【Abstract】
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.
【中文摘要】
背景：活化蛋白C可通過滅活輔因子Va及VIIIa來下調凝血酶生成。課題組發現兩例攜帶PC-G197R雜合突變的靜脈血栓患者。
目的：闡明PC-G197R突變導致血栓發生的分子發病機制。
方法：通過真核細胞表達PC-G197R蛋白，並通過相應的凝血及抗炎試驗對突變蛋白進行相關檢測。
結果：凝血酶對PC-G197R的活化作用增強約10倍，但血栓調節蛋白並不能增強凝血酶對突變蛋白C的活化。凝血酶生成試驗及APTT檢測結果均顯示，APC-G197R抗凝作用明顯減弱。內皮細胞滲透性實驗檢測顯示APC-G197R突變的抗炎作用亦顯著減弱。結構模型預測顯示APC該位點被精氨酸代替後，其側鏈影響蛋白結構進而不可逆地影響PC催化結構域中催化口袋的正確摺疊及催化反應能力。
結論：由於PC-G197R突變蛋白無明顯抗凝活性，因此按蛋白C功能分析，攜帶PC-G197R雜合突變等同於蛋白C的雜合性缺失，從而導致患者血栓形成。