RAT UTERINE STROMAL CELLS - THROMBIN RECEPTOR AND GROWTH-STIMULATION BY THROMBIN

The estrogen-stimulated maturation of the immature rat uterus is media ted by peptide growth factors whose expression is regulated by estradi ol. We present evidence that thrombin is a uterine growth factor. When an immature rat is given a single injection of estradiol, the uterus increases 50% in wet weight within 3 h through the imbibition of water and plasma proteins, including prothrombin. Tissue factor, the initia tor of coagulation, is induced 3- to 4-fold over the same time period. Thrombin is generated in situ from prothrombin through the coagulatio n cascade. It acts as a growth factor through the proteolytically acti vated thrombin receptor. Thrombin's role as a growth factor in uterine stromal cells is proven by two lines of evidence: demonstrations that the proteolytically activated thrombin receptor is present and that c ultured cells are stimulated to grow by thrombin.Thrombin receptor in the uterus is demonstrated by reverse transcription-PCR for receptor m essenger RNA by specific [I-125]peptide labeling of a membrane-bound b inding protein of about 60 kDa and by Western blot with a thrombin rec eptor antipeptide antibody. Thrombin's effectiveness as a growth facto r is shown by thrombin-stimulated growth of primary stromal cell cultu res, with maximum stimulation at 100 nM. That the effect is mediated b y the proteolytically activated thrombin receptor is shown by the inhi bition of growth by hirudin, a highly specific inhibitor of thrombin; the absence of enhanced growth with Pro-Phe-Arg-chloromethyl ketone-th rombin, an active site-inhibited thrombin derivative; and the stimulat ion of growth by the thrombin receptor-activating peptide.