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While the risk for symptomatic atherosclerotic disease increases after menopause, currently recognized risk factors do not identify ongoing disease processes in low-risk women. This study tested the hypothesis that circulating cell-derived microparticles may reflect disease processes in women defined as low risk by the Framingham risk score. The concentration and phenotype of circulating microparticles were evaluated in a cross-sectional study of apparently healthy menopausal women, screened for enrollment into the Kronos Early Estrogen Prevention Study. Microparticles were evaluated by flow cytometry, and coronary artery calcification (CAC) was scored using 64-slice computed tomography scanners. The procoagulant activity of isolated microparticles was determined with a sensitive fluorescent thrombin generation assay. Chronological age, body mass index, serum lipids, systolic blood pressure (Framingham risk score < 10%, range 1-3%), and high-sensitivity C-reactive protein did not differ significantly among women with low (0 < 35; range, 0.3-32 Agatston units) or high (>50; range, 93-315 Agatston units) CAC compared with women without calcification. The total concentration and percentage of microparticles derived from platelets and endothelial cells were greatest in women with high CAC scores. The thrombin-generating capacity of the isolated microparticles correlated with phosphatidylserine expression, which also was greatest in women with high CAC scores. The percentages of microparticles expressing granulocyte and monocyte markers were not significantly different among groups. Therefore, the characterization of platelet and endothelial microparticles may identify early menopausal women with premature CAC who would not otherwise be identified by the usual risk factor analysis. phosphatidylserine; procoagulant activity; thrombosis

Oral estrogen treatment increases thrombotic risk. Tissue factor (TF), tissue factor pathway inhibitor (TFPI), and platelet interaction with leukocytes are important determinants of thrombogenesis. Therefore, the present study was designed to define and compare platelet TF and TFPI mRNA and adhesion protein expression in platelets derived from animals treated with different types of oral estrogens. Ovariectomized pigs were treated with 17[beta]-estradiol (2 mg/day), conjugated equine estrogen (CEE; 0.625 mg/day), or raloxifene (60 mg/day) for 4 wk. Compared with intact animals, ovariectomy and treatment differentially affected populations of leukocytes: neutrophils decreased whereas lymphocytes increased significantly 4 wk after ovariectomy and with 17[beta]-estradiol and CEE treatments; eosinophils increased only with 17[beta]-estradiol treatment. Content of TF protein increased in platelets from 17[beta]-estradiol- and raloxifene-treated pigs, whereas TF mRNA was detected only in platelets from 17[beta]-estradiol- and CEE treated pigs. TFPI mRNA increased in platelets after ovariectomy and estrogen treatment. Only a trace of TFPI protein was detected, but a higher-molecular-mass protein was observed in all treatment groups. Expression of CD40 and CD40 ligand increased with ovariectomy and decreased with 17[beta]-estradiol and CEE treatments more than with raloxifene. The ratio of activated to basal P-selectin expression decreased with ovariectomy and increased with raloxifene treatments. These results suggest that estrogenic formulations may affect individual thrombotic risk by different mechanisms that regulate 'IF and platelet-leukocytic interactions. These studies provide the rationale for evaluation of interactions among platelets and TF and TFPI expression on thrombin generation during estrogen treatment in humans. conjugated equine estrogen; CD40; 17[beta]-estradiol; leukocytes; raloxifene

Estrogen receptor beta (ssER) IS the predominant estrogen receptor in platelets, Experiments were designed to define phenotypic changes in platelets with aging following deletion of ssER (ssERKO). Blood was collected from wild-type and ssERKO female mice at 4-7 (young) and 24-25 (aged) months of age. In young animals, total number of platelets, number of platelets containing RNA (reticulated platelets), aggregation, dense body adenosine triphosphate secretion, and alpha granular secretion were the same in both groups. With aging, total number of platelets decreased but reticulated platelets increased in ssERKO mice; aggregation and dense granule adenosine tri-phosphate secretion decreased whereas basal expression of fibrinogen receptors increased with age in wild-type and ssERKO mice. Basal expression of P-selectin and annexin V binding increased with aging only in ssERKO mice; thrombin did not increase expression in these mice. Therefore, deletion of ssER is associated with specific platelet functions, which are expressed only with age-associated reproductive senescence.

