A guide to murine coagulation factor structure, function, assays, and genetic alterations.

Murine blood coagulation factors and function are quite similar to those of humans. Because of this similarity and the adaptability of mice to genetic manipulation, murine coagulation factors and inhibitors have been extensively studied. These studies have provided significant insights into human hemostasis. They have also provided useful experimental models for evaluation of the pathophysiology and treatment of thrombosis. This review contains recommendations for obtaining, processing and assaying mouse blood hemostatic components, and it summarizes the extensive literature on murine coagulation factor structure and function, assays and genetic alteration. It is intended to be a convenient reference source for investigators of hemostasis and thrombosis.

No effect of C-reactive protein on early atherosclerosis development in apolipoprotein E*3-leiden/human C-reactive protein transgenic mice.

OBJECTIVE: C-reactive protein (CRP) has been associated with risk of cardiovascular disease. It is not clear whether CRP is causally involved in the development of atherosclerosis. Mouse CRP is not expressed at high levels under normal conditions and increases in concentration only several-fold during an acute phase response. Because the dynamic range of human CRP is much larger, apolipoprotein E*3-Leiden (E3L) transgenic mice carrying the human CRP gene offer a unique model to study the role(s) of CRP in atherosclerosis development. METHODS AND RESULTS: Atherosclerosis development was studied in 15 male and 15 female E3L/CRP mice; E3L transgenic littermates were used as controls. The mice were fed a hypercholesterolemic diet to induce atherosclerosis development. Cholesterol exposure did not differ between E3L/CRP and E3L mice. Plasma CRP levels were on average 10.2+/-6.5 mg/L in male E3L/CRP mice, 0.2+/-0.1 mg/L in female E3L/CRP mice, and undetectable in E3L mice. Quantification of atherosclerosis showed that lesion area in E3L/CRP mice was not different from that in E3L mice. CONCLUSIONS: This study demonstrates that mildly elevated levels of CRP in plasma do not contribute to the development of early atherosclerosis in hypercholesterolemic E3L/CRP mice.

The central roles of obesity-associated dyslipidaemia, endothelial activation and cytokines in the Metabolic Syndrome--an analysis by structural equation modelling.

HYPOTHESIS: The multi-faceted components of the metabolic syndrome now include markers of inflammation and endothelial activation. Despite this growing body of epidemiological data, standard statistical methods fail to evaluate the nature of these associations adequately. In this pilot study, we hypothesize that obesity may lead to endothelial activation which is in part mediated by dyslipidaemia and proinflammatory cytokines. These factors interact to give rise to hyperinsulinaemia, hypertension and an anti-fibrinolytic state. To test this hypothesis, we used confirmatory factor analysis and structural equation modelling to fit these data to a model designed on theoretical grounds. METHODS: Metabolic syndrome variables, cytokines (IL6 and TNFalpha), markers of inflammation and endothelial activation were measured in 107 Caucasian non-diabetic subjects aged 40-75 y. Using confirmatory factor analysis, we identified six factors to represent composite measurements of blood pressure, obesity, dyslipidaemia, hyperinsulinaemia, endothelial activation and the anti-fibrinolytic state. We fitted these variables to two separate models, one using IL-6 and the other TNFalpha as the cytokine, and examined the inter-relationships (path analysis) amongst these variables, based on the above hypothesis. RESULTS: Men were centrally more obese and had increased markers of endothelial activation, inflammation and the anti-fibrinolytic state as well as hyperinsulinaemia and dyslipidaemia, compared with women. Obesity indexes (both body mass index and waist-hip ratio) were strongly associated with multiple cardiovascular risk factors. Both IL6 and TNFalpha were correlated with age, male gender, obesity indexes and markers of endothelial activation. Only IL-6 was associated with smoking while TNFalpha was correlated with hyperinsulinaemia. In the TNFalpha model, 61% of the obesity variance was explained by male gender, 36% of TNFalpha variance by age and dyslipidaemia, 43% of dyslipidaemia variance by age and obesity, 33% of hyperinsulinaemia variance by dyslipidaemia and a non-smoking state, 29% of anti-fibrinolytic state variance by hyperinsulinaemia, 65% of endothelial activation variance by TNFalpha, dyslipidaemia and hyperinsulinaemia, 34% of blood pressure variance by hyperinsulinaemia and endothelial activation. In the IL-6 model, we observed similar relationships except that 23% of IL6 variance was explained by smoking and age. CONCLUSIONS: Using confirmatory factor analysis and structural equation modelling, we found that obesity, dyslipidemia and cytokines were the principal explanatory variables for the various components of the metabolic syndrome, with IL6 and TNFalpha having different explanatory variables and effects. These complex inter-relationships were in part mediated by hyperinsulinaemia and endothelial activation. While this hypothetical model was based on scientific evidence, supported by rigorous analysis, it requires further confirmation in large-scale prospective studies. Given the complexity of the biological system and its interactions with exogenous factors, structural equation modelling provides a useful scientific tool for hypothesis testing, complementary to the more traditional experimental and cohort studies.

