EVIDENCE THAT MEIZOTHROMBIN IS AN INTERMEDIATE PRODUCT IN THE CLOTTING OF WHOLE-BLOOD
EVIDENCE THAT MEIZOTHROMBIN IS AN INTERMEDIATE PRODUCT IN THE CLOTTING OF WHOLE-BLOOD
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DOI：
10.1161/01.atv.15.6.754
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发表时间：
1995-06-01
影响因子：
8.7
通讯作者：
MANN, KG
中科院分区：
文献类型：
--
作者：
BOVILL, EG;TRACY, RP;MANN, KG
凝血酶是在由纯化因子Xa和因子Va组成的体系中将凝血酶原转化为凝血酶时产生的一种中间体，该体系定量地组装在阴离子磷脂表面上。这种中间体的生物学意义最近受到了柠檬酸钠抗凝血浆中凝血酶明显缺失的挑战。我们分析了在玻璃管中非抗凝、未冷冻、最小操作全血凝血过程中凝血酶的形成。通过添加生物素化肽基氯甲基酮活性位点标记试剂，在0,3,5和7分钟时停止该过程。离心分离血浆/血清，用抗凝血酶原抗体免疫吸附法提取标记种。通过sds -聚丙烯酰胺梯度凝胶电泳分离纯化的凝血酶原来源物种，并在化学发光亲和素印迹上显示。减数凝血酶作为该反应的中间产物出现，并持续存在并在1分钟时间点有所增加。我们还观察到活性位点标签与β -和/或γ -凝血酶链B-1一致的低分子量物种的结合。这些降解形式的凝血酶以前还没有在生物学相关的制剂中得到证实。我们的数据清楚地表明，减数凝血酶的生成是全血凝血过程中凝血酶生成的中间产物。这些数据也代表了在生物学相关的环境和时间尺度上生成β -和γ -凝血酶的第一个实验证据。
Meizothrombin is an intermediate that is produced during the conversion of prothrombin to thrombin in systems composed of purified factor Xa and factor Va that are quantitatively assembled on an anionic phospholipid surface. The biological significance of this intermediate has recently been challenged by the apparent absence of meizothrombin during clotting of sodium citrate-anticoagulated plasma. We analyzed the formation of thrombin during coagulation of nonanticoagulated, unchilled, minimally manipulated whole blood in glass tubes. The process was stopped at 0, 3, 5, and 7 minutes by the addition of biotinylated peptidyl chloromethylketone active-site labeling reagents. Plasma/serum was separated by centrifugation, and labeled species were extracted by immunoadsorption with a polyclonal anti-prothrombin antibody. The purified prothrombin-derived species were separated by SDS-polyacrylamide gradient gel electrophoresis and visualized on a chemiluminescent avidin blot. Meizothrombin appeared as an intermediate product of this reaction and persisted with some increase through the it-minute time point. We also observed incorporation of the active-site label into a species of lower molecular weight consistent with the B-1, chain of beta- and/or gamma-thrombin. These degraded forms of thrombin have not been previously demonstrated in a biologically relevant preparation. Our data clearly establish the generation of meizothrombin as an intermediate product of thrombin generation during whole-blood clotting. The data also represent the first experimental evidence for the generation of beta- and gamma-thrombin in a biologically relevant environment and time scale.