Plasminogen Activator Inhibitor-1 Antigen Research Articles

Post-exercise Alcohol Ingestion Perturbs Blood Haemostasis During Recovery

It is known that exercise induces modification in blood haemostasis. It is, however, not known whether alcohol consumption post-exercise influences these modifications during recovery. Eleven moderately active young men were studied immediately after a standardised cycle ergometer test and during the 24-hour period of recovery. Alcohol (0.7 g/kg body mass) was given 1 hour after exercise on one test occasion, while an equal volume of alcohol-free solution was administered on the other. Exercise induced a significant increase in factor VIII activity with a significant shortening of activated partial thromboplastin time. Parallel increases in tissue plasminogen activity and antigen with a concomitant decrease in tissue plasminogen activator inhibitor-1 activity were also observed after exercise. During recovery, while the increase in factor VIII activity post-exercise persisted in both trials, fibrinolytic activity demonstrated a sharp fall. The elevated factor VIII activity was significantly higher at 5 and 22 hours during the alcohol trial compared with the control. Although no demonstrable effect of alcohol on tissue plasminogen activator activity was present from 1 hour after ingestion onward, tissue plasminogen activator antigen and tissue plasminogen activator inhibitor-1 antigen increased significantly 22 hours following alcohol ingestion. Further comparison between trials revealed a higher plasminogen activator inhibitor-1 activity 5 hours after alcohol ingestion. In conclusion, exercise-induced changes to blood haemostasis are balanced during exercise but not during recovery. Alcohol consumption after physical exercise further perturbs blood haemostasis and could constitute a thrombotic risk.

Plasminogen activator inhibitor 1, transforming growth factor-beta1, and BMI are closely associated in human adipose tissue during morbid obesity.

In adipose tissue from both obese mice and humans, plasminogen activator inhibitor 1 (PAI-1) expression has been reported to be upregulated to levels of increased plasma PAI-1. This elevated expression has been shown to be partly controlled by tumor necrosis factor (TNF)-alpha in mice. In humans, increased PAI-1 expression is associated with insulin resistance characterized by visceral fat accumulation. Therefore, the aim of this study was to investigate the expression pattern of PAI-1 and TNF-alpha (antigen and mRNA) in visceral human adipose fat in comparison with subcutaneous (SC) fat. Because transforming growth factor (TGF)-beta1 is a potent inducer of PAI-1 synthesis and has been shown to influence adipocyte metabolism, this work was extended to TGF-beta1 quantification. A total of 32 obese individuals (BMI 42 +/- 6.8 kg/m2) were investigated. Freshly collected visceral adipose tissue did not exhibit a higher content of PAI-1 or TGF-beta1 than did SC tissue. Although most of the TNF-alpha values were at the detection limit of the methods, TNF-alpha antigen was 3-fold higher and TNF-alpha mRNA was 1.2-fold higher in visceral fat. The levels of tissue TGF-beta1 antigen correlated well with those of PAI-1 antigen, regardless of the fat depot studied (SC tissue: n = 21, r = 0.72, P = 0.0006; visceral tissue: n = 20, r = 0.49, P < 0.03), and they were both significantly associated with BMI. Conversely, no relationship was observed between the levels of TNF-alpha and PAI-1 or TNF-alpha and BMI. Tissue PAI-1 levels were also significantly correlated with those of circulating PAI-1. These results describe, in severe obesity, a proportional increase in tissue PAI-1 and TGF-beta1 in visceral and SC tissues. This increased PAI-1 expression could be the result of tissue cytokine disturbances, such as elevated TGF-beta1 expression.

Fibrinolytic/hemostatic variables in arterial hypertension: response to treatment with irbesartan or atenolol