Oral oestradiol/trimegestone replacement reduces procarboxypeptidase U (TAFI): a randomized, placebo- controlled, 12-week study in early postmenopausal women.

OBJECTIVE: To investigate the effects of short-term postmenopausal oral hormone administration on plasma levels of procarboxypeptidase U (proCPU, thrombin-activatable fibrinolysis inhibitor, EC 3.4.17.20), an inhibitor of fibrinolysis, in healthy early postmenopausal women. DESIGN: A prospective, randomized, placebo-controlled study. SETTING: Outpatient clinic of the Department of Obstetrics and Gynaecology. SUBJECTS: Seventy-seven healthy early postmenopausal women were screened of whom 65 were randomized. Analyses were based on 60 participants. INTERVENTIONS: The women received oral micronized oestradiol 2 mg either alone (E2 group, n=16), or sequentially combined with dydrogesterone 10 mg (E2 + D group, n=14) or trimegestone 0.5 mg (E2 + T, n=14), or placebo (n=16) for 12 weeks. MAIN OUTCOME MEASURE: ProCPU concentrations at baseline, and at 4 and 12 weeks of treatment. RESULTS: Four weeks of E2 + T was associated with a significant decrease in the fasting proCPU concentration, which was sustained after 12 weeks [t=0: 636 +/- 57 U L(-1) (mean +/- SD); t=4: 583 +/- 63UL-1; t=12: 589 +/- 48 U L(-1); ANCOVA versus placebo: P=0.011]. The percentage change from baseline versus placebo in this group was -8.4% [95% confidence interval (CI) -15.7 to -1.1] after 4 weeks and -5.9% (95% CI -11.7 to -0.1) after 12 weeks. There were no significant changes versus placebo in the E2 group nor in the E2 + D group. CONCLUSION: Short-term treatment with E2 + T, but not E2 alone or E2 + D, lowers proCPU concentration. These findings add to accumulating evidence suggesting that different progestagens added to oestrogen replacement may differentially affect the risk of arterial and venous disease.

Progression of diabetic nephropathy: role of plasma homocysteine and plasminogen activator inhibitor-1.

Among patients with diabetic nephropathy, the decline in glomerular filtration rate (GFR) varies substantially, ranging from 2 to 20 mL/min per year. Identification of predictors of progression in diabetic nephropathy is important. Plasma total homocysteine (tHcy) rises with urinary albumin excretion rate in diabetes, and plasminogen activator inhibitor-1 (PAI-1) has been correlated with increased matrix accumulation in various glomerulopathies. In this prospective observational cohort study, we evaluated the importance of baseline tHcy and PAI-1 as predictors of the rate of decline in GFR. Baseline tHcy and PAI-1 were measured in 157 type 1 diabetic patients with diabetic nephropathy (92 men; mean age, 41 +/- 10 years; mean diabetes duration, 27 +/- 8 years; median GFR, 80 mL/min/1.73 m(2) [range, 23 to 143 mL/min/1.73 m(2)]). Hereafter, GFR was measured yearly with a plasma clearance technique for at least 3 years (median, 7 years [range, 3.0 to 8.3 years]). The mean rate of decline in GFR was 3.7 +/- 0.3 mL/min per year. A linear regression analysis revealed a borderline significant relationship between rate of decline in GFR and tHcy (P = 0.069) and PAI-1 (P = 0.087). Analysis of the rate of decline in GFR and tertiles of tHcy and PAI-1 revealed that increasing levels of tHcy were correlated with a significantly faster decline in GFR (P = 0.025), whereas increasing levels of PAI-1 were not. After adjustment for other well-established risk factors for progression of nephropathy in a multiple linear regression analysis, however, neither tHcy levels nor PAI-1 levels were independent predictors of rate of decline in GFR.

