Low PAI-1 activity in relation to inflammatory parameters, insulin profile and body mass index.

BACKGROUND AND OBJECTIVE: High plasminogen activator inhibitor type 1 (PAI-1) activity is associated with inflammatory reactions and insulin resistance, but it is unclear what regulates PAI-1 activity at the low end. The purpose of this study was to investigate if patients with low PAI-1 activity have a lack of inflammatory response or a low insulin level. DESIGN: Retrospective cohort study with internal controls. SUBJECTS: Sixty-three patients referred for investigation of bleeding tendency and with low PAI-1 activity were compared with 118 patients with normal or high PAI-1 activity. OUTCOME: Levels of C-peptide, proinsulin, high-sensitivity C-reactive protein (hs-CRP) and interleukin-6 (IL-6). Adjustments were made for body mass index (BMI), oral oestrogens and age. Low PAI-1 activity was defined as less than 1 U mL(-1). RESULTS: Body mass index in the low normal range, oral oestrogens, young age and low C-peptide were significantly associated with low PAI-1 activity and there was a trend for association with IL-6 in univariable analysis. The effect of age disappeared after correction for oral oestrogens and the effect of C-peptide and IL-6 disappeared after further adjustments. Low BMI remained as the strongest predictor of low PAI-1 activity. CONCLUSION: Patients with bleeding tendency and low PAI-1 activity have inflammatory and insulin profiles similar to those with normal or high PAI-1, whereas BMI seems to be the most important determinant.

Low PAI-1 activity in relation to the risk for perioperative bleeding complications in transurethral resection of the prostate.

INTRODUCTION: Low levels of plasminogen activator inhibitor type 1 (PAI-1) have been associated with increased risk for perioperative bleeding in some case reports. The aim of this study was to investigate prospectively whether low PAI-1 activity increases the risk for perioperative bleeding in patients undergoing transurethral resection of prostate, an organ with high fibrinolytic activity. PATIENTS AND METHODS: 62 patients with benign prostatic hyperplasia planned for transurethral resection were included. Blood samples for PAI-1 were taken together with other routine preoperative blood samples 1week before surgery but analyzed after the hospitalization. The intraoperative blood loss was determined by measuring the amount of hemoglobin in the irrigating fluid. The postoperative blood loss was estimated from calculations of hemoglobin mass (Hb mass), which is a product of hemoglobin concentration and blood volume. Hb mass was calculated before surgery and on the day of discharge, and was adjusted for intraoperative blood loss and transfused Hb mass. Bleeding complications were defined as re-operation due to bleeding, more than 40ml intraoperative bleeding/g resected prostatic tissue or postoperative blood loss corresponding to more than 100g of hemoglobin. RESULTS: Bleeding complications were observed in 3 of 4 (75%) patients with low PAI-1 levels, defined as <1U/ml, and in 16 of 58 (28%) patients with PAI-1 levels >1U/ml (P=0.082). After adjustment for resection time, resected prostatic mass and systolic blood pressure this became borderline significant (odds ratio 11.8; 95% confidence interval 1.00-139; P=0.05). CONCLUSION: Low PAI-1 activity may contribute to the risk of bleeding after transurethral resection of the prostate.

Evaluation of low PAI-1 activity as a risk factor for hemorrhagic diathesis.

BACKGROUND: Prospective studies of the epidemiology and clinical significance of low plasminogen activator inhibitor type 1 (PAI-1) activity are lacking. OBJECTIVE: To evaluate the prevalence of low PAI-1 activity in patients with a bleeding tendency in comparison with a normal population. METHODS: In 586 consecutive patients, referred because of bleeding symptoms, we added analyses of PAI-1 activity and tissue plasminogen activator complex with PAI-1 (t-PA-PAI-1) to the routine investigation, consisting of platelet count, bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen, factor VIII, von Willebrand factor activity, and antigen. Controls were 100 blood donors and 100 age- and sex-matched healthy individuals. The latter were also evaluated regarding the previous bleeding episodes. The bleeding history was classified as clinically significant or not, and the criteria were fulfilled in 75% of the patients and 18% of the healthy controls. RESULTS: The routine laboratory investigation of the patients was negative in 57%. Low PAI-1 activity, defined as <1.0 U mL(-1), was found in 23% of the patients and in 13% and 10% of the blood donors and healthy controls, respectively (odds ratio and 95% CI, 2.04; 1.11-3.77 and 2.75; 1.39-5.42, respectively). The difference remained statistically significant after the adjustment for body mass index, use of estrogens, sex and age (odds ratio for patients vs. healthy controls 3.23; 95% CI, 1.22-8.56, P = 0.019). The distribution of the 4G/5G genotypes in the patients was not different from that of two control populations. No specific symptom predicted for low PAI-1, which did not aggravate the clinical picture in association with the other hemostatic defects. Low tPA-PAI-1 was not associated with the increased bleeding tendency. CONCLUSION: Low PAI-1 activity is common in patients with a bleeding diathesis, but it is a risk factor of minor clinical importance and not associated with specific bleeding manifestations.

Association of TNF-alpha serum levels and TNFA promoter polymorphisms with risk of myocardial infarction.

