Page 254 - Haematologica - Vol. 105 n. 6 - June 2020

Coagulation & its Disorders
Ferrata Storti Foundation
Haematologica 2020 Volume 105(6):1712-1722
Ile73Asn mutation in protein C introduces a new N-linked glycosylation site on the first EGF-domain of protein C and causes thrombosis
Yeling Lu,1,2 Padmaja Mehta-D’souza,2 Indranil Biswas,2 Bruno O. Villoutreix,3 Xuefeng Wang,1 Qiulan Ding,1 and Alireza R. Rezaie2,4
1Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China; 2Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; 3Université Lille, Inserm, Institut Pasteur de Lille, U1177 - Drugs and Molecules for Living Systems, F- 59000 Lille, France and 4Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
ABSTRACT
Activated protein C exerts its anticoagulant activity by protein S- dependent inactivation of factors Va and VIIIa by limited proteol- ysis. We identified a venous thrombosis patient who has plasma protein C antigen level of 63% and activity levels of 44% and 23%, as monitored by chromogenic and clotting assays. Genetic analysis revealed the proband carries compound heterozygous mutations (c.344T>A, p.I73N and c.1181G>A, p.R352Q) in PROC. We individually expressed protein C mutations and discovered that thrombin-thrombomodulin activates both variants normally and the resulting activated protein C mutants exhibit normal amidolytic and proteolytic activities. However, while protein S-dependent catalytic activity of activated protein C- R352Q toward factor Va was normal, it was significantly impaired for activated protein C-I73N. These results suggest that the Ile to Asn sub- stitution impairs interaction of activated protein C-I73N with protein S. This conclusion was supported by a normal anticoagulant activity for activated protein C-I73N in protein S-deficient but not in normal plasma. Further analysis revealed Ile to Asn substitution introduces a new glyco- sylation site on first EGF-like domain of protein C, thereby adversely affecting interaction of activated protein C with protein S. Activated pro- tein C-R352Q only exhibited reduced activity in sub-physiological con- centrations of Na+ and Ca2+, suggesting that this residue contributes to metal ion-binding affinity of the protease, with no apparent adverse effect on its function in the presence of physiological levels of metal ions. These results provide insight into the mechanism by which I73N/R352Q mutations in activated protein C cause thrombosis in proband carrying this compound heterozygous mutation.
Introduction
Thrombin forms a complex with thrombomodulin (TM) on the endothelium to activate the vitamin K-dependent anticoagulant protein C zymogen to activated protein C (APC), thereby down-regulating thrombin generation by a feedback inhi- bition mechanism.1 Activated protein C functions as an anticoagulant by degrading the procoagulant co-factors Va and VIIIa by limited proteolysis.2 Protein C is a multi-domain glycoprotein composed of a non-catalytic light chain linked to the catalytic heavy chain by a single disulfide bond.3,4 The light chain harbors the vita- min K-dependent N-terminal γ-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor (EGF)-like domains.4 The C-terminal catalytic heavy chain with a trypsin-like substrate specificity is preceded by an activation peptide, which is removed during the activation of protein C by the thrombin-TM
Correspondence:
ALIREZA R. REZAIE
ray-rezaie@omrf.org
QIULAN DING
qiulan_ding@hotmail.com
Received: May 15,2019. Accepted: August 7, 2019. Pre-published: August 8, 2019.
doi:10.3324/haematol.2019.227033
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