Influence of plasma triglyceride and plasma cholesterol levels on the clearance rate of fibrinogen.

Increased plasma levels of fibrinogen are associated with a higher risk of cardiovascular disease. It has been suggested that lipid levels may influence the fibrinogen levels by a mechanism other than the synthesis rate, for example a decreased clearance rate. We performed a pilot study to explore this possibility. Twelve male Wistar rats were fed for four weeks with a control low fat/low cholesterol diet, a high fat/high cholesterol diet, and a high fat/high cholesterol diet with an additional 0.5% cholic acid. Labeled 125I fibrinogen was injected, and blood was sampled repeatedly. From the plasma radioactivity of the samples, fibrinogen halflife time was calculated for each animal. Our results suggest that plasma lipids lengthen the fibrinogen halflife times, although the differences were not statistically significant in this small study. Our final conclusion from this study is that lipids may have an effect on the turnover rate of fibrinogen and possibly affect fibrinogen levels through this mechanism.

Polylysine as a vehicle for extracellular matrix-targeted local drug delivery, providing high accumulation and long-term retention within the vascular wall.

We present the first steps in the elaboration of an approach of extracellular matrix-targeted local drug delivery (ECM-LDD), designed to provide a high concentration, ubiquitous distribution, and long-term retention of a drug within the vessel wall after local intravascular delivery. The approach is based on the concept of a bifunctional drug comprising a "therapeutic effector" and an "affinity vehicle," which should bind to an abundant component of the vessel wall. The aim of the present study was to select molecules suitable for the role of affinity vehicles for ECM-LDD and to study their intravascular delivery and retention ex vivo and in an animal model. By use of fluorescence microscopy, the following molecules were selected on the basis of strong binding to cross sections of human vessels: protamine, polylysine, polyarginine, a glycosaminoglycan-binding peptide from vitronectin, and a synthetic dendrimer. With polylysine as a prototypic affinity vehicle, we showed that after intravascular delivery, polylysine was concentrated throughout a luminal layer of the vascular wall to an extremely high concentration of 20 g/L and was retained therein for at least 72 hours of perfusion without noticeable losses. Low molecular weight (fluorescein) and high molecular weight (hirudin) compounds could be chemically conjugated to polylysine and were retained in the vessel wall after intravascular delivery of the conjugates. In conclusion, by use of the ECM-LDD method, an extremely high concentration and long-term retention of locally delivered drug can be reached. Polycationic molecules can be considered as potential affinity vehicles for ECM-LDD.

Endothelial function in patients with hyperthyroidism before and after treatment with propranolol and thiamazol.

Hyperthyroidism is associated with a higher incidence of arterial thromboembolism; increasing age, atrial fibrillation, and mitral valve abnormalities are risk factors. However, the contribution of endogenous coagulation parameters is unclear. Because thyroid hormone influences receptor and transcription factors, it can be expected that it will influence proteins involved in coagulation processes synthetised in many cells. Fourteen hyperthyroid patients were studied untreated, after 1 week of treatment with propranolol, and after therapeutic treatment with thiamazol. Fourteen matched controls were used for comparison. On each occasion, endothelial marker proteins, coagulation/fibrinolysis factors, and inflammatory (liver) markers were measured. Excess thyroid hormone was associated with elevated levels of most endothelium-associated proteins. In addition, plasma fibronectin and fibrinogen were increased, while plasminogen was decreased. No evidence was found that hyperthyroidism was associated with coagulation/fibrinolysis activation, or with increased levels of the inflammation markers interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) or C-reactive protein (CRP). Propranolol treatment only lowered the von Willebrand factor propeptide, and slightly increased plasminogen. Treatment with thiamazol returned all parameters to normal. Hyperthyroidism increased the plasma levels of most endothelial marker proteins, and of some liver-synthetized proteins. No evidence for coagulation/fibrinolysis activation was found. However, it appears that endothelial activation, which is indicative of a procoagulant state, is present in hyperthyroidism. This may explain the association between hyperthyroidism and thromboembolism especially if other risk factors are present. von Willebrand factor II (vWF:Ag-II) levels may be suitable markers to evaluate acute changes in endothelial function because this parameter responds more rapidly to changes in endothelial function than other factors.