Changes in platelet functions could contribute to thrombotic risk associated with estrogen treatments. This study was designed to test the hypothesis that three clinically relevant estrogenic treatments affect platelet function comparably. Adult female pigs were ovariectomized and randomized to either no treatment or treatment with oral 17[beta]-estradiol (2 mg/day), conjugated equine estrogen (0.625 mg/day), or raloxifene (60 mg/day) for 4 wk. Platelet turnover, aggregation, and secretion were assessed before and after treatment. Platelet turnover and mRNA increased significantly only in pigs treated with 17[beta]-estradiol. Expression of estrogen receptors increased with ovariectomy and decreased with all treatments. Platelet aggregation and secretion of ATP, platelet-derived growth factor, and matrix metalloproteinase-2 increased with ovariectomy. All treatments reduced both aggregation and secretion. Expression of mRNA for constitutive endothelial nitric oxide synthase (eNOS), but not eNOS protein, increased with ovariectomy. Only eNOS mRNA decreased with all treatments, but only treatment with 17[beta]-estradiol increased secretion of nitric oxide from intact platelets. Platelets from 17[beta]-estradiol-treated animals caused relaxation of coronary arteries, which was sensitive to inhibition of nitric oxide. Although three different estrogenic treatments reversed increases in platelet aggregation caused by ovariectomy, only 17[beta]-estradiol increased platelet RNA and release of platelet-derived nitric oxide. These differences reflect transcriptional and posttranscriptional regulation of protein synthesis in bone marrow megakaryocytes and circulating platelets. hormones; matrix metalloproteinase; nitric oxide; thrombosis

Differences in the aggregation and release of growth factors including matrix metalloproteinases (MMPs) after loss of ovarian hormones could contribute to an exaggerated response to injury in arteries of ovariectomized animals. Therefore, experiments were designed to compare aggregation, dense granular ATP release, expression of MMPs (MMP-2, MMP-9, and MMP-14) and tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) in circulating platelets from sexually mature (7 mo old) gonadally intact and ovariectomized (4 wk) female pigs. Numbers of circulating platelets did not change after ovariectomy, but the percentage of reticulated platelets increased significantly. Platelet aggregation and dense granular ATP secretion also increased significantly with ovariectomy. In platelet lysates, active MMP-2 increased, whereas MMP-14 significantly decreased, after ovariectomy; the expression of TIMP-1, TIMP-2, and P-selectin did not change. These results suggest that platelet turnover, aggregation, and ATP secretion increase with ovariectomy. Also, ovarian hormones selectively regulate the expression and activity of MMPs in porcine platelets. Increased platelet aggregation and activity of MMP-2 would alter platelet-platelet and platelet-vessel wall interactions, contributing to an exaggerated response to injury with loss of ovarian hormones. ATP secretion; estrogen; hemostasis; tissue inhibitors of matrix metalloproteinase; thrombosis

Experiments were designed to determine how ovariectomy modulates mitogenic factors in platelets and how these factors affect proliferation of coronary arterial smooth muscle. Platelet-derived growth factors (PDG[F.sub.AB] and PDG[F.sub.BB]), transforming growth factors (TGF-[[beta].sub.1] and TGF-[[beta].sub.2]), and vascular endothelial growth factor (VEG[F.sub.165]) were quantified in platelet lysates and platelet-poor plasma from adult gonadally intact and ovariectomized female pigs by ELISA. Proliferation of cultured coronary arterial smooth muscle cells (SMCs) from both groups of pigs was determined in response to autologous or heterologous platelet lysates. Platelet concentrations of PDG[F.sub.BB], but not PDG[F.sub.AB], TGF-[[beta].sub.1], and TGF-[[beta].sub.2], increased with ovariectomy. VEG[F.sub.165] was not detected in platelets from either group. Proliferation of SMCs from ovariectomized females was significantly greater on exposure to autologous or heterologous platelet lysates than proliferation of SMCs from intact females. These results indicate that ovariectomy increases concentrations of PDG[F.sub.BB] in platelets. Higher levels of PDG[F.sub.BB] in platelets in synergy with other platelet-derived products could contribute to increased proliferative arterial response to injury after ovariectomy. estrogen; hormone; megakaryocytes; platelet-derived growth factor; transforming growth factor

Research has been conducted on the mitochondrial protein frataxin which is required for iron homeostasis. Results indicate that recombinant yeast frataxin and iron form a hydrophilic complex detectable by both protein and iron staining on the nondenaturing polyacrylamide gels.