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Published on: April 1, 2019
Gly197Arg mutation in protein C causes recurrent thrombosis in a heterozygous carrier
Yeling Lu1,2, Hemant Giri2, Bruno O Villoutreix3
1Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
Insights
A Gly197 to Arg mutation in protein C causes venous thrombosis by impairing activated protein C (APC) anticoagulant and anti-inflammatory functions. This mutation results in a functionally inactive protein, similar to a protein C knockout.
Area of Science:
- Biochemistry
- Hematology
- Molecular Biology
Background:
- Activated protein C (APC) regulates thrombin generation by inactivating factors Va and VIIIa.
- Two protein C-deficient patients with heterozygous Gly197 to Arg (G197R) mutation in PROC presented with venous thrombosis.
Purpose of the Study:
- To elucidate the molecular basis of the clotting defect associated with the G197R mutation in protein C.
- To characterize the functional consequences of the G197R mutation on protein C's anticoagulant and anti-inflammatory activities.
Main Methods:
- Expressed protein C-G197R in mammalian cells.
- Assessed protein C-G197R activation by thrombin and thrombomodulin (TM).
- Evaluated anticoagulant activity in plasma and purified systems using thrombin generation and aPTT assays.
- Determined anti-inflammatory activity via an endothelial cell permeability assay.
- Utilized structural modeling to predict the impact of the mutation on protein structure and function.
Main Results:
- Protein C-G197R activation by thrombin was enhanced ~10-fold but not promoted by TM.
- APC-G197R showed no significant anticoagulant activity in various assay systems.
- Anti-inflammatory activity of APC-G197R was markedly impaired.
- Structural modeling indicated significant local and catalytic pocket distortions due to the Arg substitution.
Conclusions:
- The G197R mutation renders protein C functionally inactive, akin to a heterozygous knockout.
- This loss of function contributes to thrombosis in affected patients.
- The findings highlight the critical role of protein C in hemostasis and inflammation.
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 anti-inflammatory 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 because it showed no activity in an aPTT assay. Anti-inflammatory 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.
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