Prognostic implications of retinopathy and a high plasma von Willebrand factor concentration in type 2 diabetic subjects with microalbuminuria.

BACKGROUND: Microalbuminuria in subjects with type 2 diabetes may be heterogeneous with respect to clinical features, renal histology, and prognosis. There may be at least two types of microalbuminuria in diabetes, namely with and without generalized endothelial dysfunction. We investigated whether, among microalbuminuric subjects with type 2 diabetes, the presence of generalized endothelial dysfunction, as indicated by the presence of retinopathy or a high plasma von Willebrand factor (vWf) level, has prognostic implications. METHODS: In 173 type 2 diabetic subjects of a population-based cohort, we assessed the urinary albumin-to-creatinine ratio, the plasma vWf level, and the presence of retinopathy. The main outcome was cardiovascular mortality. RESULTS: The absolute difference in 7 years' cardiovascular mortality between microalbuminuric (albumin-to-creatinine ratio 2.0-30.0 mg/mmol) and normoalbuminuric subjects was higher in the presence as compared to the absence of retinopathy (55.6 vs 11.1%). The age- and sex-adjusted relative risk (95% confidence interval) of cardiovascular mortality, as compared to normoalbuminuric subjects without retinopathy, was 1.1 (0.1-9.2) for normoalbuminuric subjects with retinopathy, 1.8 (0.5-6.7) for microalbuminuric subjects without retinopathy, and 9.8 (3.1-30.9) for microalbuminuric subjects with retinopathy. The absolute difference in risk of 7 years' cardiovascular mortality between microalbuminuric and normoalbuminuric subjects was higher in the presence as compared to the absence of a high (>1.89 IU/ml) vWf level (49.8 vs 16.4%). The age- and sex-adjusted relative risk of cardiovascular mortality, as compared to normoalbuminuric subjects without a high vWf level, was 1.5 (0.4-5.5) for normoalbuminuric subjects with a high vWf level, 2.6 (0.7-9.6) for microalbuminuric subjects without a high vWf level, and 12.0 (2.9-49.5) for microalbuminuric subjects with a high vWf level. These differences in risk of cardiovascular mortality did not change materially after further adjustment for known duration of diabetes, hypertension, creatinine clearance, level of glycated haemoglobin and high-density lipoprotein cholesterol, and presence of cardiovascular disease. Analysis of all-cause instead of cardiovascular mortality showed a similar difference in risk of mortality between microalbuminuric subjects with or without retinopathy or a high vWf level. CONCLUSIONS: Among type 2 diabetic subjects with microalbuminuria, the presence of retinopathy or a high plasma vWf level affects the risk of cardiovascular death. Although larger studies are necessary, these findings support the concept that microalbuminuria in type 2 diabetes can occur in the absence or the presence of generalized endothelial dysfunction, and that the latter is a much more 'malignant' condition than the former.

Short-term effect of surgical trauma on rat peritoneal fibrinolytic activity and its role in adhesion formation.

