Val325Met mutation affects N-glycosylation and impairs the function of protein C.

BACKGROUND: Protein C (PC) is a key anticoagulant protein that regulates hemostatic balance. Hereditary PC deficiency increases the risk of thrombosis. We identified the same mutation, c.1099G>A:p.V325M (V325M), in the PROC gene in four unrelated patients with thrombotic events and one patient with recurrent pregnancy loss, prompting further investigation. OBJECTIVE: The objective of this study was to decipher the molecular basis of the clotting defect in patients carrying the V325M mutation. METHODS: We expressed and purified both wild-type and mutant PC/activated protein C (APC) in mammalian cells and characterized their properties in established coagulation and cytoprotective assay systems. RESULTS: Functional characterization of the recombinant mutant APC revealed multiple defects, including reduced catalytic efficiency toward chromogenic substrates, decreased affinity for Na⁺, impaired anticoagulant activity in both purified and plasma systems, and diminished barrier-protective function. In parallel, electrophoretic analysis showed that the mutant APC exhibited an altered isoform distribution compared with wild-type; this difference was eliminated upon treatment with PNGase F for deglycosylation. CONCLUSION: Our studies demonstrate that the V325M mutation directly impairs the anticoagulant and cytoprotective functions of PC, together with altered N‑glycosylation patterns, providing a molecular mechanism for the prothrombotic phenotype observed in the carriers with this mutation.