The protein C ω‐loop substitution Asn2Ile is associated with reduced protein C anticoagulant activity

Abstract

Summary 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 ω‐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.

Purpura fulminans: recognition, diagnosis and management

“…Evidence for this diagnosis is strengthened by serological evidence of recent Varicella infection or if coagulation assay tests for lupus anticoagulant are positive. Definitive diagnosis requires specialist interpretation of PC and PS assay results because some rare heritable variants may be difficult to identify in commercially available laboratory tests 26. Diagnosis of severe heritable PC or PS deficiency may be confirmed by demonstration of homozygous or compound heterozygous mutations in the PROC or PROS1 genes 27 28…”

“…Control of long-term anticoagulation with vitamin K antagonists is often challenging in younger children. There is limited experience of long-term anticoagulation with low-molecular weight heparin in children with severe heritable PC deficiency which may circumvent some of these difficulties 26 45…”

“…This usually requires PC replacement therapy either alone, or in combination with coumarins or low-molecular weight heparin 18. In some cases, recurrent PF may be prevented with anticoagulant drugs alone 26 45. Liver transplantation offers long term cure of heritable PC deficiency 47…”

Marked discrepancy between coagulometric Protein C activity assays with the pro‐thrombotic Protein C Asn2Ile substitution

“…We have previously reported two siblings with a PROC c.131C>T nucleotide substitution predictive of an Asn2Ile substitution in the PC Gla domain. This substitution was associated with purpura fulminans when co‐inherited with a partial type I PC deficiency caused by an independent heterozygous nucleotide substitution on the other PROC allele (Preston et al. , 2009).…”

Protein C and protein S deficiencies: similarities and differences between two brothers playing in the same game

“…Missense mutations are the most frequently reported types; the resulting amino acid change involving the Gla-domain or the pro-peptide result in defective calcium and phospholipid binding (49)(50)(51)(52)(53)(54)(55)(56)(57). Mutations in the serine protease domain result in defective protease activity or decreased substrate binding (58)(59)(60).…”