BACKGROUND: Fibrin deposition, the primary step in the formation of post-surgical adhesions, is the result of a disbalance between the fibrin-forming and the fibrin-dissolving capacity of the peritoneum. Literature data suggest a transient reduction in local plasminogen activator activity after peritoneal trauma, which results in a reduction of fibrinolysis and permits deposited fibrin to become organized into fibrous, permanent adhesions. In the present study, the fibrinolytic parameters tissue-type plasminogen activator (tPA; antigen and activity) and plasminogen activator inhibitor type-1 (PAI-1; antigen and activity) were measured in peritoneal fluid, in peritoneal biopsies and in plasma to establish the time course of changes in fibrinolytic activity. DESIGN: A standardized peritoneal adhesion model in the rat. OUTCOME MEASURES: Analysis, over a 72-h period following surgical trauma. of the main fibrinolytic parameters in peritoneal lavage, in biopsies of damaged and undamaged peritoneum, and in plasma, and determination of fibrin and fibrin(ogen)-degradation products in peritoneal lavage fluid. RESULTS: At all time intervals, tPA antigen was found to be about six-fold increased in peritoneal lavage after surgical trauma. This significant rise in tPA antigen was accompanied by a large increase in its main inhibitor PAI-1, resulting in tPA activity levels similar to, or slightly higher than, those found in control animals. tPA activity was lowest at 4 h and increased thereafter. Also in biopsies from damaged peritoneum, tPA antigen was significantly increased. Tissue tPA activity was also lowest at 4 h, after which it increased, significantly so at 24 and 72 h. Similar, though smaller, changes were seen in the biopsies from undamaged areas of the peritoneal wall in operated rats. PAI-1 (antigen and activity) was not detected in peritoneal biopsies. Fibrin-related material (especially fibrin monomer/fibrinogen, an indicator of forming fibrin) in peritoneal fluid was slightly increased at 4 h, and abundantly present at 16 and 24 h, returning to control levels at 72 h. Fibrin degradation products were always present. From 2 h onward, adhesions were found. CONCLUSIONS: In contrast to the view that adhesions are formed as a result of a reduced fibrinolytic activity, our results demonstrate that tPA activity remained unchanged or slightly increased after surgical trauma, and point to increased fibrin formation rather than diminished fibrinolytic activity as the main cause of fibrin deposition after peritoneal trauma. Therapies directed at prevention of adhesion formation should therefore aim at avoiding massive fibrin production and at promoting fibrinolytic activity during the early period after trauma.

The influence of improved glycaemic control with insulin and sulphonylureas on acute phase and endothelial markers in Type II diabetic subjects.

AIMS/HYPOTHESIS: Improved glycaemic control might reduce both microvascular and macrovascular complications of Type II diabetes (non-insulin-dependent) mellitus. To explore such possible mechanisms, we investigated the effects of intensive treatment on markers of endothelial dysfunction and of acute phase activation, using both sulphonylureas and insulin. METHODS: In a randomised cross-over study we gave sulphonylureas or insulin each for a period of 16 weeks to 22 poorly controlled Type II diabetic subjects who were being treated by diet. There was a 4 week washout period between each treatment. Subjects were studied at baseline and at the end of each treatment. RESULTS: Treatment with sulphonylureas and insulin resulted in similar improvements in glycaemic control (glycated haemoglobin, baseline: 11.8 [(SD 2.2)%; after sulphonylureas: 8.6 (1.2)%,p < 0.001; after insulin: 8.6 (1.2)%, p < 0.001] and in insulin sensitivity ¿metabolic clearance rate of glucose, baseline: median 1.75 [interquartile (IQ) range 1.41, 2.27] ml x kg(-1) x min(-1); after sulphonylureas: 2.41 (1.82, 3.01) ml x kg(-1) x min(-1), p = 0.001; after insulin: 2.23 (1.92, 2.75) ml x kg(-1) min(-1), p = 0.027¿. There were no significant changes in concentrations of endothelial markers von Willebrand factor, cellular fibronectin, thrombomodulin, tissue plasminogen activator, soluble E-selectin or soluble intercellular adhesion molecule-1 or in urinary albumin excretion rate after either treatment period. Concentrations of C-reactive protein were not significantly influenced by sulphonylureas but fell after insulin [baseline: median 4.50 (IQ range 1.37, 6.44) microg x ml(-1); sulphonylureas: 2.69 (0.88, 9.65) microg x ml(-1) (p = 0.53); insulin: 2.07 (1.16, 5.24) microg x ml(-1) (p = 0.017)]. There were, however, no significant effects of either treatment on circulating concentrations of fibrinogen (p = 0.28-0.34) or of the proinflammatory cytokines interleukin-6 or tumour necrosis factor-alpha (p = 0.65-0.79). CONCLUSION/INTERPRETATION: Markers of endothelial dysfunction and concentrations of proinflammatory cytokines in Type II diabetes are not influenced by improved glycaemic control over 16 weeks. Improved metabolic control with insulin could, however, be associated with reduced concentrations of the acute phase marker C-reactive protein.

Use of fibrinolytic agents in the prevention of postoperative adhesion formation.

OBJECTIVE: To review the events leading to the formation of adhesions, to describe the development of fibrinolytic agents, to review more than a century of research on the use of fibrinolytic agents in adhesion prevention, and to look at future aspects of adhesion prevention. RESULTS: A better understanding of the pathogenesis of adhesion formation has resulted in the use of fibrinolytic agents in their prevention. Fibrinolytic agents promote fibrinolytic activity during the early period after peritoneal trauma during which an increased formation of fibrin is seen in combination with a deficiency of endogenous fibrinolytic activity. Initially, chemical attacks on fibrin (fibrolysin and hypertonic glucose), foreign digestive ferments (pepsin, trypsin, and papain), and stimulation of intraperitoneal leukocytosis (amniotic fluid) were used. Development of new thrombolytic agents was soon followed by experiments in animal adhesion models and clinical studies to examine their antiadhesion properties. Plasmin preparations (plasmin, actase, and fibrinolysin) and plasmin activators (streptokinase, urokinase, and tissue-type plasminogen activator) were found to be efficacious in preventing adhesion formation in the greater part of reviewed animal and clinical studies. CONCLUSION(S): From the current literature, it can be concluded that postoperative intraperitoneal administration of thrombolytic agents can significantly decrease adhesion formation. Given the large number of experimental studies in animals, future studies should focus on the clinical use of fibrinolytic agents in the prevention of postsurgical adhesion formation.

Increased levels of soluble vascular cell adhesion molecule 1 are associated with risk of cardiovascular mortality in type 2 diabetes: the Hoorn study.

Membrane-bound vascular cell adhesion molecule 1 (VCAM-1) allows the tethering and rolling of monocytes and lymphocytes as well as firm attachment and transendothelial migration of leukocytes. Soluble forms of VCAM (sVCAM-1) may serve as monitors of increased expression of membrane-bound VCAM-1 and thus may reflect progressive formation of atherosclerotic lesions. Levels of sVCAM-1 have been found to be increased among type 2 diabetic as compared with nondiabetic subjects. To study the association of plasma sVCAM-1 concentration and risk of cardiovascular and all-cause mortality among nondiabetic and diabetic subjects, we investigated an age-, sex-, and glucose-tolerance-stratified sample (n = 631) of a population-based cohort aged 50-75 years that was followed prospectively. Plasma levels of sVCAM-1 were determined in frozen -70 degrees C baseline samples. After 7.4 years (mean) of follow-up, 107 (17%) subjects had died (42 of cardiovascular causes). In the entire group, increased sVCAM-1 levels were significantly associated with increased risk of cardiovascular mortality (relative risks [RRs] per 100 ng/ml sVCAM-1 increase, 1.10 [1.05-1.15] after adjustment for age, sex, and glucose tolerance status). This RR was somewhat diminished by further adjustment for the presence of hypertension and cardiovascular disease; levels of total, HDL, and LDL cholesterol and homocysteine; the presence of microalbuminuria (a putative marker of endothelial dysfunction); levels of von Willebrand factor (a marker of endothelial dysfunction) and C-reactive protein (a marker of low-grade inflammation); and estimates of glomerular filtration rate. However, the RR remained statistically significant. The RR among type 2 diabetic subjects was 1.13 (1.07-1.20) per 100 ng/ml sVCAM-1 increase after adjustment for age and sex, which was somewhat higher but not significantly different from the RR in nondiabetic subjects (P value for interaction term, 0.12). Further adjustment for other risk factors gave similar results. In conclusion, levels of sVCAM-1 are independently associated with the risk of cardiovascular mortality in type 2 diabetic subjects and therefore might be useful for identifying subjects at increased cardiovascular risk. Increased plasma sVCAM-1 levels may reflect progressive formation of atherosclerotic lesions, or sVCAM-1 itself may have bioactive properties related to cardiovascular risk. Our data, however, argue against the hypotheses of sVCAM-1 levels simply being a marker of endothelial dysfunction, of low-grade inflammation, or of an impaired renal function.

