title: Protein C Thr315Ala variant results in gain of function but manifests as type II deficiency in diagnostic assays.
abstract: Protein C (PC) is a vitamin K-dependent plasma glycoprotein, which upon activation by thrombin in complex with thrombomodulin (TM), regulates the coagulation cascade through a feedback loop inhibition mechanism. PC deficiency is associated with an increased risk of venous thromboembolism (VTE). A recent cohort study aimed at establishing a normal PC range identified a healthy PC-deficient subject whose PC antigen level of 65% and activity levels of 50% (chromogenic assay) and 36% (clotting assay) were markedly low. The proband has a negative family history of VTE. Genetic analysis revealed the proband has a heterozygous missense mutation in which Thr-315 of the PC heavy chain has been substituted with Ala. We expressed this mutant in HEK-293 cells and purified it to homogeneity. A similar decrease in both anticoagulant and anti-inflammatory activities of the activated protein C mutant was observed in plasma- and cell-based assays. Interestingly, we discovered if functional assays were coupled to PC activation by the thrombin-TM complex, the variant exhibits improved activities in all assays. Sequence analysis revealed Thr-315 is a consensus N-linked glycosylation site for Asn-313 and that its elimination significantly (∼four- to fivefold) improves the maximum velocity of PC activation by the thrombin-TM complex, explaining the basis for the proband's negative VTE pedigree.
authors: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China; and.
authors: Qiulan
authors: Q
authors: Ding
authors: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO.
authors: Likui
authors: L
authors: Yang
authors: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO.
authors: Peyman
authors: P
authors: Dinarvand
authors: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China; and.
authors: Xuefeng
authors: X
authors: Wang
authors: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO.
authors: Alireza R
authors: AR
authors: Rezaie
mesh_terms: D000328:Adult; D001777:Blood Coagulation; D001780:Blood Coagulation Tests; D015331:Cohort Studies; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D017354:Point Mutation; D011486:Protein C / Q000235:genetics* / Q000378:metabolism; D020151:Protein C Deficiency / Q000097:blood / Q000175:diagnosis* / Q000235:genetics* / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D018180:Thrombomodulin / Q000378:metabolism
chemical_list: D011486:Protein C; D018180:Thrombomodulin; D013917:Thrombin
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