Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland 4059, Australia.
Biochemistry. 2011 Oct 4;50(39):8454-62. doi: 10.1021/bi201203y. Epub 2011 Sep 9.
Perioperative bleeding is a cause of major blood loss and is associated with increased rates of postoperative morbidity and mortality. To combat this, antifibrinolytic inhibitors of the serine protease plasmin are commonly used to reduce bleeding during surgery. The most effective and previously widely used of these is the broad range serine protease inhibitor aprotinin. However, adverse clinical outcomes have led to use of alternative serine lysine analogues to inhibit plasmin. These compounds suffer from low selectivity and binding affinity. Consequently, a concerted effort to discover potent and selective plasmin inhibitors has developed. This study used a noncombinatorial peptide library to define plasmin's extended substrate specificity and guide the design of potent transition state analogue inhibitors. The various substrate binding sites of plasmin were found to exhibit a higher degree of cooperativity than had previously been appreciated. Peptide sequences capitalizing on these features produced high-affinity inhibitors of plasmin. The most potent of these, Lys-Met(sulfone)-Tyr-Arg-H [KM(O(2))YR-H], inhibited plasmin with a K(i) of 3.1 nM while maintaining 25-fold selectivity over plasma kallikrein. Furthermore, 125 nM (0.16 μg/mL) KM(O(2))YR-H attenuated fibrinolysis in vitro with an efficacy similar to that of 15 nM (0.20 μg/mL) aprotinin. To date, this is the most potent peptide inhibitor of plasmin that exhibits selectivity against plasma kallikrein, making this compound an attractive candidate for further therapeutic development.
围手术期出血是大量失血的一个原因，并与术后发病率和死亡率的增加有关。为了应对这一问题，通常使用丝氨酸蛋白酶纤溶酶的纤维蛋白溶解抑制剂来减少手术过程中的出血。其中最有效且以前广泛使用的是广谱丝氨酸蛋白酶抑制剂抑肽酶。然而，不良的临床结果导致使用替代的丝氨酸赖氨酸类似物来抑制纤溶酶。这些化合物的选择性和结合亲和力较低。因此，人们一直在努力开发有效的、选择性的纤溶酶抑制剂。本研究使用非组合肽文库来确定纤溶酶的扩展底物特异性，并指导设计有效的过渡态类似物抑制剂。纤溶酶的各种底物结合位点被发现比以前认为的具有更高程度的协同性。利用这些特征的肽序列产生了对纤溶酶具有高亲和力的抑制剂。其中最有效的是 Lys-Met(sulfone)-Tyr-Arg-H [KM(O(2))YR-H]，对纤溶酶的抑制常数（Ki）为 3.1 nM，而对血浆激肽释放酶的选择性保持在 25 倍。此外，125 nM（0.16 μg/mL）KM(O(2))YR-H 在体外抑制纤维蛋白溶解的效果与 15 nM（0.20 μg/mL）抑肽酶相似。迄今为止，这是对血浆激肽释放酶具有选择性的最有效的纤溶酶肽抑制剂，这使得该化合物成为进一步治疗开发的有吸引力的候选物。