Oral, but not transdermal, administration of estrogens lowers tissue-type plasminogen activator levels in humans without affecting endothelial synthesis.

Oral estrogen administration decreases plasma levels of tissue-type plasminogen activator (tPA), which may be explained by a decrease in endothelial tPA synthesis, an increase in its hepatic clearance, or both. In the present study, we determined (1) differences between oral (ie, via the liver) ethinyl estradiol and transdermal (ie, systemic) 17beta-estradiol administration on plasma antigen levels of tPA and plasminogen activator inhibitor type-1 before and after 4 months of hormone administration and (2) effects on endothelial tPA synthesis, by measuring the local increase in plasma tPA during venous occlusion of the upper extremity. Thirty transsexual males (median age 32 years, range 20 to 44 years ) were randomly assigned to either oral ethinyl estradiol (n=15) or transdermal 17beta-estradiol (n=15); both treatments included the antiandrogen cyproterone acetate (CA). Ten males were treated with CA alone. Seventeen transsexual females (median age 27 years, range 18 to 37 years) were treated with intramuscular testosterone esters. Only oral ethinyl estradiol plus CA but neither transdermal 17beta-estradiol plus CA, nor oral CA, nor parenteral testosterone lowered plasma tPA and plasminogen activator inhibitor-1 (P<0.001 for both). tPA release during venous occlusion was not affected by oral ethinyl estradiol plus CA in males (P=0.52) or by parenteral testosterone in females (P=0.89). These data are consistent with a previous observation, in rodents, that the decrease in tPA after oral estrogen administration can be explained by an increase in hepatic tPA clearance, leaving endothelial tPA synthesis unchanged, and suggest that these mechanisms also explain the decrease in tPA in humans.

Plasminogen activator inhibitor-1 and apolipoprotein E gene polymorphisms and diabetic angiopathy.

BACKGROUND: A point mutation in the plasminogen activator inhibitor-1 (PAI-1) gene and a three-allelic variation in the apolipoprotein-E (ApoE) gene have been suggested as risk factors for the development of diabetic micro- and macrovascular complications. METHODS: We studied 198 type 1 diabetic patients with diabetic nephropathy [121 men, age (mean+/-SD) 41+/-10 years, diabetes duration 28+/-8 years] and 192 patients with persistent normoalbuminuria (118 men, age 43+/-10 years, diabetes duration 27+/-9 years). RESULTS: Male patients with nephropathy had elevated plasma PAI-1 levels [geometric mean (95% CI)], 70 (62-79) ng/ml, compared with normoalbuminuric men, 43 (38-47) ng/ml, P<0.001. Even though nephropathic patients with the 4G4G genotype tended to have higher plasma PAI-1 levels, P=0.06, no difference in allele frequency (4G/5G) was seen between patients with and without nephropathy: 0.538/0.462 vs 0.539/0.461, respectively. Nor did ApoE allele frequencies (epsilon2/epsilon3/epsilon4) differ between nephropathic and normoalbuminuric patients: 0.099/0.749/0. 152 vs 0.081/0.745/0.174, respectively. Genotype distributions were also similar, n.s. Coronary heart disease was more prevalent (36%) among nephropathic patients carrying the atherogenic epsilon4-allele compared with 12% in patients with the epsilon3,epsilon3 genotype, P<0.001. No associations between diabetic retinopathy and PAI-1 or ApoE polymorphisms were observed, n.s. CONCLUSIONS: The ApoE polymorphism may accelerate the development of coronary heart disease often seen in Caucasian patients with type 1 diabetes and diabetic nephropathy, a condition characterized by elevated plasma PAI-1 in men. Neither the PAI-1 nor the ApoE gene polymorphism contributes to the genetic susceptibility to diabetic nephropathy or retinopathy.

Impaired procoagulant-anticoagulant balance during hormone replacement therapy? A randomised, placebo-controlled 12-week study.

UNLABELLED: In this randomised, placebo-controlled 12-week study, sixty healthy postmenopausal women received either placebo (N = 16) or daily 2 mg micronised oestradiol, either unopposed (N = 16, E2 group) or combined with a progestagen for 14 days of each cycle (N = 28, E2+P group). RESULTS: As compared to placebo, plasma levels of AT III were reduced only in the E2 group (approximately 28%), plasma levels of protein C decreased only in the E2+P group (approximately 4%) and plasma levels of protein S decreased in both the E2 and E2+P group (approximately 21%). In both the E2 and E2+P groups, the plasma levels of factor VII (antigen and activity) showed a borderline significant increase (approximately 10%), whereas no significant change was observed in active factor VII. Plasma levels of tissue-type plasminogen activator (approximately 22%), urokinase plasminogen activator (approximately 25%) and plasminogen activator inhibitor type-1 (approximately 43%) decreased in the E2 and E2+P groups, whereas those of plasminogen increased (approximately 12%). Treatment was associated with an increase in levels of prothrombin fragment 1+2 (approximately 31%), but levels of thrombin-antithrombin III complexes, and of plasmin-alpha2-antiplasmin complexes and total fibrin(ogen) degradation products did not change significantly. CONCLUSION: Short-term E2 and E2+P treatment is associated with a shift in the procoagulant-anticoagulant balance towards a procoagulant state. A substantial proportion of women do not have a net increase in fibrinolytic activity. These data may be relevant in explaining the increased risk of venous thromboembolism associated with ERT and HRT, and possibly also in explaining the negative results of the Heart and Estrogen/progestin Replacement Study.

Simvastatin improves disturbed endothelial barrier function.

BACKGROUND: Recent clinical trials have established that inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) reduce the risk of acute coronary events. These effects of statins cannot be fully explained by their lipid-lowering potential. Improved endothelial function may contribute to the positive effects of statin treatment. METHODS AND RESULTS: In the present study, we report that simvastatin reduces endothelial barrier dysfunction, which is associated with the development of atherosclerosis. Treatment of human umbilical vein endothelial cells for 24 hours with 5 micromol/L simvastatin reduced the thrombin-induced endothelial barrier dysfunction in vitro by 55+/-3%, as assessed by the passage of peroxidase through human umbilical vein endothelial cell monolayers. Similar effects were found on the thrombin-induced passage of (125)I-LDL through human aortic endothelial cell monolayers. This reduction in barrier dysfunction by simvastatin was both dose and time dependent and was accompanied by a reduction in the thrombin-induced formation of stress fibers and focal adhesions and membrane association of RhoA. Simvastatin treatment had no effect on intracellular cAMP levels. In Watanabe heritable hyperlipidemic rabbits, treatment for 1 month with 15 mg/kg simvastatin reduced vascular leakage in both the thoracic and abdominal part of the aorta, as evidenced by the Evans blue dye exclusion test. The decreased permeability was not accompanied by a reduction of oil red O-stainable atherosclerotic lesions. CONCLUSIONS: These data show that simvastatin, in a relatively high concentration, improves disturbed endothelial barrier function both in vitro and in vivo. The data also support the beneficial effects of simvastatin in acute coronary events by mechanisms other than its lipid-lowering effect.

Unopposed estrogen increases total plasma factor VII, but not active factor VII--a short-term placebo-controlled study in healthy postmenopausal women.

Estrogen therapy may increase the risk of arterial thromboembolism, at least in the short term. In a randomized, double-blind and placebo-controlled study in 25 healthy postmenopausal women (52.5 +/- 2.8 years), we therefore examined the short-term effect of unopposed estrogen on the fasting and fat-load-stimulated plasma levels of total factor VII versus active factor VII. Plasma total factor VII was measured by use of a chromogenic assay; plasma active FVII by a recently developed method using truncated tissue factor. As compared to placebo, 8 weeks of oral 17beta-estradiol (2 mg daily) increased the mean fasting and postprandial plasma levels of total factor VII by 17 and 21% points, respectively (both P < 0.01 ), but did not affect the fasting and/or postprandial plasma levels of active factor VII (mean change both 0.05 ng/mL; P > 0.35). Furthermore, the change in the fasting level of total factor VII after therapy was not associated with the change in the fasting level of active factor VII (r = 0.27; P = 0.21). These findings argue against the idea that elevated levels of total factor VII underlie an increased risk of arterial thromboembolism in postmenopausal women using unopposed estrogen replacement.