A patient with hematuria in our clinic was diagnosed with urolithiasis. Analysis of the patient’s plasma clotting-time indicated that both APTT (52.6 s) and PT (19.4 s) are prolonged and prothrombin activity is reduced to 12.4% of normal, though the patient exhibited no abnormal bleeding phenotype and a prothrombin antigen level of 87.9%. Genetic analysis revealed the patient is homozygous for prothrombin Y510N mutation. We expressed and characterized the prothrombin-Y510N variant in appropriate coagulation assays and found that the specificity constant for activation of the mutant zymogen by factor Xa is impaired ~5-fold. Thrombin generation assay using patient’s plasma and prothrombin-deficient plasma supplemented with either wild-type or prothrombin-Y510N revealed that both peak height and time to peak for the prothrombin mutant are decreased however the endogenous thrombin generation potential is increased. Further analysis indicated that the thrombin mutant exhibits resistance to antithrombin and is inhibited by the serpin with ~12-fold slower rate constant. Protein C activation by thrombin-Y510N was also decreased ~10-fold, however, thrombomodulin overcame the catalytic defect. The Na+-concentration-dependence of the amidolytic activities revealed that the dissociation constant for the interaction of Na+ with the mutant has been elevated ~20-fold. These results suggest that Y510 (Y184a in chymotrypsin numbering) belongs to network of residues involved in binding Na+. A normal protein C activation by thrombin-Y510N suggests that thrombomodulin modulates the conformation of the Na+-binding loop of thrombin. The clotting defect of thrombin-Y510N appears to be compensated by its markedly lower reactivity with antithrombin, explaining patient’s normal hemostatic phenotype.

中文翻译：

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抗凝血酶抗性可挽救纯合子凝血酶原-Tyr510N 凝血酶原血症的凝血缺陷

我们门诊一位出现血尿的患者被诊断为尿石症。对患者血浆凝血时间的分析表明，APTT（52.6 秒）和 PT（19.4 秒）均延长，凝血酶原活性降至正常水平的 12.4%，但患者未表现出异常出血表型且凝血酶原抗原水平为 87.9 %。遗传分析显示该患者是凝血酶原 Y510N 突变的纯合子。我们在适当的凝血测定中表达并表征了凝血酶原-Y510N 变体，发现 Xa 因子激活突变酶原的特异性常数受损约 5 倍。使用患者血浆和补充有野生型或凝血酶原-Y510N 的凝血酶原缺陷血浆进行的凝血酶生成测定显示，凝血酶原突变体的峰高和达峰时间均降低，但内源性凝血酶生成潜力增加。进一步分析表明，凝血酶突变体表现出抗凝血酶抗性，并被丝氨酸蛋白酶抑制剂抑制，速率常数降低约 12 倍。凝血酶-Y510N 对蛋白 C 的激活也降低了约 10 倍，然而，血栓调节蛋白克服了催化缺陷。酰胺分解活性的 Na+ 浓度依赖性表明，Na+ 与突变体相互作用的解离常数已升高约 20 倍。这些结果表明 Y510（糜蛋白酶编号中的 Y184a）属于参与结合 Na+ 的残基网络。由凝血酶-Y510N 激活的正常蛋白 C 表明凝血调节蛋白调节凝血酶 Na+ 结合环的构象。凝血酶-Y510N 的凝血缺陷似乎被其与抗凝血酶的显着较低反应性所补偿，这解释了患者的正常止血表型。