Journal Article

Distinct pathogenic mechanisms underlying two protein C variants (p.Arg211Gln and p.Val367Met) in a thrombophilic family: integrated functional and structural analyses

Wang, Beili, Zhang, Huayang, Gu, Meixiu, Guo, Wei, Pan, Baishen, Wang, Chong, Zhang, Li, Jiang, Huiqin, Shi, Zhenyu, Zhao, Ying

2025

Overview

Background Hereditary protein C deficiency (PCD) increases thrombotic risk, but the molecular mechanisms of distinct missense variants remain incompletely defined. Two siblings from a family with recurrent deep vein thrombosis and a strong family history of thrombotic events were found to carry compound heterozygous PROC variants. Objectives To elucidate the biosynthetic and functional consequences of two PROC missense variants, c.632G > A (p.Arg211Gln) and c.1099G > A (p.Val367Met), and their combined impact on anticoagulant capacity. Methods Two siblings with recurrent deep vein thrombosis were analyzed. Family segregation analysis was performed on multiple heterozygous carriers to support genotype-phenotype correlations. Recombinant protein C variants were expressed in HEK293T cells to assess expression efficiency and intracellular accumulation. Zymogen activation by thrombin-thrombomodulin and APC anticoagulant activity were evaluated using FVa degradation, clotting, and thrombin generation assays. Structural modeling and Na⁺-dependent enzymatic assays were performed to explore variant-specific mechanistic defects. Results Both variants exhibited partial expression defects, with p.Val367Met showing significantly increased intracellular accumulation and reduced secretion efficiency. p.Arg211Gln selectively impaired zymogen activation without affecting APC function (type IIa deficiency), whereas p.Val367Met preserved activation but reduced Na⁺-dependent catalytic efficiency (type IIb deficiency). Structural modeling revealed that Arg211 substitution disrupts activation peptide conformation, while Val367Met perturbs the Na⁺-binding loop adjacent to the S1 pocket. Co-inheritance of both resulted in synergistic anticoagulant impairment, leading to markedly elevated thrombin generation. Conclusions These findings demonstrate that PCD pathophysiology can arise from combined biosynthetic and functional defects, with distinct structural perturbations differentially affecting zymogen processing and protease activity. Integrated genetic, biochemical, and structural analyses, in the context of family history and clinical phenotype, are essential for accurate variant interpretation. Understanding the specific molecular mechanisms of PROC variants can inform clinical decision-making, guide personalized anticoagulant therapy, and help prevent thrombotic events in affected individuals. Therapeutic strategies targeting zymogen activation or proteostasis may offer potential avenues to mitigate thrombotic risk in hereditary PCD.

• Publisher: BioMed Central,Springer Nature B.V
• Subject: Adult / Antibodies / Anticoagulants / Antigens / Bioinformatics / Biomedical and Life Sciences / Biomedicine / Clotting / Decision making / Efficiency / Enzymes / Female / Genetic analysis / Genotype & phenotype / Genotypes / HEK293 Cells / Human Genetics / Humans / Intracellular / Laboratories / Male / Models, Molecular / Molecular modelling / Mutation, Missense / Pedigree / Phenotypes / Plasma / Plasmids / Proenzymes / Protein C - chemistry / Protein C - genetics / Protein C - metabolism / Protein C Deficiency - genetics / Protein deficiency / Protein expression / Proteins / Proteomics / Siblings / Structure-function relationships / Thrombin / Thrombin - metabolism / Thrombosis / Venous Thrombosis - genetics