Abstract
Abstract
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.V325 M (V325 M), in the PROC gene in four unrelated patients with thrombotic events and one patient with recurrent pregnancy loss, prompting further investigation.
The objective of this study was to decipher the molecular basis of the clotting defect in patients carrying the V325 M mutation.
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.
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.
Our studies demonstrate that the V325 M mutation directly impairs the anticoagulant and cytoprotective functions of PC, together with altered Nglycosylation patterns, providing a molecular mechanism for the prothrombotic phenotype observed in the carriers with this mutation.