Effect of prolonged physical exercise on the fibrinolytic system

The effect of a test marathon race on plasma fibrinolytic activity (FA) was studied in 16 endurance athletes before, immediately after, 3 h, and 31 h after the run. Tissue plasminogen activator (t-PA) activity increased about 31-fold immediately after the run. Similar increases were found in t-PA antigen concentration. Plasminogen activator inhibitor (PAI) was not detectable immediately after the race and was significantly decreased 3 h (P less than 0.05) and 31 h (P less than 0.01) later. B beta 15-42 peptide increased by 0.63 pmol.ml-1 (P less than 0.001), D-dimer by 68.3 ng.ml-1 (P less than 0.05). Euglobulin lysis time (ELT) was reduced from 109 to 18 min (P less than 0.001). The increased t-PA activity and t-PA antigen concentration disappeared in the course of the first 3 h after exertion. ELT also reached its pre-exercise levels at this time. Thirty-one hours after the race ELT and t-PA antigen levels were slightly but significantly reduced (P less than 0.05), whereas B beta 15-42 peptide remained increased (P less than 0.05). t-PA activity was unchanged compared with pre-exercise values. It seems that the exercise-induced FA is mainly caused by the marked increase of t-PA antigen and t-PA activity.     

Concentration of plasminogen activators and inhibitor in the human endometrium at different phases of the menstrual cycle.

The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor (PAI-1) have been determined in endometrial curettings obtained from 46 subfertile women during proliferative, early or late secretory phases of the menstrual cycle. t-PA activity and antigen concentrations was significantly higher (P < 0.001) in late secretory endometrium than in proliferative or early secretory endometrium. Higher concentrations of PAI-1 antigen (P < 0.05) were also noted in late secretory phase than in proliferative and early secretory endometrium. However, u-PA concentration was not significantly different and no PAI activity could be demonstrated in the menstrual phases studied. Zymography studies confirmed the presence of both t-PA and u-PA in the endometrium. Ovarian hormonal patterns may therefore influence the activity of plasminogen activators especially of t-PA in the endometrium during various phases of the menstrual cycle.     

Insulin sensitivity and regular alcohol consumption: large,prospective, cross sectional population study (Bruneck study)

OBJECTIVES: To assess the relation between regular alcohol consumption and insulin sensitivity, and to estimate the importance of insulin in the association of alcohol with multiple vascular risk factors and cardiovascular disease. DESIGN: Prospective and cross sectional study of a large randomly selected population sample. SETTING: Part of the Bruneck study 1990-5 (Bolzano province, Italy). SUBJECTS: 820 health, non-diabetic women and men aged 40-79 years. MAIN OUTCOME MEASURE: Concentrations of fasting and post-glucose insulin, cholesterol, apolipoproteins, triglycerides, Lp(a) lipoprotein glucose, fibrinogen, and antithrombin III; blood pressure; insulin resistance estimated by the homeostasis model assessment. RESULTS: Fasting insulin concentrations in those who did not drink alcohol and subjects reporting low (1-50 g/day), moderate (51-99 g/day), and heavy (> or = 100 g/day) alcohol intake were 12.4, 10.0, 8.7, and 7.1 mU/l (P < 0.001). Likewise, post-glucose insulin concentrations and estimates for insulin resistance assessed by the homeostasis model assessment decreased significantly with increasing amounts of regular alcohol consumption. These trends were independent of sex, body mass index, physical activity, cigarette smoking, medication, and diet (P < 0.001). Regular alcohol intake predicted multiple changes in vascular risk factors over a five year period including increased concentrations of high density lipoprotein cholesterol and apolipoprotein A I; higher blood pressure; and decreased concentration of antithrombin III. These associations were in part attributable to the decrease in insulin concentrations observed among alcohol consumers. CONCLUSIONS: Low to moderate amounts of alcohol, when taken on a regular basis, improve insulin sensitivity. Insulin is a potential intermediate component in the association between alcohol consumption and vascular risk factors (metabolic syndrome).     

