats and cynomolgus monkeys [8,9]. As part of these studies we investigated the safety profile of HSA-NEPv and observed that the molecule had an unexpected effect on coagulation. We explored this effect on coagulation and found that administration of HSA-NEPv increased both prothrombin time (PT) and activated partial thromboplastin time (APTT) in rats and cynomolgus monkeys compared with controls (Figs 1 and 2)
Mean PT and APTT increased from 12.8 ± 1 to 19.9 ± 3 s and from 17.9 ± 2 to 29.3 ± 3 s, respectively, the day after the first administration of HSA-NEPv at the highest dose level (143 mg/kg; ÃÃP < 0.01)
Summary
Neprilysin (NEP) is an integral type II membrane-bound zinc-dependent peptidase that degrades a number of physiological peptides that are involved in processes such as blood pressure regulation and nociception [1]. NEP Inhibition of Coagulation interests: All authors at the time of performing the work were salaried employees of MedImmune or AstraZeneca and have a theoretical conflict of interest. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. We have engineered a variant of NEP (NEP G399V/ G714K; “NEPv”) that has increased specificity and activity on Aβ and shown that while peripheral administration of a human serum albumin fusion of this variant (HSA-NEPv) reduces plasma Aβ, the level of the peptide in the brain is not affected [8,9]. NEP has been implicated as a human skin fibroblast elastase [12]
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