title: Neprilysin Inhibits Coagulation through Proteolytic Inactivation of Fibrinogen.
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.
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Milstein Building, Granta Park, Cambridge, United Kingdom.
authors.forename: Matthew
authors.initials: M
authors.lastname: Burrell
authors.affiliation: Biologics Safety Assessment, MedImmune, Aaron Klug Building, Granta Park, Cambridge, United Kingdom.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Henderson
authors.affiliation: Cardiovascular and Metabolic Disease iMed, AstraZeneca R&D, SE-431 83 Mölndal, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Ravnefjord
authors.affiliation: Pharmaceutical Development, AstraZeneca R&D, SE-431 83 Mölndal, Sweden.
authors.forename: Fritz
authors.initials: F
authors.lastname: Schweikart
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Milstein Building, Granta Park, Cambridge, United Kingdom.
authors.forename: Susan B
authors.initials: SB
authors.lastname: Fowler
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Milstein Building, Granta Park, Cambridge, United Kingdom.
authors.forename: Susanne
authors.initials: S
authors.lastname: Witt
authors.affiliation: Cardiovascular and Metabolic Disease iMed, AstraZeneca R&D, SE-431 83 Mölndal, Sweden.
authors.forename: Kenny M
authors.initials: KM
authors.lastname: Hansson
authors.affiliation: Antibody Discovery and Protein Engineering, MedImmune, Milstein Building, Granta Park, Cambridge, United Kingdom.
authors.forename: Carl I
authors.initials: CI
authors.lastname: Webster
mesh_terms: D000544:Alzheimer Disease / Q000097:blood / Q000150:complications / Q000188:drug therapy*; D016229:Amyloid beta-Peptides / Q000378:metabolism; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D016657:Cerebral Amyloid Angiopathy / Q000097:blood / Q000188:drug therapy* / Q000235:genetics; D005337:Fibrin / Q000187:drug effects / Q000378:metabolism; D005340:Fibrinogen / Q000037:antagonists & inhibitors; D006801:Humans; D008252:Macaca fascicularis; D015260:Neprilysin / Q000008:administration & dosage* / Q000009:adverse effects / Q000235:genetics; D010314:Partial Thromboplastin Time; D059748:Proteolysis / Q000187:drug effects; D011517:Prothrombin Time; D051381:Rats; D012709:Serum Albumin / Q000008:administration & dosage / Q000009:adverse effects / Q000235:genetics*; D013925:Thromboplastin / Q000235:genetics
chemical_list: D016229:Amyloid beta-Peptides; D012709:Serum Albumin; D005337:Fibrin; D005340:Fibrinogen; D013925:Thromboplastin; D015260:Neprilysin