Effects of thrombin on steroid-modulated cultured endometrial stromal cell fibrinolytic potential.
By virtue of their unique chronic expression of tissue factor, the primary initiator of hemostasis, decidualized endometrial stromal cells are capable of significant thrombin generation after vascular disruption. In addition to its potent procoagulant effects, thrombin modifies endothelial and glomerular cell fibrinolytic activity. Therefore, we evaluated whether thrombin affected the expression of endometrial stromal cell urokinase-type (uPA) and tissue-type (tPA) plasminogen activators and their primary inhibitor, type 1 plasminogen activator inhibitor (PAI-1), and whether ovarian steroids modulated putative thrombin effects. Confluent stromal cell cultures were incubated in a defined medium containing vehicle control, 10(-8) mol/L estradiol (E2), 10(-7) mol/L medroxyprogesterone acetate (MPA), or E2 plus MPA for 4 days. The medium was then collected and exchanged for medium containing the corresponding steroids with or without thrombin and the specific thrombin inhibitor, D-phenyl-alanyl-propyl-arginine-chloromethyl ketone, for an additional 24 h. The conditioned medium was then collected and analyzed for immunoreactive (ir) uPA, tPA, and PAI-1 by enzyme-linked immunosorbent assay and for PA activity by chromogenic assay, whereas Northern analysis of the cells was employed to evaluate the expression of thrombin receptor, uPA, tPA, and PAI-1 messenger ribonucleic acid (mRNA) species. The latter studies revealed that confluent cultures incubated in defined medium expressed the 3.45-kilobase thrombin receptor message. Steady state levels of thrombin receptor mRNA were unaffected by exogenous steroids. Thrombin added in the absence of exogenous steroids elevated concentrations of ir tPA, uPA, and PAI-1 compared with control cultures. Conversely, in the absence of added thrombin, MPA added alone or together with E2 inhibited levels of ir tPA and uPA while stimulating PAI-1 levels despite the lack of a response to E2 alone. Interestingly, thrombin counteracted this progestin inhibition of tPA and uPA expression and augmented the progestin-enhanced expression of PAI-1. Northern analysis revealed that steady state levels of tPA and uPA mRNA were also enhanced by thrombin in both control and steroid-containing cultures. Net PA activity reflects the balance between PA and PAI-1. In the absence of thrombin, there is virtually no detectable tPA activity and minimal uPA activity in progestin-exposed cultures. However, thrombin elicited significant increases in tPA and uPA activity in control and E2-treated cultures. Despite the molar excess of PAI-1 in MPA-treated and E2- plus MPA-treated cultures, thrombin reversed progestin inhibition of PA activity. Predictably, the addition of D-phenyl-alanyl-propyl-arginine-chloromethyl ketone, blocked the effects of thrombin on PAI-1, tPA, and uPA protein and mRNA expression and PA activity. In summary, thrombin enhances endometrial stromal cell fibrinolytic and extracellular matrix-degrading protease activity in vitro. Such processes occurring in vivo would probably play a role in menstruation and abnormal uterine bleeding.
Department of Obstetrics and Gynecology, New York University School of Medicine, New York 10016, USA.
C J
CJ
Lockwood
G
G
Krikun
S
S
Aigner
F
F
Schatz
D002478:Cells, Cultured; D004717:Endometrium / Q000166:cytology* / Q000187:drug effects; D005260:Female; D005342:Fibrinolysis / Q000187:drug effects*; D012739:Gonadal Steroid Hormones / Q000494:pharmacology*; D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D018179:Receptors, Thrombin / Q000032:analysis; D017154:Stromal Cells / Q000502:physiology; D013917:Thrombin / Q000494:pharmacology*; D010959:Tissue Plasminogen Activator / Q000032:analysis / Q000235:genetics; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis / Q000235:genetics
D012739:Gonadal Steroid Hormones; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D018179:Receptors, Thrombin; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator