Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
Thromb Haemost. 2012 May;107(5):972-84. doi: 10.1160/TH11-09-0604. Epub 2012 Feb 28.
The variant serpin α1-PI M358R inhibits thrombin and other proteases such as activated protein C (APC) and factor XIa. We previously described recombinant proteins HAPI M358R (α1-PI M358R containing an N-terminal extension corresponding to residues 1-75 of heparin cofactor II) and HAPI RCL5 (HAPI M358R with F352-I356 and I360 substituted for the corresponding residues of antithrombin), with enhanced selectivity for thrombin over APC inhibition. We tested the hypotheses that these recombinant proteins would limit thrombosis in three mouse models, and that the HAPI chimeric proteins would be more effective than α1-PI M358R. Recombinant serpins were purified from Escherichia coli by nickel chelate and ion exchange affinity chromatography, and administered to mice intravenously. HAPI RCL5 reduced incorporation of radiolabelled fibrin(ogen) into thrombi in the ferric chloride-injured vena cava in a dose-dependent manner; HAPI M358R was less effective and α1-PI M358R was without effect. In a model of murine endotoxaemia, HAPI RCL5 was more effective than α1-PI M358R in reducing radiolabelled fibrin(ogen) deposition in heart and kidneys; immunohistochemistry of tissue sections showed lesser staining with anti-fibrin(ogen) antibodies with both treatments. In the ferric chloride-injured murine carotid artery, administration of both recombinant serpins was equally effective in lengthening the vessel's time to occlusion. Our results show that the antithrombotic efficacy of the recombinant serpins correlates with their potency as thrombin inhibitors, since HAPI RCL5 inhibits thrombin, but not factors Xa, XIa, XIIa, or neutrophil elastase, more rapidly than α1-PI M358R.
变异丝氨酸蛋白酶抑制剂 α1-PI M358R 可抑制凝血酶和其他蛋白酶，如激活蛋白 C (APC) 和因子 XIa。我们之前描述了重组蛋白 HAPI M358R（含有肝素辅因子 II 1-75 位残基 N 端延伸的 α1-PI M358R）和 HAPI RCL5（用 F352-I356 和 I360 取代抗凝血酶相应残基的 HAPI M358R），它们对凝血酶的抑制作用比对 APC 的抑制作用更具选择性。我们检验了以下假设：这些重组蛋白将在三种小鼠模型中限制血栓形成，并且 HAPI 嵌合蛋白将比 α1-PI M358R 更有效。重组丝氨酸蛋白酶通过镍螯合和离子交换亲和层析从大肠杆菌中纯化，并通过静脉内给药给予小鼠。HAPI RCL5 以剂量依赖性方式减少氯化铁损伤的腔静脉中放射性标记纤维蛋白原（ogen）掺入血栓的量；HAPI M358R 的效果较差，而 α1-PI M358R 则无效。在小鼠内毒素血症模型中，HAPI RCL5 在减少心脏和肾脏放射性标记纤维蛋白原（ogen）沉积方面比 α1-PI M358R 更有效；两种治疗方法的组织切片免疫组织化学染色显示抗纤维蛋白原（ogen）抗体染色较少。在氯化铁损伤的小鼠颈总动脉中，两种重组丝氨酸蛋白酶的给药同样有效，可延长血管闭塞时间。我们的结果表明，重组丝氨酸蛋白酶的抗血栓功效与其作为凝血酶抑制剂的效力相关，因为 HAPI RCL5 比 α1-PI M358R 更快地抑制凝血酶，但不抑制因子 Xa、XIa、XIIa 或中性粒细胞弹性蛋白酶。
