Tissue prothrombinase activity in myometrium from timed-pregnant rats.
Tissue prothrombinase activity in myometrium from timed-pregnant rats.
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定时妊娠大鼠子宫肌层组织凝血酶原酶活性。
DOI：
10.1016/j.jsgi.2006.07.009
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Engle,Daniel
中科院分区：
文献类型：
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作者：
Phillippe,Mark;Bradley,DianaF;Phillippe,Kathrynn;Engle,Daniel
Fg 12和凝血酶在炎症诱导的早产中可能发挥作用。这些研究旨在证明功能凝血酶原酶在大鼠子宫的酶活性，与表达ofFg 12在此tissue.MethodsMyometrial和其他组织从非妊娠和定时妊娠大鼠均质化的Tris缓冲盐水。基于显色凝血酶底物的动力学代谢测定凝血酶原酶活性。将匀浆与凝血酶原孵育，然后加入凝血酶底物。通过将组织活性与使用0.01至0.04单位的活性凝血酶产生的标准曲线进行比较来确定凝血酶活性。还进行了蛋白质印迹研究以确认子宫肌层匀浆中的组织凝血酶原酶活性。凝血酶原与组织匀浆孵育，然后终止与十二烷基硫酸钠（SDS）加载buffer.ResultsProthrombinase活性在子宫肌层组织中观察到的反应是0.047至0.011 U凝血酶/10分钟/μg蛋白。热变性和钙去除消除凝血酶原酶活性，而添加因子V增强活性。Western印迹证实存在凝血酶原、预期的凝血酶前片段和凝血酶。与酶研究一致，凝血酶带在与妊娠和非妊娠大鼠的子宫肌层匀浆孵育后形成。与此相反，凝血酶带是不明显的钙，热变性和治疗与丝氨酸蛋白酶inhibitors.ConclusionsIn summary，这些研究证实了功能活跃的凝血酶原酶活性在大鼠子宫肌层，支持的假设thatFg 12是在这个组织中表达。
ObjectiveFg12and thrombin potentially play roles during inflammation-induced preterm delivery. These studies sought to demonstrate functional prothrombinase enzyme activity in rat uterus, consistent with the expression ofFg12in this tissue.MethodsMyometrial and other tissue obtained from non-pregnant and timed-pregnant rats was homogenized in Trisbuffered saline. Prothrombinase activity was determined based on the kinetic metabolism of a chromogenic thrombin substrate. Homogenates were incubated with prothrombin followed by the addition of the thrombin substrate. Thrombin activity was determined by comparing tissue activity to a standard curve generated using 0.01 to 0.04 units of active thrombin. Western blot studies were also performed to confirm tissue prothrombinase activity in myometrial homogenates. Prothrombin was incubated with tissue homogenates; the reactions were then terminated with sodium dodecyl sulfate (SDS) loading buffer.ResultsProthrombinase activity in myometrial tissue was observed to be 0.047 to 0.011 U thrombin/10 min/μg protein. Heat denaturation and calcium removal eliminated prothrombinase activity, whereas the addition of Factor V enhanced activity. The Western blots confirmed the presence of prothrombin, the anticipated prethrombin fragments, and thrombin. Consistent with the enzyme studies, the thrombin band formed upon incubation with myometrial homogenates from pregnant and nonpregnant rats. In contrast, the thrombin band was not apparent with removal of calcium, heat denaturation and treatment with serine protease inhibitors.ConclusionsIn summary, these studies have confirmed functionally active prothrombinase activity in rat myometrium, supporting the hypothesis thatFg12is expressed in this tissue.