Gly197Arg mutation in protein C causes recurrent thrombosis in a heterozygous carrier

BACKGROUND: Activated protein C (APC) downregulates thrombin generation by inactivating procoagulant cofactors Va and VIIIa by limited proteolysis. We identified two protein C-deficient patients both of whom carry a heterozygous Gly197 to Arg (G197R) mutation in PROC and experience venous thrombosis. OBJECTIVE: The objective of this study was to determine the molecular basis of the clotting defect in patients carrying the G197R mutation. METHODS: We expressed protein C-G197R in mammalian cells and characterized its properties in established coagulation and antiinflammatory assay systems. RESULTS: The activation of protein C-G197R by thrombin was improved ~10-fold, however, its activation by thrombin was not promoted by thrombomodulin (TM). In a tissue factor-mediated thrombin generation assay, the addition of soluble TM to protein C-deficient plasma, supplemented with protein C-G197R, did not have a significant inhibitory effect on thrombin generation parameters. APC-G197R did not exhibit a significant anticoagulant activity in either purified or plasma-based assay systems. APC-G197R was essentially inactive since it showed no activity in an aPTT assay. Antiinflammatory activity of APC-G197R was also dramatically impaired as determined by an endothelial cell permeability assay. Structural modeling predicted that the side-chain of Arg cannot be accommodated at this site of APC without a major distortion of the local structure that appears to propagate and adversely affect the reactivity/folding of the catalytic pocket. CONCLUSION: The G197R mutation in patients appears to be functionally equivalent to a heterozygous protein C knockout with half of the protein having no significant activity and thus causing thrombosis.