Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
Institute for Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Blood. 2019 Nov 7;134(19):1658-1669. doi: 10.1182/blood.2019000481.
The contact system produces the inflammatory peptide bradykinin and contributes to experimental thrombosis. C1 esterase-inhibitor (C1INH) deficiency or gain-of-function mutations in factor XII (FXII) cause hereditary angioedema, a life-threatening tissue swelling disease. C1INH is a relatively weak contact system enzyme inhibitor. Although α1-antitrypsin (α1AT) does not naturally inhibit contact system enzymes, a human mutation (M358R; α1AT-Pittsburgh) changes it into a powerful broad-spectrum enzyme inhibitor. It blocks the contact system, but also thrombin and activated protein C (APC), making it an unattractive candidate for therapeutic contact system blockade. We adapted the reactive center loop of α1AT-Pittsburgh (AIPR/S) to overcome these obstacles. Two α1AT variants (SMTR/S and SLLR/S) strongly inhibit plasma kallikrein, activated FXII, and plasmin. α1AT-SMTR/S no longer inhibits thrombin, but residually inhibits APC. In contrast, α1AT-SLLR/S residually inhibits thrombin, but no longer APC. Additional modification at the P1' position (S→V) eliminates residual inhibition of thrombin and APC for both variants, while retaining their properties as contact system inhibitors. Both α1AT-SMTR/V and -SLLR/V are superior to C1INH in reducing bradykinin production in plasma. Owing to their capacity to selectively block contact system-driven coagulation, both variants block vascular occlusion in an in vivo model for arterial thrombosis. Furthermore, both variants block acute carrageenan-induced tissue edema in mice. Finally, α1AT-SLLR/V, our most powerful candidate, suppresses epithelial leakage of the gut in a mouse model of colitis. Our findings confirm that redesign of α1AT strongly alters its inhibitory behavior and can be used for the treatment of contact system-mediated thrombosis and inflammation.
接触系统产生炎症肽缓激肽，并有助于实验性血栓形成。C1 酯酶抑制剂（C1INH）缺乏或因子 XII（FXII）获得性功能突变导致遗传性血管水肿，这是一种危及生命的组织肿胀疾病。C1INH 是一种相对较弱的接触系统酶抑制剂。虽然α1-抗胰蛋白酶（α1AT）天然不抑制接触系统酶，但人类突变（M358R；α1AT-匹兹堡）将其转变为强大的广谱酶抑制剂。它阻断接触系统，但也阻断凝血酶和活化蛋白 C（APC），使其成为治疗性接触系统阻断的不理想候选物。我们适应了α1AT-匹兹堡的反应中心环（AIPR/S），以克服这些障碍。两种α1AT 变体（SMTR/S 和 SLLR/S）强烈抑制血浆激肽释放酶、活化的 FXII 和纤溶酶。α1AT-SMTR/S 不再抑制凝血酶，但仍抑制 APC。相比之下，α1AT-SLLR/S 仍抑制凝血酶，但不再抑制 APC。在 P1'位置（S→V）的进一步修饰消除了两种变体对凝血酶和 APC 的残余抑制作用，同时保留了它们作为接触系统抑制剂的特性。在降低血浆中缓激肽产生方面，两种α1AT-SMTR/V 和 -SLLR/V 均优于 C1INH。由于它们能够选择性地阻断接触系统驱动的凝血，两种变体均可在动脉血栓形成的体内模型中阻断血管闭塞。此外，两种变体均可阻断急性角叉菜胶诱导的小鼠组织水肿。最后，我们最强大的候选物α1AT-SLLR/V 抑制了结肠炎小鼠模型中的肠道上皮渗漏。我们的研究结果证实，α1AT 的重新设计强烈改变了其抑制行为，可用于治疗接触系统介导的血栓形成和炎症。
