Nielsen Vance G, Cohen Jack B, Malayaman S Nini, Nowak Mathew, Vosseller Keith
Department of Anesthesiology bDepartment of Biochemistry, Drexel University College of Medicine, Broad & Vine Streets, Philadelphia, PA 19102, USA.
Blood Coagul Fibrinolysis. 2011 Jul;22(5):443-7. doi: 10.1097/MBC.0b013e328345c069.
The objective of this study was to determine how carbon monoxide directly modifies fibrinogen utilizing liquid chromatography-mass spectrometry (LC-MS/MS) by examining fibrinogen exposed to carbon monoxide releasing molecule-2 [tricarbonyldichlororuthenium (II) dimer; CORM-2]. Purified fibrinogen was exposed to 0, 25, 50 or 100 μmol/l CORM-2 for 5 min at 37°C and then stored at -80°C before analyses with LC-MS/MS. In a second series of experiments, normal plasma was exposed to 0 or 100 μmol/l CORM-2 in the absence or presence of the nitric oxide donor sodium nitroprusside and hydroquinone (an organic reductant) to compete with carbon monoxide binding to a putative heme group found on fibrinogen. Coagulation was activated with tissue factor (n=8 per condition). Thrombus growth was monitored with thrombelastography for 15 min. LC-MS/MS did not detect any direct modifications of amino acids in fibrinogen, but detection of small regions of both the alpha and gamma chains was lost following exposure to CORM-2 and endoproteinase digestion with trypsin and Glu-C. An ion with the same m/z and expected retention time as heme was found in the purified fibrinogen. Exposure of plasma to nitric oxide/hydroquinone significantly decreased CORM-2-mediated enhancement of coagulation without affecting the coagulation kinetics of plasma not exposed to CORM-2. Carbon monoxide derived from CORM-2 likely modifies fibrinogen via modulation of a fibrinogen-associated heme group(s). Whereas the precise molecular location of heme attachment and three-dimensional conformational change secondary to carbon monoxide exposure remain to be determined, fibrinogen appears to be a carbon monoxide sensing molecule.
本研究的目的是通过检测暴露于一氧化碳释放分子 -2[二氯三羰基钌（II）二聚体；CORM -2]的纤维蛋白原，利用液相色谱 - 质谱联用技术（LC-MS/MS）来确定一氧化碳如何直接修饰纤维蛋白原。将纯化的纤维蛋白原在37°C下暴露于0、25、50或100 μmol/l的CORM -2中5分钟，然后在-80°C下储存，再用LC-MS/MS进行分析。在第二系列实验中，将正常血浆在不存在或存在一氧化氮供体硝普钠和对苯二酚（一种有机还原剂）的情况下暴露于0或100 μmol/l的CORM -2，以竞争一氧化碳与纤维蛋白原上假定的血红素基团的结合。用组织因子激活凝血（每种条件下n = 8）。用血栓弹力图监测血栓形成15分钟。LC-MS/MS未检测到纤维蛋白原中氨基酸的任何直接修饰，但在暴露于CORM -2并用胰蛋白酶和Glu-C进行内切蛋白酶消化后，α链和γ链的小区域检测信号丢失。在纯化的纤维蛋白原中发现了一个与血红素具有相同m/z和预期保留时间的离子。血浆暴露于一氧化氮/对苯二酚可显著降低CORM -2介导的凝血增强作用，而不影响未暴露于CORM -2的血浆的凝血动力学。源自CORM -2的一氧化碳可能通过调节与纤维蛋白原相关的血红素基团来修饰纤维蛋白原。虽然血红素附着的确切分子位置以及一氧化碳暴露后继发的三维构象变化仍有待确定，但纤维蛋白原似乎是一种一氧化碳传感分子。