Induction of Urokinase-Type Plasminogen Activator in Rat Facial Nucleus by Axotomy of the Facial Nerve

Abstract: The response of plasminogen activator activity in the CNS to peripheral nerve axotomy was examined in vivo. After transection of the rat facial nerve, a transient increase in plasminogen activator activity was observed in the facial nucleus on the operated side with maximal activity 3–5 days after lesion. This activity was inhibited by the urokinase-specific inhibitor amiloride but not by antibodies against tissue plasminogen activator. The molecular mass of the induced form of plasminogen activator was estimated to be ∼48 kDa. An in vitro assay of plasminogen hydrolysis also demonstrated an increase in amiloride-sensitive plasminogen activator activity in facial nerve extracts following facial nerve axotomy. These data indicate that the plasminogen activator activity induced in the facial nucleus following axotomy of facial motoneurons is of the urokinase type. It is suggested that the urokinase-type plasminogen activator might play a role in the events accompanying injury and regeneration in the facial nucleus following motoneuron lesion.     

Association between alcohol consumption and symptom severity and quality of life in patients with fibromyalgia

Introduction

Although alcohol consumption is a common lifestyle behavior with previous studies reporting positive effects of alcohol on chronic pain and rheumatoid arthritis, no studies to this date have examined alcohol consumption in patients with fibromyalgia. We examined the association between alcohol consumption and symptom severity and quality of life (QOL) in patients with fibromyalgia.

Methods

Data on self-reported alcohol consumption from 946 patients were analyzed. Subjects were grouped by level of alcohol consumption (number of drinks/week): none, low (≤3), moderate (>3 to 7), and heavy (>7).Univariate analyses were used to find potential confounders, and analysis of covariance was used to adjust for these confounders. Tukey HSD pairwise comparisons were used to determine differences between alcohol groups.

Results

Five hundred and forty-six subjects (58%) did not consume alcohol. Low, moderate, and heavy levels of alcohol consumption were reported for 338 (36%), 31 (3%), and 31 patients (3%), respectively. Employment status (P <0.001), education level (P = 0.009), body mass index (P = 0.002) and opioid use (P = 0.002) differed significantly among groups with drinkers having higher education, a lower BMI, and a lower frequency of unemployment and opioid use than nondrinkers. After adjusting for these differences, the measures including the number of tender points (P = 0.01), FIQ total score (P = 0.01), physical function (P <0.001), work missed (P = 0.005), job ability (P = 0.03), and pain (P = 0.001) differed across groups, as did the SF-36 subscales of physical functioning (P <0.001), pain index (P = 0.002), general health perception (P = 0.02), social functioning (P = 0.02), and the physical component summary (P <0.001). Pairwise comparison among the 4 groups showed that the moderate and low alcohol drinkers had lower severity of fibromyalgia symptoms and better physical QOL than nondrinkers.

Conclusions

Our study demonstrates that low and moderate alcohol consumption was associated with lower fibromyalgia symptoms and better QOL compared to no alcohol consumption. The reasons for these results are unclear. Since recent studies have demonstrated that γ-Aminobutyric Acid (GABA) levels are low in fibromyalgia, and alcohol is known to be a GABA-agonist, future studies should examine whether alcohol could have a salutary effect on pain and other symptoms in fibromyalgia.     

Purification of epidermal plasminogen activator inhibitor

A plasminogen activator inhibitor was purified from human cornified cell extract by DEAE-Sepharose, Sephacryl S-200, and high-performance liquid chromatographies on hydroxyapatite HPHT and anion-exchanger Mono Q at pH 7.2 and 8.0. The purified inhibitor showed Mr 43,000 and pI 5.2 50% inhibition of fibrinolytic activity (1.5 IU) of urokinase and tissue-type plasminogen activator was attained by 0.60 ng and 11.0 ng purified inhibitor, respectively. Synthetic substrate assay demonstrated slow tight-binding inhibition to both urokinase and tissue-type plasminogen activator. The inhibitor did not inactivate plasmin, thrombin, glandular kallikrein or trypsin.     

Alcohol consumption stimulates early steps in reverse cholesterol transport.

Alcohol consumption is associated with increased HDL cholesterol levels, which may indicate stimulated reverse cholesterol transport. The mechanism is, however, not known. The aim of this study was to evaluate the effects of alcohol consumption on the first two steps of the reverse cholesterol pathway: cellular cholesterol efflux and plasma cholesterol esterification. Eleven healthy middle-aged men consumed four glasses (40 g of alcohol) of red wine, beer, spirits (Dutch gin), or carbonated mineral water (control) daily with evening dinner, for 3 weeks, according to a 4 x 4 Latin square design. After 3 weeks of alcohol consumption the plasma ex vivo cholesterol efflux capacity, measured with Fu5AH cells, was raised by 6.2% (P < 0.0001) and did not differ between the alcoholic beverages. Plasma cholesterol esterification was increased by 10.8% after alcohol (P = 0.008). Changes were statistically significant after beer and spirits, but not after red wine consumption (P = 0.16). HDL lipids changed after alcohol consumption; HDL total cholesterol, HDL cholesteryl ester, HDL free cholesterol, HDL phospholipids and plasma apolipoprotein A-I all increased (P < 0.01). In conclusion, alcohol consumption stimulates cellular cholesterol efflux and its esterification in plasma. These effects were mostly independent of the kind of alcoholic beverage     

Isolation and interrelationships of the multiple molecular tissue-type and urokinase-type plasminogen activator forms produced by cultured human umbilical vein endothelial cells

Primary and early subcultures (1st- to 3rd passage) of human umbilical vein endothelial cells produce tissue-type plasminogen activator (t-PA) antigen, consisting only of a major Mr 110,000 t-PA form. Later subcultures (greater than 4th passage) produce increasing amounts of t-PA antigen, consisting of a major Mr 110,000 and a minor Mr 68,000 form as well as increasing amounts of urokinase-type plasminogen activator (u-PA) antigen, consisting of a minor Mr 95,000 and major Mr 54,000 form. All of the major plasminogen activator forms were purified to homogeneity from 72 h serum-free conditioned media (3 liters, 1-1.8 x 10(9) cells) by a combination of immunoaffinity and gel filtration chromatography. Typically, 4th to 6th passage cultures produced/secreted t-PA-type proteins consisting of an inactive Mr 110,000 (220 IU/mg) and active Mr 68,000 (76,500 IU/mg) form representing about 39 and 8%, respectively, of the total starting sodium dodecyl sulfate stable t-PA activity, and u-PA-type proteins consisting of an inactive Mr 95,000 (700 IU/mg) and active Mr 54,000 (81,000 IU/mg) form representing about 9 and 38%, respectively, of the total starting sodium dodecyl sulfate stable u-PA activity. The isolated Mr 68,000 t-PA and Mr 54,000 u-PA proteins, exist only as two-chain forms in the absence of aprotinin and as mixtures of single- and two-chain proteins in the presence of aprotinin. Treatment with nucleophilic agents completely dissociated the Mr 110,000 t-PA and Mr 95,000 u-PA proteins into their respective Mr 68,000 t-PA and Mr 54,000 u-PA activity forms and a common Mr 46,000 protein, confirming the enzyme-inhibitor complex nature of these inactive plasminogen activator forms.     

Limited prognostic value of c-erbB-2 compared to uPA and PAI-1 in primary breast carcinoma

In a retrospective study of 488 women with primary breast cancer, after a median follow-up of 10 years, we sought interactions between disease-free survival (DFS) and overall survival (OS) and tumor antigen levels of two components of the plasminogen system, urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1, and the transmembrane growth factor receptor c-erbB-2. We used ELISAs (American Diagnostica, Greenwich, CT, USA) to quantify uPA and PAI-1 antigen levels in cytosols, and a double monoclonal antibody-based assay (EIA) (Ciba Corning Diagnostics, Alameda, CA, USA) to quantify c-erbB-2 in membrane extracts of the same tissues. Weak positive correlations were found between uPA and c-erbB-2 (r(s) = 0.146; p = 0.001) and between PAI-1 and c-erbB-2 (r(s) = 0.154; p < 0.001). In the overall population, using univariate analyses, c-erbB-2 overexpression and high uPA and PAI-1 antigen levels (> 300 IU/mg, > 1.40 ng/mg and > 5.53 ng/mg, respectively) were significantly associated with shorter DFS (p = 0.003, p < 0.001 and p < 0.001, respectively) and OS (p < 0.001 in all cases). Using multivariate analyses, PAI-1, node status and tumor size were independent predictors of DFS and c-erbB-2 was retained in the model only for OS. In the node-negative subgroup, PAI-1 was the strongest significant survival predictor both for OS (p = 0.003; HR 2.52) and DFS (p < 0.001; HR 2.39). This study shows that in primary breast cancer c-erbB-2 offers no additional prognostic information when uPA and/or PAI-1 are candidates in the multivariate analyses.     

Isolation and characteristics of urokinase-type plasminogen activator from a culture of human embryo lung fibroblasts

Plasminogen activator from conditioned medium of human embryonal lung fibroblasts was purified by phosphocellulose P11 chromatography, followed by p-aminobenzamidine-agarose chromatography. Two forms of plasminogen activators were separated by chromatography on the heparin-sepharose. The high molecular weight form (53 kDa) with specific activity 130 000 IU/mg consists of two polypeptide chains (31 kDa and 20 kDa) and exhibits strong affinity for fibrin-celite, lysine-sepharose and heparin-sepharose. The low molecular weight form (32 kDa, 190 000 IU/mg) also binds to these sorbents, but more weakly, and its properties are very similar to those of low molecular weight urokinase. Activity of both forms of plasminogen activators are inhibited by monoclonal antibodies against urokinase. A number of enzymological chromatographic and immunological properties indicates, that the plasminogen activator from lung fibroblasts is of urokinase type.     

Involvement of the plasminogen activation system in cow endometritis

The objectives of this study were to investigate the: (a) presence and activity of components of the "plasminogen activators/plasmin" system in dairy cows with or without endometritis; (b) variations in enzyme activity according to the degree of endometritis; and (c) associations between these enzymes and changes in endometrial histology after intrauterine antibiotic treatment. Endometrial biopsies were collected from anestrus (no palpable ovarian structures and milk progesterone <1 ng/ml) Holstein cows, 30-40 days postpartum. On the basis of a vaginoscopic examination, rectal palpation of the cervix and uterus, and endometrial histology, there were 92 cows with endometritis and 20 cows without endometritis. After biopsy collection, each cow was given an intrauterine infusion of 1.5x10(6) IU of procaine penicillin G. In cows with endometritis, genital tract examinations and biopsies were repeated 2 weeks later. Both plasminogen activators (PAs), tissue type (t-PA) and urokinase (u-PA), were immunologically identified in all uterine biopsies. Plasminogen activator activity (PAA) increased, whereas plasminogen activator inhibition (PAI) and plasmin inhibition (PI) decreased in proportion to the degree of inflammation. Two weeks after intrauterine treatment, PAA had decreased significantly in all cows that had reduced severity of endometrial inflammation and had increased significantly in all cows with increased severity of inflammation. The change in the degree of inflammation depended upon plasminogen activator activity; cows with higher PAA were more likely to improve. In conclusion, there was evidence for a role of the plasminogen activation proteolytic system in bovine endometritis.     

Randomised controlled trial of general practitioner intervention in patients with excessive alcohol consumption.

OBJECTIVE: To determine effectiveness of advice from general practitioners to heavy drinkers to reduce their excessive alcohol consumption (35 U or more a week for men, 21 U or more for women). DESIGN: Randomised, controlled double blind trial over 12 months with interim assessment at six months. SETTING: Group practices (n = 47; list size averaging 10,000) recruited from Medical Research Council''s general practice research framework, mostly in rural or small urban settings. PATIENTS: Patients recruited after questionnaire survey. Of total of 2571 (61.2%) of 4203 patients invited for interview who attended, 909 (35.4%) stated that in past seven days they had drunk above the limits set for study and had not received advice; they were randomised to control and treatment groups. INTERVENTIONS: Patients in treatment group were interviewed by general practitioner (who had had a training session) and received advice and information about how to reduce consumption and also given a drinking diary. END POINT: Study aimed at detecting a reduction in proportion of men with excessive alcohol consumption of 30% in treatment group and 20% in control group (for women 40% and 20%, respectively) with a power of 90% at 5% level of significance. In addition, corroborative measures such as estimation of gamma-glutamyltransferase activity were included. MEASUREMENTS AND MAIN RESULTS: At one year a mean reduction in consumption of alcohol of 18.2 (SE 1.5) U/week had occurred in treated men compared with a reduction of 8.1 (1.6) U/week in controls (p less than 0.001). The proportion of men with excessive consumption at interview had dropped by 43.7% in the treatment group compared with 25.5% in controls (p less than 0.001). A mean reduction in weekly consumption of 11.5 (1.6) U occurred in treated women compared with 6.3 (2.0) U in controls (p less than 0.05), with proportionate reductions of excessive drinkers in treatment and control groups of 47.7% and 29.2% respectively. Reduction in consumption increased significantly with number of general practitioner interventions. At one year the mean value for gamma-glutamyltransferase activity had dropped significantly more in treated men (-2.4 (0.9)IU/l) than in controls (+1.1(1.0)IU/l; t = 2.7, p less than 0.01). Reduction in gamma-glutamyltransferase activity tended to increase with number of intervention sessions in men. Changes in gamma-glutamyltransferase activity in women and changes in other indicators in both sexes did not differ significantly between treatment and control groups. CONCLUSIONS: If the results of this study were applied to the United Kingdom intervention by general practitioners could each year reduce to moderate levels the alcohol consumption of some 250000 men and 67500 women who currently drink to excess. General practitioners and other members of the primary health care team should therefore be encouraged to include counselling about alcohol consumption in their preventive activities.     

Intravenous tissue plasminogen activator and size of infarct,left ventricular function,and survival in acute myocardial infarction.

STUDY OBJECTIVE--To assess effect of intravenous recombinant tissue type plasminogen activator on size of infarct, left ventricular function, and survival in acute myocardial infarction. DESIGN--Double blind, randomised, placebo controlled prospective trial of patients with acute myocardial infarction within five hours after onset of symptoms. SETTING--Twenty six referral centres participating in European cooperative study for recombinant tissue type plasminogen activator. PATIENTS--Treatment group of 355 patients with acute myocardial infarction allocated to receive intravenous recombinant plasminogen activator. Controls comprised 366 similar patients allocated to receive placebo. INTERVENTION--All patients were given aspirin 250 mg and bolus injection of 5000 IU heparin immediately before start of trial. Patients in treatment group were given 100 mg recombinant tissue plasminogen activator over three hours (10 mg intravenous bolus, 50 mg during one hour, and 40 mg during next two hours) by infusion. Controls were given placebo by same method. Full anticoagulation treatment and aspirin were given to both groups until angiography (10-22 days after admission). beta Blockers were given at discharge. END POINT--Left ventricular function at 10-22 days, enzymatic infarct size, clinical course, and survival to three month follow up. MEASUREMENTS AND MAIN RESULTS--Mortality was reduced by 51% (95% confidence interval -76 to 1) in treated patients at 14 days after start of treatment and by 36% (-63 to 13) at three months. For treatment within three hours after myocardial infarction mortality was reduced by 82% (-95 to -31) at 14 days and by 59% (-83 to -2) at three months. During 14 days in hospital incidence of cardiac complications was lower in treated patients than controls (cardiogenic shock, 2.5% v 6.0%; ventricular fibrillation, 3.4% v 6.3%; and pericarditis, 6.2% v 11.0% respectively), but that of angioplasty or artery bypass, or both was higher (15.8% v 9.6%) during the first three months. Bleeding complications were commoner in treated than untreated patients. Most were minor, but 1.4% of treated patients had intracranial haemorrhage within three days after start of infusion. Enzymatic size of infarct, determined by alpha hydroxybutyrate dehydrogenase concentrations, was less (20%, 2p = 0.0018) in treated patients than in controls. Left ventricular ejection fraction was 2.2% higher (0.3 to 4.0) and end diastolic and end systolic volumes smaller by 6.0 ml (-0.2 to -11.9) and 5.8 ml (-0.9 to -10.6), respectively, in treated patients. CONCLUSION--Recombinant tissue type plasminogen activator with heparin and aspirin reduces size of infarct, preserves left ventricular function, and reduces complications and death from cardiac causes but at increased risk of bleeding complications4+     

ATF-PAI2CD融合蛋白的生物学功能

为了解尿激酶型纤溶酶激活物 (urokinase typeplaminogenactivator,uPA)的氨基末端片段 (amino terminalfrag ment,ATF)和纤溶酶激活物抑制剂 2 (plasminogenactivatorinhibitortype 2 ,PAI 2 )突变体PAI 2CD融合蛋白在大肠杆菌的表达情况及进一步研究其生物学活性、将ATF PAI2CD融合基因与大肠杆菌表达载体pLY 4重组得到表达质粒pZWE ATF PAI2CD ,以其转化大肠杆菌JF112 5 ,经温度诱导 ,ATF PAI2CD获得较高水平表达 ,融合蛋白质以包涵体形式存在 ,占菌体总蛋白质的 15 %。包涵体经洗涤、8mol L尿素溶解、稀释复性及离子交换色谱一步分离 ,纯度达 90 % ,分子量与理论值相符。每升发酵液得重组融合蛋白质 5 0mg。经牛奶板法检测具有纤溶抑制活性 ,比活性达 12 0 0 0IU mg ;应用放射竞争法证实融合蛋白质能与肿瘤细胞表面的uPA受体特异性结合。双功能融合蛋白质的纤溶抑制活性与野生型PAI 2 (或PAI 2突变体 ,PAI 2CD)基本一致 ,与肿瘤细胞表面的uPA受体的结合能力同pro uPA。     

Alcohol consumption,serum low density lipoprotein cholesterol concentration,and risk of ischaemic heart disease: six year follow up in the Copenhagen male study.

OBJECTIVES: To investigate the interplay between use of alcohol, concentration of low density lipoprotein cholesterol, and risk of ischaemic heart disease. DESIGN: Prospective study with controlling for several relevant confounders, including concentrations of other lipid fractions. SETTING: Copenhagen male study, Denmark. SUBJECTS: 2826 men aged 53-74 years without overt ischaemic heart disease. MAIN OUTCOME MEASURE: Incidence of ischaemic heart disease during a six year follow up period. RESULTS: 172 men (6.1%) had a first ischaemic heart disease event. There was an overall inverse association between alcohol intake and risk of ischaemic heart disease. The association was highly dependent on concentration of low density lipoprotein cholesterol. In men with a high concentration (> or = 5.25 mmol/l) cumulative incidence rates of ischaemic heart disease were 16.4% for abstainers, 8.7% for those who drank 1-21 beverages a week, and 4.4% for those who drank 22 or more beverages a week. With abstainers as reference and after adjustment for confounders, corresponding relative risks (95% confidence interval) were 0.4 (0.2 to 1.0; P<0.05) and 0.2 (0.1 to 0.8; P<0.01). In men with a concentration <3.63 mmol/l use of alcohol was not associated with risk. The attributable risk (95% confidence interval) of ischaemic heart disease among men with concentrations > or = 3.63 mmol/l who abstained from drinking alcohol was 43% (10% to 64%). CONCLUSIONS: In middle aged and elderly men the inverse association between alcohol consumption and risk of ischaemic heart disease is highly dependent on the concentration of low density lipoprotein cholesterol. These results support the suggestion that use of alcohol may in part explain the French paradox.     

The active and the inactive plasminogen activator inhibitor from human endothelial cell conditioned medium are immunologically and functionally related to each other

In human endothelial cell conditioned medium a fast-acting inhibitor of tissue-type plasminogen activator and urokinase has been detected. Moreover, an inactive inhibitor of these plasminogen activators is present, that can be activated by denaturing agents such as sodium dodecyl sulphate (SDS). The mutual relationship between these inhibitors was studied. The fast-acting plasminogen activator inhibitor from human endothelial cell conditioned medium was purified in a complex with tissue-type plasminogen activator by immune adsorption, using an immobilized anti-tissue-type plasminogen activator antibody. With the complex as an antigen, specific antibodies were raised against this inhibitor in rabbits. The antiserum immunoreacted with both the inactive and the fast-acting plasminogen activator inhibitor. Endothelial cell conditioned medium (containing the inactive plasminogen activator inhibitor) was treated with SDS and the inhibitory activity that emerged was purified. The SDS-generated product formed complexes with tissue-type plasminogen activator with the same molecular mass as those formed with the fast-acting inhibitor. Moreover, the inhibitory activity generated by SDS treatment showed the same kinetic behaviour with tissue-type plasminogen activator as did the fast-acting inhibitor. These data show that the fast-acting and the inactive plasminogen activator inhibitor are immunologically and functionally related to each other, and probably represent different molecular forms of the same protein.     

Dehydroepiandrosterone therapy in men with angiographically verified coronary heart disease: the effects on plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) and fibrinogen plasma concentrations

INTRODUCTION: The aim of this study was to analyze the influence of DHEA therapy on fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) plasma concentrations in men with decreased serum DHEA-S levels and angiographically verified coronary heart disease (CHD). MATERIAL AND METHODS: The study included thirty men aged 41-60 years (mean age 52 +/- 0.90 yr) with serum DHEA-S concentration < 2000 mg/l, who were randomized into a double-blind, placebo-controlled, cross-over trial. Subjects completed the 80 days study of 40 days of 150 mg oral DHEA daily or placebo, and next groups were changed after 30 days of wash-out. Fasting early morning blood samples were obtained at baseline and after each treatment to determine serum hormones levels (testosterone, DHEA-S, LH, FSH and estradiol) and also fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) plasma concentrations. RESULTS: Administration of DHEA was associated with 4.5-fold increase in DHEA-S levels. Estrogen levels significantly increased after DHEA from 22.1 +/- 0.7 pg/ml to 26.4 +/- 1.6 pg/l (mean +/- SEM; p < 0.05), while testosterone levels did not changed. Fibrinogen concentrations significantly decreased in DHEA group from 4.5 +/- 0.3 g/l to 3.83 +/- 0.2 g/l (p < 0.05 vs. placebo). Changes of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) were not statistical significant (respectively: 8.37 +/- 0.4 ng/ml vs. 8.93 +/- 0.5 ng/ml and 82.3 +/- 6.3 ng/ml vs. 92.7 +/- 9.1 ng/ml (mean +/- SEM; NS vs. placebo). Tolerance of the treatment was good and no adverse effects were observed. CONCLUSIONS: DHEA therapy in dose of 150 mg daily during 40 days in men with DHEAS levels < 2000 mg/l and angiographically verified coronary heart disease (CHD) was connected with significant decreasing of fibrinogen concentration and increasing of estradiol levels, and did not influence on plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) plasma concentrations.     

Separation and characterization of nonphosphorylated and serine-phosphorylated urokinase. Catalytic properties and sensitivity to plasminogen activator inhibitor type 1.

Urokinase synthesized by human A431 epidermoid carcinoma cells is phosphorylated on serine (Mastronicola, M. R., Stoppelli, M. P., Migliaccio, A., Auricchio, F., and Blasi, F. (1990) FEBS Lett. 266, 109-114). To test the possibility that phosphorylation may have specific effects on urokinase function, the phosphorylated and nonphosphorylated forms of urokinase were separated by Fe(3+)-Sepharose chromatography. Both forms exhibit indistinguishable Km and kcat for plasminogen activation. On the other hand, their sensitivity toward the specific plasminogen activator inhibitor type 1 is different as assessed by measuring both the stability of the covalent complex and the residual enzymatic activity. Phosphorylated urokinase was 50% inhibited at a concentration of plasminogen activator inhibitor type 1 4-fold higher than nonphosphorylated urokinase (0.7 versus 0.15 nM). Furthermore about 10% of phosphorylated urokinase was resistant to plasminogen activator inhibitor type 1 at a concentration as high as 20 nM. Thus, phosphorylation affects urokinase sensitivity to plasminogen activator inhibitor type 1, therefore resulting in a net, although indirect, increase of urokinase activity. These results suggest the existence of a novel cellular regulatory mechanism of extracellular proteolysis.     

Oxygen modulates the release of urokinase and plasminogen activator inhibitor-1 by retinal pigment epithelial cells

The aims of this study were to examine the effect of oxygen, in the presence or absence of exogenous growth factors, on the release of plasminogen activators and plasminogen activator inhibitor-1 by cultured human retinal pigment epithelial cells. Antigen and activity levels of urokinase, tissue plasminogen activator and plasminogen activator inhibitor were measured in conditioned media after cells were exposed to three different oxygen environments: hypoxia, normoxia and hyperoxia. Overall proteolytic balance was determined by zymography. The effects of exogenous basic fibroblast growth factor and transforming growth factor-beta were also examined. it was found that retinal pigment epithelial cells released urokinase, tissue plasminogen activator and plasminogen activator inhibitor in measurable quantities. After 48 h, urokinase levels were highest at normoxia, reaching 7.2ng/10(6) cells (+/-2.0 SEM), whereas plasminogen activator inhibitor 1 levels were highest at hyperoxia, reaching 67.5ng/10(6) cells (+/-3.7 SEM). Tissue plasminogen activator levels were minimal (<0.5ng/10(6) cells) and unaffected by both oxygen and growth factors. Overall proteolytic activity was also greatest at normoxia. Fibroblast growth factor stimulated urokinase production dose-dependently, but plasminogen activator inhibitor only minimally. Transforming growth factor-beta stimulated plasminogen activator inhibitor production dose-dependently but urokinase only at higher concentrations. These results suggest that both oxygen tension and growth factors may interact to modulate the proteolytic properties of the human retinal pigment epithelium.     

Regulation of extracellular plasminogen activator by human fibroblasts. The role of protease nexin

When the plasminogen activator urokinase was radioiodinated and incubated at 40 ng/ml in medium conditioned by human foreskin (HF) cells, within 30 min over 80% of the added plasminogen activator was complexed to cell-released protease nexin (PN). The urokinase complexed to PN had little if any activity. Incubation of purified PN with urokinase confirmed that PN is an inhibitor of this plasminogen activator. However, a widely used plasminogen-dependent fibrinolysis assay for plasminogen activator indicated that abundant endogenous plasminogen activator activity co-existed with PN in HF cell-conditioned medium. The source of this activity was electrophoretically and immunologically indistinguishable from urokinase. Furthermore, gel exclusion chromatography showed that about 90% of the urokinase antigen detected in conditioned medium had a molecular weight similar to that of free active urokinase. These paradoxical findings are resolved by evidence that this "PN-resistant urokinase-like" plasminogen activator is actually urokinase proenzyme that is activated by plasmin or conditions in the fibrinolysis assay for plasminogen activator. It is shown that the activated form of HF cell plasminogen activator is sensitive to inhibition by PN. PN may thus be an important component in the cellular regulation of endogenous plasminogen activator activity.