No abstract

Selective oestrogen receptor modulators (SERMs) are structurally diverse non-steroidal compounds that bind to oestrogen receptors and produce oestrogen agonist effects in some tissues and oestrogen antagonist effects in others. SERMs are being evaluated for a number of oestrogen-related diseases, including post-menopausal osteoporosis, hormone-dependent cancers, and cardiovascular disease. Several compounds that exhibit a SERM profile are currently available for clinical use, including clomiphene, tamoxifen, and toremifene (which are triphenylethylenes) and raloxifene (a benzothiophene). Clomiphene is used for the induction of ovulation in sub-fertile women attempting pregnancy. Tamoxifen and toremifene are both used to treat breast cancer. Tamoxifen may have beneficial effects on bone mineral density and serum lipids. The effects of toremifene on serum lipids are similar to that of tamoxifen. Both compounds have stimulatory effects on the endometrium. Raloxifene, indicated for the treatment and prevention of post-menopausal osteoporosis, has beneficial effects on bone mineral density and serum lipids, but does not increase the risk of endometrial hyperplasia or endometrial cancer. Recently, raloxifene was shown to reduce the incidence of vertebral fractures in otherwise healthy women with osteoporosis; in the same study, a reduced incidence of breast cancer was also observed. Similar to oestrogens, SERMs increase the incidence of venous thromboembolism. Several newer compounds that exhibit a SERM profile are also in clinical development, including other triphenylethylenes (droloxifene, idoxifene) and benzothiophenes (LY353381.HCl), benzopyrans (EM-800), and naphthalenes (CP-336,156).

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A heparin cofactor II (HCII) mutant with an Arg substituted for Leu 444 at the P1 position (L444R-rHCII) was previously found to have altered proteinase specificity (Derechin, V. M., Blinder, M. A., and Tollefsen, D. M. (1990) J. Biol. Chem. 265, 5623-5628). The present study characterizes the effect of glycosaminoglycans on the substrate versus inhibitor activity of L444R-rHCII. Heparin increased the stoichiometry of inhibition of L444R-rHCII with ␣-thrombin (compared with minus glycosaminoglycan) but decreased it with R93A,R97A,R101A-thrombin, a mutant thrombin that does not bind glycosaminoglycans. Dermatan sulfate decreased the stoichiometry of inhibition of L444R-rHCII with both proteinases. SDS-polyacrylamide gel electrophoresis showed no proteolysis of L444R-rHCII when incubated with R93A,R97A,R101A-thrombin in the absence or the presence of glycosaminoglycan or with ␣-thrombin and dermatan sulfate. In contrast, greater than 75% of the L444R-rHCII was converted to a lower molecular weight form when incubated with ␣-thrombin/heparin. A time course of ␣-thrombin inhibition by L444R-rHCII/heparin showed a rapid but transient inhibition with approximately 80% of the ␣-thrombin activity being regained after 6 h of incubation. In contrast, all other combinations of inhibitor, proteinase, and glycosaminoglycan resulted in complete and sustained inhibition of the proteinase. Heparin fragments of 8 -20 polysaccharides in length rapidly accelerated L444R-rHCII inhibition of both ␣-thrombin and R93A,R97A,R101A-thrombin. After extended incubations, R93A,R97A,R101A-thrombin was completely inhibited by L444R-rHCII with all the heparin fragments, but approximately 30 -50% of ␣-thrombin activity remained with fragments long enough to bridge HCII-thrombin. These results collectively indicate that ternary complex formation, mediated by heparin, increases L444R-rHCII inactivation by ␣-thrombin. Serine proteinase inhibitors (serpins)1 are a highly conserved family of proteins whose primary role is to regulate the activity of a wide variety of serine proteinases involved in processes such as blood coagulation, fibrinolysis, inflammation, and cancer metastasis (1-3). Serpins inhibit their target proteinase by functioning as suicide substrates. The serpin contains a peptide bond, termed P1-P1Ј by the nomenclature of Schechter and Berger (4), within an exposed region, called the reactive site loop, that is recognized as a substrate by the proteinase (2). The initial binding of the serpin and proteinase results in the formation of a Michaelis complex, which can then partition between a kinetically stable intermediate, resulting in proteinase inhibition, and a cleaved inactive inhibitor (3, 5, 6). Although the sequence of the amino acids in the reactive site loop and on the P1-P1Ј residues in particular determines in part the proteinase specificity of the serpin, they also determine the partitioning of the serpin into an inhibitor versus substrate pathway with a given proteinase. Heparin cofactor II (HCII), protein C inhibitor, pr...

Raloxifene and CEE have beneficial effects on bone density and bone turnover, although effects of CEE are more marked. Raloxifene and CEE produce different patterns of lipid responses and have distinct adverse effect profiles.