title: Gly74Ser mutation in protein C causes thrombosis due to a defect in protein S-dependent anticoagulant function.
abstract: Protein C is a vitamin K-dependent serine protease zymogen in plasma which upon activation by thrombin in complex with thrombomodulin (TM) down-regulates the clotting cascade by a feedback loop inhibition mechanism. Activated protein C (APC) exerts its anticoagulant function through protein S-dependent degradation of factors Va and VIIIa. We recently identified a venous thrombosis patient whose plasma level of protein C antigen is normal, but its anticoagulant activity is only 34 % of the normal level. Genetic analysis revealed that the proband and her younger brother carry a novel heterozygous mutation c.346G>A, p.Gly74Ser (G74S) in PROC. Thrombin generation assay indicated that the TM-dependent anticoagulant activity of the proband's plasma has been significantly impaired. We expressed protein C-G74S in mammalian cells and characterised its properties in established coagulation assays. We demonstrate that the protein C variant can be normally activated by the thrombin-TM complex and the resulting APC mutant also exhibits normal amidolytic and proteolytic activities toward both FVa and FVIIIa. However, it was discovered the protein S-dependent catalytic activity of APC variant toward both procoagulant cofactors has been significantly impaired. Protein S concentration-dependence of FVa degradation revealed that the capacity of APC variant to interact with the cofactor has been markedly impaired. The same results were obtained for inactivation of FVa-Leiden suggesting that the protein S-dependent activity of APC variant toward cleavage of Arg-306 site has been adversely affected. These results provide insight into the mechanism through which G74S substitution in APC causes thrombosis in the proband carrying this mutation.
authors.forename: Changming
authors.initials: C
authors.lastname: Chen
authors.forename: Likui
authors.initials: L
authors.lastname: Yang
authors.forename: Bruno O
authors.initials: BO
authors.lastname: Villoutreix
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Wang
authors.forename: Qiulan
authors.initials: Q
authors.lastname: Ding
authors.affiliation: Alireza R. Rezaie, PhD, Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA, Tel: +1 405 271 4711, E-mail: Ray-Rezaie@omrf.org, or, Qiulan Ding, PhD, Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, No. 197 Ruijin Second Road, Shanghai, 200025 China, Tel.: +86 21 54667770, Fax: +86 21 64333548, E-mail: qiulan_ding@hotmail.com.
authors.forename: Alireza R
authors.initials: AR
authors.lastname: Rezaie
mesh_terms: D019943:Amino Acid Substitution; D000925:Anticoagulants / Q000097:blood*; D005260:Female; D057809:HEK293 Cells; D006579:Heterozygote; D006801:Humans; D008297:Male; D050505:Mutant Proteins / Q000097:blood* / Q000737:chemistry / Q000235:genetics*; D020125:Mutation, Missense; D010375:Pedigree; D011486:Protein C / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020151:Protein C Deficiency / Q000097:blood* / Q000235:genetics*; D017293:Protein S / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D018180:Thrombomodulin / Q000097:blood; D020246:Venous Thrombosis / Q000097:blood / Q000235:genetics
chemical_list: D000925:Anticoagulants; D050505:Mutant Proteins; D011486:Protein C; D017293:Protein S; D011994:Recombinant Proteins; C546931:THBD protein, human; D018180:Thrombomodulin; D013917:Thrombin
keywords: Protein C; factor VIIIa; factor Va; protein S; thrombosis