title: The protein C omega-loop substitution Asn2Ile is associated with reduced protein C anticoagulant activity.
abstract: We report a kindred with heritable protein C (PC) deficiency in which two siblings with severe thrombosis showed a composite type I and IIb PC deficiency phenotype, identified using commercial PC assays (proband: PC antigen 42 u/dl, amidolytic activity 40 u/dl, anticoagulant activity 9 u/dl). The independent PROC nucleotide variations c.669C>A (predictive of Ser181Arg) and c.131C>T (predictive of Asn2Ile) segregated with the type I and type IIb PC deficiency phenotypes respectively, but co-segregated in the siblings with severe thrombosis. Soluble thrombomodulin (sTM)-mediated inhibition of plasma thrombin generation from an individual with PC-Asn2Ile was lower (endogenous thrombin potential (ETP) 56 +/- 1% that of ETP determined without sTM) than control plasma (ETP 15 +/- 2%) indicating reduced PC anticoagulant activity. Recombinant APC-Asn2Ile exhibited normal amidolytic activity but impaired anticoagulant activity. Protein S (PS)-dependent anticoagulant activity of recombinant APC-Asn2Ile and binding of recombinant APC-Asn2Ile to endothelial protein C receptor (EPCR) were reduced compared to recombinant wild-type APC. Asn2 lies within the omega-loop of the PC/APC Gla domain and this region is critical for calcium-induced folding and subsequent interactions with anionic phospholipids, EPCR and PS. The disruption of these interactions in this naturally-occurring PC variant highlights their collective importance in mediating APC anticoagulant activity in vivo.
authors.affiliation: Haemostasis Research Group, Institute of Molecular Medicine, Trinity College Dublin, St James Hospital, Dublin, Ireland.
authors.forename: Roger J S
authors.initials: RJ
authors.lastname: Preston
authors.forename: Colin
authors.initials: C
authors.lastname: Morse
authors.forename: Sherina L
authors.initials: SL
authors.lastname: Murden
authors.forename: Sara Kate
authors.initials: SK
authors.lastname: Brady
authors.forename: James S
authors.initials: JS
authors.lastname: O'Donnell
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Mumford
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D019943:Amino Acid Substitution; D001324:Autoantigens / Q000097:blood; D001777:Blood Coagulation / Q000235:genetics*; D001780:Blood Coagulation Tests; D002648:Child; D005260:Female; D005838:Genotype; D006801:Humans; D008297:Male; D010375:Pedigree; D010641:Phenotype; D011486:Protein C / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D020151:Protein C Deficiency / Q000150:complications / Q000235:genetics* / Q000378:metabolism; D055665:Purpura Fulminans / Q000235:genetics; D013917:Thrombin / Q000096:biosynthesis; D013927:Thrombosis / Q000209:etiology / Q000235:genetics
chemical_list: D001324:Autoantigens; D011486:Protein C; D013917:Thrombin