Tissue prothrombinase activity in myometrium from timed-pregnant rats.

Abstract

Fgl2 and 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 of Fgl2 in this tissue. Myometrial and other tissue obtained from non-pregnant and timed-pregnant rats was homogenized in Tris-buffered 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. Prothrombinase activity in myometrial tissue was observed to be 0.047 to 0.077 U thrombin/10 min/microg 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. In summary, these studies have confirmed functionally active prothrombinase activity in rat myometrium, supporting the hypothesis that Fgl2 is expressed in this tissue.