Essential hypertension is often accompanied by abnormalities of the coagulation/fibrinolytic system, predisposing to a procoagulant state. The aim of the present study was to compare the effects of atenolol (β 1-blocker agent) and irbesartan (angiotensin II type 1 receptor antagonist) on plasma levels of hemostatic/fibrinolytic and endothelial function markers in a cohort of previously untreated hypertensives. Fifty-four patients were randomly assigned to atenolol 25 to 150 mg (26 patients) or irbesartan 75 to 300 mg (28 patients). The plasma levels of plasminogen activator inhibitor-1 antigen, thrombomodulin, tissue factor pathway inhibitor antigen, fibrinogen, and factor XII were determined before and after 6 months of therapy. Age, gender distribution, body mass index, lipid profile, and baseline values of the measured markers were similar in both groups. Baseline values for systolic and diastolic blood pressure, as well as the reduction after treatment, were not significantly different between the two groups. Treatment with irbesartan was associated with a significant decrease in the levels of all the parameters. Similar findings were observed in the atenolol group, except for factor XII and tissue factor pathway inhibitor levels, which were not significantly decreased in this group. The reduction, however, of fibrinogen, plasminogen activator inhibitor-1, and thrombomodulin was significantly greater in the irbesartan than in the atenolol group. In conclusion, the results indicated that, despite an equally controlled blood pressure, 6-month therapy with irbesartan was associated with a more favorable modification of hemostatic/fibrinolytic status than atenolol.

Brain injury and cerebrovascular fibrin deposition correlate with reduced antithrombotic brain capillary functions in a hypertensive stroke model.

Hemostasis factors may influence the pathophysiology of stroke. The role of brain hemostasis in ischemic hypertensive brain injury is not known. We studied ischemic injury in spontaneously hypertensive rats in relation to cerebrovascular fibrin deposition and activity of different hemostasis factors in brain microcirculation. In spontaneously hypertensive rats subjected to transient middle cerebral artery occlusion versus normotensive Wistar-Kyoto (W-K) rats, infarct and edema volumes were increased by 6.1-fold (P < 0.001) and 5.8-fold (P < 0.001), respectively, the cerebral blood flow (CBF) reduced during middle cerebral artery occlusion (MCAO) by 55% (P < 0.01), motor neurologic score increased by 6.9-fold (P < 0.01), and cerebrovascular fibrin deposition increased by 6.8-fold (P < 0.01). Under basal conditions, brain capillary protein C activation and tissue plasminogen activator activity were reduced in spontaneously hypertensive rats compared with Wistar-Kyoto rats by 11.8-fold (P < 0.001) and 5.1-fold (P < 0.001), respectively, and the plasminogen activator inhibitor-1 antigen and tissue factor activity were increased by 154-fold (P < 0.00001) and 74% (P < 0.01), respectively. We suggest that hypertension reduces antithrombotic mechanisms in brain microcirculation, which may enhance cerebrovascular fibrin deposition and microvascular obstructions during transient focal cerebral ischemia, which results in greater neuronal injury.

Oversulfated fucoidan inhibits the basic fibroblast growth factor-induced tube formation by human umbilical vein endothelial cells: its possible mechanism of action

We have previously demonstrated that chemically oversulfated fucoidan (OSF) but not native fucoidan (NF) effectively suppresses the tube structure formation by human umbilical vein endothelial cells (HUVEC) on the basement membrane preparation, Matrigel. In this study, using more defined systems where basic fibroblast growth factor (bFGF) induces the tube formation by HUVEC on collagen gel, we investigated the mechanism responsible for the inhibition of angiogenesis by OSF in vitro. Unlike NF and desulfated fucoidan (desF), OSF potently inhibited the bFGF-induced HUVEC migration and tube formation. ELISA for tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in the culture media indicated that OSF increased the bFGF-induced release of PAI-1 antigen, but not of t-PA antigen. Analyses of the binding of bFGF to HUVEC surfaces and the following protein tyrosine phosphorylation revealed that OSF could promote the cell binding and autophosphorylation of 140 and 160 kDa receptors. In heparitinase-treated HUVEC, contrarily, the bFGF binding and PAI-1 release were decreased by OSF. These results suggest that OSF is a highly sulfated unique polysaccharide that can promote the binding of bFGF to the heparan sulfate molecules required for binding to the high affinity receptors with tyrosine kinase activity. The resultant increase in PAI-1 release may play a key role for the prevention of cell migration accompanied by matrix proteolysis.

Plasminogen activator inhibitor 2 and urokinase-type plasminogen activator in plasma and leucocytes in patients with severe sepsis.

Proteins influencing plasminogen activation to plasmin, namely plasminogen activators tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) and their principal inhibitors, plasminogen activator inhibitor 1 (PAI-1) and PAI-2, were measured in the plasma, the polymorph and mononuclear cell fractions taken from patients with major sepsis who were entering a general intensive care unit. The purpose of this study was to elucidate the factors favouring the persistence of fibrin in the microvasculature and thus contributing to multiple organ failure. Levels of u-PA antigen in plasma rose in sepsis and u-PA activity, not detectable in normal plasma, appeared. Levels of u-PA antigen in the cell fractions fell concomitantly. t-PA antigen in plasma and in the mononuclear cell fraction rose in sepsis, but t-PA activity was not detectable. Plasma PAI-1 antigen levels were strikingly raised in sepsis, presumably accounting for the complete neutralization of t-PA activity. PAI-2 antigen, not normally detected in plasma, appeared in the plasma of some patients, whereas it disappeared from the cellular fractions. Appearance of PAI-2 in plasma was associated with non-survival of the patient. The observations indicate that all the agents involved in plasminogen activation are released into the plasma in major sepsis. The levels of PAI-1 reached were quantitatively sufficient to suppress all activity of the released t-PA, but the inhibitors did not prevent expression of u-PA activity in the circulation. Circulating active u-PA and PAI-2 in the plasma of patients with severe sepsis may represent material originating from leucocytes. Leucocyte release of these agents within fibrin deposits may influence the persistence of fibrin and thus the development of multiple organ failure.

Effect of tumor necrosis factor alpha and transforming growth factor beta 1 on plasminogen activator inhibitor-1 secretion from subcutaneous and omental human fat cells in suspension culture

Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are characteristic of the obese state and may contribute to the association between obesity and cardiovascular disease. In this study, we measured the rate of secretion of PAI-1 antigen in isolated subcutaneous and omental abdominal adipocytes from severely obese and non-obese individuals and studied the effect of selected cytokines on PAI-1 release using a suspension culture technique. PAI-1 secretion was approximately 2-fold greater in isolated fat cells from the obese versus non-obese subjects. In addition, PAI-1 mRNA levels were higher in adipose tissue samples from obese versus non-obese individuals (P < .05). PAI-1 release was also approximately 2-fold greater in omental versus subcutaneous adipocytes from both obese and non-obese subjects (each P < .05). A 24-hour exposure to 1 nmol/L tumor necrosis factor alpha (TNF-alpha) slightly increased PAI-1 release from both subcutaneous and omental adipocytes (30% +/- 21% and 17% +/- 18%, respectively, nonsignificant [NS]). Transforming growth factor beta 1 (TGF-beta1) induced a significant dose-dependent increase of PAI-1 release into the medium. Exposure to 400 pmol/L TGF-beta1 of subcutaneous and omental fat cells from both obese and non-obese individuals elevated PAI-1 secretion by 2-fold. These data provide evidence that human fat cells release a substantial amount of PAI-1 in a depot-specific manner and that TGF-beta1 particularly contributes to the regulation of PAI-1 secretion.

Fibrinolytic function in diuretic-induced volume depletion

Accumulating evidence suggests that the renin-angiotensin system (RAS) may participate in the regulation of fibrinolytic function. In clinical studies, however, angiotensin-converting enzyme (ACE) inhibitors and the angiotensin II receptor antagonist losartan have failed to consistently affect endogenous fibrinolysis. Because such an effect may depend on the degree of prestimulation of the RAS, we have studied parameters of fibrinolytic function in 15 healthy volunteer subjects during baseline (day 1) and after 10 days of treatment with 25 mg of hydrochlorothiazide (HCT)/day (day 11). On the last day of the study (day 12), a single oral dose of 50 mg of losartan was given to the volunteers in addition to HCT and fibrinolytic function was assessed at the peak effect of losartan (5 h later). Plasma renin activity (PRA) was significantly stimulated during diuretic treatment (1.35 ± 0.21 v 0.34 ± 0.06 ng mL −1 · h −1 [ P < .001]) and further increased after losartan (6.39 ± 1.16 ng mL −1 · h −1 [ P < .001]). No effects of either the diuretic or losartan could be observed on tissue-type plasminogen activator (t-PA) antigen concentration and activity. However, 10 days of treatment with HCT significantly increased plasminogen activator inhibitor-1 (PAI-1) antigen (26.8 ± 5.8 v 21.1 ± 3.4 ng/mL [p = .037]). In addition, PAI-1 activity was also tentatively raised by HCT treatment (5.48 ± 1.82 v 3.88 ± 0.79 IU/mL [ P = .067]). In spite of the marked further rise in PRA after losartan, the stimulation of PAI-1 antigen and activity was blunted by losartan (24.4 ± 3.6 ng/mL and 4.55 ± 0.99 IU/mL, respectively). Our results demonstrate that volume depletion induced by HCT treatment is associated with a rise in PAI-1. Acute administration of losartan is capable of blunting this effect, suggesting that the angiotensin II type 1 receptor may participate in this effect of angiotensin II.

Angiotensin converting enzyme insertion/deletion polymorphism is associated with plasma antigen levels of plasminogen activator inhibitor-1 in healthy Japanese population

Plasminogen activator inhibitor-1 (PAI-1) has a central role in the regulation of the fibrinolytic enzyme system. An elevated plasma PAI-1 level is associated with thrombotic disorders. In vitro and in vivo studies indicate that the renin-angiotensin system is involved in the regulation of PAI-1. A 287-bp insertion/deletion (I/D) polymorphism in the gene-encoding angiotensin converting enzyme (ACE) is associated with cardiovascular disorders. We evaluated the association between the ACE I/D polymorphism and plasma PAI-1 antigen levels in 110 healthy Japanese male subjects. Subjects with the D-allele of the gene-encoding ACE had higher levels of PAI-1 (26.3 +/- 14.7 ng/ml, mean +/- standard deviation) compared with those without (21.0 +/- 12.0; P = 0.0491). A multiple linear regression model with independent variables (age, body-mass index, total cholesterol level, triglyceride level, ACE I/D genotype, and PAI-1 genotype due to a single guanine I/D polymorphism in the PAI-1 gene) demonstrated that the triglyceride level (P = 0.0059) and ACE I/D genotype (P = 0.0372) were independent predictors of plasma PAI-1 antigen levels in a subset of the subjects without diabetes mellitus that were not taking lipid-lowering drugs. These findings suggest that the ACE I/D polymorphism is a genetic factor for the regulation of plasma PAI-1 antigen levels in the healthy Japanese population.

Angiotensin converting enzyme insertion/deletion polymorphism is associated with plasma antigen levels of plasminogen activator inhibitor-1 in healthy Japanese population

Plasminogen activator inhibitor-1 (PAI-1) has a central role in the regulation of the fibrinolytic enzyme system. An elevated plasma PAI-1 level is associated with thrombotic disorders. In vitro and in vivo studies indicate that the renin-angiotensin system is involved in the regulation of PAI-1. A 287-bp insertion/deletion (I/D) polymorphism in the gene-encoding angiotensin converting enzyme (ACE) is associated with cardiovascular disorders. We evaluated the association between the ACE I/D polymorphism and plasma PAI-1 antigen levels in 110 healthy Japanese male subjects. Subjects with the D-allele of the gene-encoding ACE had higher levels of PAI-1 (26.3 ± 14.7 ng/ml, mean ± standard deviation) compared with those without (21.0 ± 12.0; P = 0.0491). A multiple linear regression model with independent variables (age, body-mass index, total cholesterol level, triglyceride level, ACE I/D genotype, and PAI-1 genotype due to a single guanine I/D polymorphism in the PAI-1 gene) demonstrated that the triglyceride level (P = 0.0059) and ACE I/D genotype (P = 0.0372) were independent predictors of plasma PAI-1 antigen levels in a subset of the subjects without diabetes mellitus that were not taking lipid-lowering drugs. These findings suggest that the ACE I/D polymorphism is a genetic factor for the regulation of plasma PAI-1 antigen levels in the healthy Japanese population.

HMG CoA reductase inhibitors reduce plasminogen activator inhibitor-1 expression by human vascular smooth muscle and endothelial cells.

The clinical benefit of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may derive from a qualitative, functional change in atherosclerotic lesions in addition to their lipid-lowering properties. We examined whether statins altered expression of the major determinants of fibrinolytic balance, plasminogen activator inhibitor-1 (PAI-1), and tissue-type plasminogen activator (tPA) in human vascular smooth muscle (SMC) and endothelial (EC) cells. Simvastatin reduced levels of PAI-1 antigen released from SMCs and ECs stimulated with platelet-derived growth factor or transforming growth factor-beta (IC(50) approximately 1 micromol/L). Levels of EC-derived tPA increased 2-fold over the same concentrations of simvastatin that inhibited release of PAI-1. Simvastatin's inhibitory effect was mimicked by C3 exoenzyme and prevented by geranylgeranyl pyrophosphate, but not by farnesyl pyrophosphate, suggesting the involvement of geranylgeranyl-modified intermediates. Decreased PAI-1 antigen was correlated with reduced mRNA transcription and activity of the PAI-1 promoter. By inhibiting expression of PAI-1 from SMCs and ECs while increasing expression of tPA from ECs, simvastatin may alter the local fibrinolytic balance within the vessel wall toward increased fibrinolytic capacity that, in turn, would reduce thrombotic risk after plaque rupture.

Diurnal changes of fibrinolysis in patients with liver cirrhosis and esophageal varices.

Variceal bleeding, whose triggering mechanisms are largely unknown, occurs with a circadian rhythmicity, with 2 peaks, one greater, in the evening, and one smaller, in the early morning. We assessed some clotting and hemodynamic parameters, possibly involved in variceal hemorrhage, over a 24-hour period, at 4-hour intervals, in 16 patients with cirrhosis and esophageal varices and in 9 controls. At each time interval, tissue plasminogen activator (tPA) and tPA inhibitor-1 (PAI-1) antigens and activities and total euglobulin fibrinolytic activity were determined and portal-vein flow velocity, volume, and congestion index were measured by duplex-Doppler. Significant circadian rhythms were searched for by least-squares and cosinor methods. tPA activity showed a circadian rhythm in cirrhosis, with a peak of 2.85 times the trough value, calculated at 18:42, and remained over 2.5-fold until shortly after 22:00. Total fibrinolytic activity showed a similar pattern, which was statistically significant also in controls. tPA and PAI antigens also showed significant circadian rhythm both in controls and cirrhotics, with higher values in the morning. Among the portal hemodynamic parameters only the congestion index showed significant rhythmic changes and only in cirrhosis, with the highest values in the late evening, but with limited diurnal excursion (+/- 5.5%). In conclusion, we showed the existence of a circadian rhythm of fibrinolysis in cirrhosis, whose temporal distribution might suggest a role of fibrinolysis in variceal hemorrhage on the basis of the comparison to the known chronorisk of variceal bleeding.

Association of blood pressure with fibrinolytic potential in the Framingham offspring population.

Hypertension is an established risk factor for acute coronary events. Because fibrinolytic and hemostatic factors are also associated with cardiovascular disease, we examined the relations of systolic and diastolic blood pressures (SBP and DBP) to levels of plasminogen activator inhibitor antigen, tissue plasminogen activator antigen, fibrinogen, factor VII, von Willebrand factor, fibrinogen, and plasma viscosity in subjects of the Framingham Offspring Study. We studied 1193 men and 1459 women after the exclusion of subjects with known cardiovascular disease and those receiving anticoagulant or antihypertensive therapy. Linear regression models were used to evaluate SBP and DBP as predictors of fibrinolytic and hemostatic factor levels in separate sex models, with adjustment for age, body mass index, smoking, diabetes, total cholesterol, HDL, triglycerides, alcohol intake, and estrogen use (in women). In both sexes, levels of plasminogen activator inhibitor and tissue plasminogen activator antigen were positively related to SBP and DBP (P<0.001). Plasma viscosity was positively related to SBP (P=0.008) and DBP (P=0.001) in women only. There was no association between SBP or DBP and fibrinogen, factor VII, or von Willebrand factor in either sex. These data suggest that impaired fibrinolysis may play an important role in the pathogenesis of cardiovascular disease in hypertensive patients.

Hyperinsulinemia, hyperglycemia, and impaired hemostasis: the Framingham Offspring Study.

Increased risk for cardiovascular disease in persons with glucose intolerance (impaired glucose tolerance and type 2 diabetes mellitus) is not fully explained by concomitant elevations in traditional atherosclerosis risk factors. Hyperinsulinemia associated with glucose intolerance may increase risk directly, or its effect could be mediated through impaired hemostatic function. To evaluate associations between fasting insulin levels and hemostatic factors in subjects with normal and impaired glucose homeostasis. Cross-sectional analysis conducted between January 1991 and June 1995. The population-based Framingham Offspring Study. A total of 1331 men and 1631 women aged 26 to 82 years, without diagnosed diabetes or cardiovascular disease and classified as having normal glucose tolerance (80.2%) or glucose intolerance (impaired glucose tolerance and impaired fasting glucose combined, 15.2%; previously undiagnosed diabetes, 4.7%) using an oral glucose tolerance test. Trends across quintiles of fasting insulin in levels of plasminogen activator inhibitor 1 (PAI-1) antigen, tissue-type plasminogen activator (tPA) antigen, von Willebrand factor (vWF) antigen, factor VII antigen, fibrinogen, and plasma viscosity. We stratified analyses by sex and glucose tolerance status and adjusted hemostatic factor levels for obesity, lipid levels, and traditional cardiovascular disease risk factors. Mean levels of all hemostatic factors (except for fibrinogen in men) increased across fasting insulin quintiles among subjects with normal glucose tolerance (P<.001 for trend). Levels of PAI-1 and tPA antigens, but not other hemostatic factors, were higher comparing subjects with glucose intolerance with those with normal glucose tolerance (P<.001). Among subjects with glucose intolerance, levels of PAI-1 and tPA antigen in men and women (P<.01 for trend) and vWF antigen in men (P<.05 for trend) increased significantly across insulin quintiles, but levels of factor VII antigen, fibrinogen, and plasma viscosity did not increase. Elevated levels of fasting insulin are associated with impaired fibrinolysis and hypercoagulability in subjects with normal glucose tolerance. Hyperinsulinemia is associated primarily with impaired fibrinolysis in subjects with glucose intolerance. Excess risk for cardiovascular disease associated with hyperinsulinemia and glucose intolerance may be mediated in part by enhanced potential for acute thrombosis.

Increased plasminogen activator inhibitor-1 activity in offspring of type 2 diabetic patients: lack of association with plasma insulin levels.

To determine whether a dysregulation of the fibrinolytic system exists in normal glucose tolerant offspring of type 2 diabetic patients. In this cross-sectional study, 32 offspring of type 2 diabetic patients and 26 subjects with no family history of diabetes were studied. With respect to the metabolic parameters, plasma fasting and 2-h postload (75 g glucose) glucose and insulin levels, total cholesterol, triglycerides, and HDL cholesterol concentrations were determined. To evaluate the status of hemostatic factors, fibrinogen, tissue plasminogen activator (tPA) antigen level, plasminogen activator inhibitor-1 (PAI-1) antigen level, and PAI-1 activity were assessed. The statistical analyses included the Mann-Whitney U test to check the significance of differences between variables in the two groups and Spearman's rank correlation tests to check the interrelationships between the hemostatic and metabolic parameters in the offspring group. All subjects had normal glucose tolerance according to the American Diabetes Association criteria. Plasma fasting and postload insulin concentrations were significantly higher in offspring compared with control group (P<0.00001 and P<0.01, respectively). Plasma fasting and postload glucose, fibrinogen, tPA antigen, total cholesterol, and BMI were comparable between the groups. The offspring had significantly higher waist-to-hip ratio (WHR) (P = 0.03), higher triglycerides (P = 0.01), and lower HDL cholesterol (P<0.01) compared with the control group. PAI-1 antigen level and PAI-1 activity were higher in the offspring (P = 0.05 and P = 0.04, respectively). In the offspring group, PAI-1 activity was correlated with plasma PAI-1 antigen level (r = 0.40, P = 0.02), fibrinogen (r = 0.45, P = 0.01), and HDL cholesterol (r = -0.36, P = 0.04). However, tPA antigen level, fasting and postload plasma glucose and insulin, total cholesterol, triglycerides, WHR, and BMI did not correlate with PAI-1 activity. These data suggest that normal glucose tolerant offspring of type 2 diabetic subjects have elevated PAI-1 activity indicating to hypofibrinolysis in this group. The elevated PAI-1 activity has no association with plasma insulin concentration.

Job strain and risk indicators for cardiovascular disease in young female nurses.

This study examined the possible effects of job demands, decision latitude, and job-related social support on risk indicators for cardiovascular disease (CVD) in 165 female nurses. Job strain was measured with the Job Content Questionnaire; CVD risk was measured with insulin, total cholesterol, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), fibrinogen, tissue-type plasminogen activator (tPA) antigen, tPA activity, plasminogen activator inhibitor-1 antigen, and blood pressure. Multivariate analysis of covariance and regression analyses revealed no effects of either job strain or social support on these risk indicators. All risk indicators deteriorated with age and body mass index. Oral contraceptive use improved fibrinolytic potential and increased HDL-C but had adverse effects on TG levels. Results suggest that in healthy young women job strain is not associated with an unfavorable metabolic or fibrinolytic risk profile.

Immunoassays for the Quantitation of Porcine PAI-1 Antigen and Activity in Biological Fluid Samples

Two monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) for the quantitation of porcine plasminogen activator inhibitor-1 (PAI-1) antigen and activity in plasma were constructed and validated. The intra-assay, interassay, and interdilution coefficients of variation were 4.3, 13, and 8%, respectively, for the antigen ELISA and 5, 16, and 11% for the activity assay. Assay recoveries, in the antigen ELISA. of either latent or active recombinant porcine PAI-1 (10 and 50 ng/ml) added to plasma were 86 +/- 9% and 92 +/- 22%, respectively, for the latent form and 89 +/- 99% and 87 +/- 7% for the active form (mean +/- SD, n = 3 to 4). In the immunofunctional assay, recoveries for the same concentrations of active PAI-1 were 108 +/- 16% and 92 +/- 21%, respectively. In male porcine plasma the level of PAI-1 antigen was 31 +/- 11 ng/ ml and the activity, 34 +/- 16 ng/ml (mean +/- SD, n = 10). In female plasma PAI-1 antigen levels were 20 +/- 5.2 ng/ml and the PAI-1 activity 42 +/- 17 ng/ml (n = 13). A linear correlation was found between PAI-1 antigen and activity levels in male (r = 0.60) and female (r = 0.70) plasma. Immunodepletion resulted in a decrease of >95% of the original PAI-1 antigen or activity levels. Incubation of plasma samples at 37 degrees C for 16 h resulted in a significant decrease (70 to 85%) of PAI-1 activity. Under these conditions (37 degrees C, 16 h) PAI-1 antigen levels remained unchanged in males whereas the response of the female samples in the PAI-1 antigen assay increased two-fold. In lysed platelet-rich plasma males had 990 +/- 470 ng/ml antigen and 160 +/- 80 ng/ml activity and females, 920 +/- 500 ng/ml antigen and 150 +/- 98 ng/ml activity corresponding to 2.1 +/- 0.77 fg PAI-1 antigen per platelet. Only 16% of PAI-1 released from platelets was found to be active. Linear correlations between PAI-1 antigen and activity were found for both males (r = 0.61) and females (r = 0.67). The assays are both sensitive and specific and may, therefore, aid the elucidation of the pathophysiological role of PAI-1 in swine experimental models of atherosclerosis and other thrombotic disorders.

Increased Fibrinolytic Activity during Use of Oral Contraceptives Is Counteracted by an Enhanced Factor XI-independent down Regulation of Fibrinolysis

The effect of oral contraceptives (OC) on fibrinolytic parameters was investigated in a cycle-controlled cross-over study in which 28 non-OC using women were randomly prescribed either a representative of the so-called second (30 microg ethinylestradiol, 150 microg levonorgestrel) or third generation OC (30 microg ethinylestradiol, 150 microg desogestrel) and who switched OC after a two month wash out period. During the use of OC, the levels of tissue-type plasminogen activator (tPA) activity, plasminogen, plasmin-alpha2-antiplasmin complexes and D-dimer significantly increased (by 30 to 80%), while the levels of plasminogen activator inhibitor- (PAI-1) antigen, PAI-1 activity and tPA antigen significantly decreased (25 to 50%), suggesting an increase in endogenous fibrinolytic activity. These OC-induced changes were not different between the two contraceptive pills. TAFI (thrombin-activatable fibrinolysis inhibitor) levels increased on levonorgestrel, and even further increased on desogestrel. A clot lysis assay that probes both fibrinolytic activity and the efficacy of the coagulation system to generate thrombin necessary to down regulate fibrinolysis via TAFI showed no change of the clot lysis time during OC use. This finding suggests that the OC-induced increase in endogenous fibrinolytic activity is counteracted by an increased capacity of the coagulation system to down regulate fibrinolysis via TAFI. Indeed we observed that during OC use there was a significant increase of F1+2 generation during clot formation. When these assays were performed in the presence of an antibody against factor XI, we observed that the clot lysis time was significantly increased during OC use and that the increase in F1+2 generation during OC therapy was due to a factor XI-independent process, which was significantly higher on desogestrel than on levonorgestrel. These data indicate that the OC-induced inhibition of endogenous fibrinolysis takes place in a factor XI-independent way and is more pronounced on desogestrel than on levonorgestrel-containing OC.

The Effect of Red Wine on the Fibrinolytic System and the Cellular Activation Reactions before and after Exercise

The effect of red wine drinking was tested on fibrinolytic parameters and blood cells in nine healthy students at rest and after acute exercise. The subjects were randomly assigned in a crossover design to one of three treatment regimes: control situation, low-dose wine group, and high-dose wine group. Blood samples were drawn just prior to experimental start, at 2 and 4 hours, and the next morning at 8:00 a.m., at 8:30 a.m. just after exercise, and 2 hours after exercise. The fibrinolytic potential was measured by whole blood clot lysis time (WBCLT), tissue plasminogen activator, and plasminogen activator inhibitor-1 (PAI-1) antigens in plasma. A whole blood system was used to test the reactivity of blood cells by stimulating hirudinized blood with 5 ng/mL lipopolyusaccharide (LPS) for 2 hours at 37°C and measurements of tissue necrosis factor α and interleukin-8 (IL-8) in the plasma. Intake of red wine caused impaired fibrinolysis shown by prolonged WBCLT (3.6, 20.7, and 55.7%, respectively, for control, low- and high-dose wine groups) due to increase in PAI-1 antigen (−0.8, 4.8, and 11.0 ng/mL, respectively, in the three groups). There was no effect of the red wine the next morning on the fibrinolytic system. A strong correlation was observed between WBCLT and PAI-1 antigen (p<0.0001). Acute exercise caused an immediate rise in both tissue plasminogen activator antigen and PAI-1 antigen levels and WBCLT was significantly shortened. In contrast to that of the wine groups, 2 hours after exercise WBCLT was prolonged in the control group, but not significantly so. Thus the red wine has a negative effect on the fibrinolytic system during rest, but may have a positive effect after strenuous exercise. The red wine had no immediate effect on LPS-induced tissue necrosis factor α or IL-8 production, although there was a tendency for higher cytokine production in the control group compared to the wine groups during and just after intake of wine. The next morning after exercise, the LPS-induced IL-8 production increased 137, 89, and 96%, respectively, in control, low-, and high-dose wine groups, probably due to a rise in epinephrine and activation of platelets. Although not significantly so, there was a tendency for red wine intake in the evening to suppress the reactivity of the cells after physical exercise the subsequent morning. It is suggested that the negative effect of red wine ingestion may be due to the toxic effect of ethanol on hepatocytes or adipose tissue and subsequent release of PAI-1, whereas the positive effect may be due to the red wine suppression of platelet activation and release of PAI-1 from activated platelets. It is proposed that at least part of the beneficial effect of red wine ingestion may be associated with the downregulation of cytokine production.

Work stress and metabolic and hemostatic risk factors.

A high level of work stress has been associated with cardiovascular disease. However, the pathophysiological mechanisms underlying this association remain unclear. This study examined the effect of work stress on a cluster of metabolic and hemostatic risk factors. Blood was collected three times, on the first, third, and fifth day of a work week, from 124 middle-aged, white-collar workers. Metabolic measures were insulin, glucose, triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and total cholesterol. Hemostatic measures were fibrinogen, tissue-type plasminogen activator activity, tissue-type plasminogen activator antigen, and type 1 plasminogen activator inhibitor antigen. Chronic work stress was defined according to Siegrist's model as 1) a combination of high effort and low reward at work (effort-reward imbalance) or 2) high overcommitment (an exhaustive work-related coping style). Overcommitment, but not imbalance or the imbalance-overcommitment interaction, was associated with an impaired fibrinolytic system, as reflected in decreased tissue-type plasminogen activator activity levels and increased type 1 plasminogen activator inhibitor antigen levels on all three measurement occasions. After controlling for body mass index, total cholesterol, triglycerides, high-density lipoprotein/low-density lipoprotein cholesterol ratio, glucose, and insulin, the relation between overcom-mitment and the fibrinolytic factors was attenuated but remained significant. The results suggest that individuals with an exhaustive coping style at work have an impaired fibrinolytic capacity that is possibly due to the effects of chronic stress on insulin resistance.