Komissarov Andrey A, Andreasen Peter A, Declerck Paul J, Kamikubo Yuichi, Zhou Aiwu, Gruber András
Department of Chemistry, Portland State University, P. O. Box 751, Portland, OR 97207-0751, USA.
Thromb Res. 2008;122(3):397-404. doi: 10.1016/j.thromres.2007.10.012. Epub 2007 Nov 28.
Activated protein C (APC) reduces mortality in severe sepsis. Protecting APC in the circulatory system from inactivation by serine protease inhibitors (serpins) could improve its therapeutic efficiency. Significantly elevated levels of a serpin plasminogen activator inhibitor 1 (PAI-1) correlate with a lethal outcome in severe sepsis and disseminated intravascular coagulation. Intermolecular mechanisms were employed to redirect the reaction between APC and PAI-1 from the inhibitory to the substrate pathway, which results in the catalytic neutralization of the serpin.
The effects of anti-PAI-1 monoclonal antibodies (mAbs) and vitronectin, as well as their fragments, on the kinetics and stoichiometry of the reaction between PAI-1 and APC were studied using SDS PAGE and fluorescence spectroscopy.
MAbs with epitopes at alpha-helix F redirected 70-80% of the reaction between PAI-1 and APC, to the substrate pathway. Vitronectin and its SMB domain did not affect the stoichiometry of acyl-enzyme formation, but enhanced the effect of mAbs. While vitronectin induced a more than two-fold increase in the rate of the reaction between PAI-1 and APC, neither mAbs (mAb fragments), nor SMB domain of vitronectin affected it.
Ligands interacting with alpha-helix F of PAI-1 demonstrated a potential for the protection of APC from inactivation by PAI-1. Since the mechanism of proteinase/serpin interaction is universal, a similar design and approach could be employed for enhancing the inactivation of other serpins in order to preserve APC activity in the circulation. Rational pharmacological targeting of the inhibitors of APC could have therapeutic utility.
活化蛋白C（APC）可降低严重脓毒症的死亡率。保护循环系统中的APC不被丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制因子）灭活可提高其治疗效果。丝氨酸蛋白酶抑制因子纤溶酶原激活物抑制剂1（PAI-1）水平显著升高与严重脓毒症和弥散性血管内凝血的致死结局相关。采用分子间作用机制将APC与PAI-1之间的反应从抑制途径重定向至底物途径，从而导致丝氨酸蛋白酶抑制因子的催化失活。
使用SDS-PAGE和荧光光谱法研究抗PAI-1单克隆抗体（mAb）和玻连蛋白及其片段对PAI-1与APC之间反应动力学和化学计量学的影响。
表位位于α-螺旋F的mAb将PAI-1与APC之间70%-80%的反应重定向至底物途径。玻连蛋白及其SMB结构域不影响酰基酶形成的化学计量，但增强了mAb的作用。虽然玻连蛋白使PAI-1与APC之间的反应速率提高了两倍多，但mAb（mAb片段）和玻连蛋白的SMB结构域均未对其产生影响。
与PAI-1的α-螺旋F相互作用的配体显示出保护APC不被PAI-1灭活的潜力。由于蛋白酶/丝氨酸蛋白酶抑制因子相互作用的机制具有普遍性，因此可采用类似的设计和方法来增强其他丝氨酸蛋白酶抑制因子的失活，以维持循环中APC的活性。对APC抑制剂进行合理的药理学靶向可能具有治疗效用。
