Trp83 is vital to the interaction with phospholipids in γ-carboxyglutamic acid domain of protein C

Activated protein C is an important physiological anticoagulant protein. It can downregulate thrombin generation by inactivating coagulation factors Va and VIIIa, thereby exerting an anticoagulant effect. We identified that several families presenting with thrombosis harbored heterozygous mutations at the identical Trp83 residue of PROC, c.247T>C, p.Trp83Arg (W83R) and c.248G>C, and p.Trp83Ser (W83S). While the mutations were previously reported in a large-scale screening of the Chinese population, functional studies to elucidate the pathogenic mechanisms remain uninvestigated.

This study aims to evaluate the contribution of PC-W83R and PC-W83S mutations to thrombosis risk and to elucidate the specific pathogenic mechanisms.

We expressed the recombinant PC-W83R and PC-W83S in mammalian cells and characterized their properties in established coagulation and anti-inflammatory assay systems.

Both PC-W83R and PC-W83S were expressed at levels comparable to that of PC-WT. The activation rate of PC-W83R by thrombin was higher than that of WT, whereas the activation rate of PC-W83S was comparable to WT. However, when activated by the thrombin and thrombomodulin complex, both PC-W83R and PC-W83S variants exhibited reduced activation rates when compared with PC-WT. The catalytic efficiency toward the chromogenic substrate was comparable to that of the wild type. Both mutants exhibited reduced interaction with phospholipids and led to a dramatically attenuated capacity for FVa inhibition. Additionally, the anti-inflammatory activity of activated protein C-W83R and activated protein C-W83S was also impaired.

The W83R and W83S mutations impair the binding of PC to phospholipids, consequently leading to a substantial reduction in its anticoagulant function, which increases the thrombosis risk in patients.