Elevated levels of tumor necrosis factor-alpha (TNF-alpha), and presence of polymorphisms of the TNFA gene have been implicated in cardiovascular disease pathogenesis. We explored the relationship between polymorphisms in the TNFA gene (-1031C/T, -863C/A -857T/C, -308G/A, -238G/A), protein levels of TNF-alpha and their association to myocardial infarction (MI) using a sample of 1213 post-MI patients and 1561 healthy controls. MI risk was higher among men with elevated TNF-alpha levels, with the highest compared to the lowest TNF-alpha quartile giving a 70% risk increase (OR [95% CI]: 1.7 [1.1; 2.6]). Obese subjects who also had elevated TNF-alpha levels were at even higher risk for MI (OR [95% CI]: 3.4 [2.1; 5.6]). Higher TNF-alpha levels were seen among smokers (but not among non-smokers) carrying the -857T allele. Furthermore, a rare haplotype occurred more frequently among the cases than the controls. Elevated TNF-alpha levels are associated with increased MI risk. Obese subjects with elevated TNF-a levels, and carriers of polymorphisms in or near TNFA are particularly susceptible to the hazards of smoking, results which may have implications for cardiovascular preventive measures.

Identification of a PAI-1 binding site in vitronectin.

Active PAI-1 (plasminogen activator inhibitor 1) is bound to vitronectin in plasma and in the extracellular matrix. In this study we aimed at identifying the PAI-1 binding site in vitronectin, which at present is a matter of dispute. Vitronectin was cleaved with trypsin and the fragments were tested for inhibitory effect on the PAI-1/vitronectin interaction using vitronectin-coated microtiter plates. Intact vitronectin and the tryptic digest of vitronectin both caused a 50% reduction in PAI-1 binding at a concentration of about 2 nmol/I. Gel-filtration on Sephadex G-50 superfine of the tryptic peptides resulted in one main peak of inhibitory activity. The elution volume, Kav, was 0.55 indicating (a) medium-size peptide(s). The peptide was further purified by reverse-phase HPLC. Structural analysis revealed that it constituted the 45 NH2-terminal amino-acid residues in vitronectin. The NH2-terminal vitronectin peptide caused a 50% decrease in PAI-1 binding to the vitronectin-coated microtiter plates at a concentration of about 13 nmol/l. Thus, the peptide is a little less effective in this respect than intact vitronectin. Reduced and S-carboxymethylated peptide had no effect on the interaction. The NH2-terminal vitronectin fragment increased the stability of active PAI-1 by about 60%, which is a little less than with intact vitronectin. The peptide also prevented PAI-1 from oxidation with chloramine T. The half-life was prolonged about 4-fold as compared to about 30-fold with intact vitronectin.

Purification of high and low molecular weight plasminogen activator inhibitor 1 from fibrosarcoma cell-line HT 1080 conditioned medium.

Functionally active (high-Mr) and inactive (low-Mr) plasminogen activator inhibitor 1 (PAI) have been purified from fibrosarcoma cell-line HT 1080 conditioned medium, containing 1% fetal calf serum. The two forms were first purified by affinity chromatography on heparin-Sepharose and then separated from each other by gel filtration on Sephadex G-150. The final purification was achieved by affinity chromatography on insolubilized monoclonal antibodies towards human PAI. Alternatively, the low-Mr form was purified by chromatography on carboxymethyl-cellulose. Low-Mr PAI purified in this way, could be almost fully reactivated by treatment with guanidinium chloride. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by immunoblotting revealed that the low-Mr form contained nothing but PAI at an Mr of about 50,000. In addition to PAI, the high-Mr form contained a component, which was not antigenically related to PAI. This compound had a molecular weight of about 75,000 and its NH2-terminal amino acid sequence corresponded to that of human vitronectin. We conclude that the high-Mr form of PAI constitutes a complex between 50,000 Mr PAI and vitronectin from fetal calf serum.

Complex formation between plasminogen activator inhibitor 1 and vitronectin in purified systems and in plasma.

Functionally active PAI-1 is bound to a discrete binding or carrier protein in plasma, which was recently identified as vitronectin. In the present study, the interaction between PAI-1 and vitronectin has been studied in purified systems and in plasma by agarose gel electrophoresis using non-denaturing conditions and by crossed immunoelectrophoresis using an antiserum produced towards purified PAI-1/vitronectin complex. Both methods revealed a clearly distinguishable complex with electrophoretic mobility in between the parent molecules. Virtually all of the purified vitronectin, which did not contain any appreciable amounts of polymerized material, and almost all of the vitronectin in plasma, had the capacity to form a complex with PAI-1. The results suggested a stoichiometry of 1:1 as the most likely ratio between the two molecules in the complex. In contrast to functionally active PAI-1, latent or chloramine T-inactivated PAI-1 did not form such a complex with vitronectin.

Purification and characterization of human C1-esterase inhibitor.

A new purification method for C1-esterase inhibitor is described, which is essentially a three-step procedure: precipitation with poly(ethylene glycol), chromatography on DEAE-cellulose and hydrophobic interaction chromatography on hexyl-Sepharose. The final product is a single-chain glycoprotein with a molecular weight of about 100 000 and NH2-terminal asparagine. The molecule is fully active as judged by complex formation with C1s. Two of its three disulphide bridges can be easily reduced and S-carboxymethylated under non-denaturing conditions without loss of activity. However, at high dithioerythritol concentration the third disulphide bridge is also cleaved and accompanied by loss of the activity, indicating that this disulphide bridge is involved in maintaining the conformation around the reactive site in the inhibitor.

Circular dichroism studies on alpha 2-antiplasmin and its interactions with plasmin and plasminogen.

Spectropolarimetric studies of alpha 2-antiplasmin in the far ultraviolet region indicates a content of 16% alpha-helix, 18% beta-structure and 66% random coil. Two of its three disulphide bridges are reduced under non-denaturing conditions without major changes in conformation of functionality. Cleavage of the third disulphide bridge requires denaturing agents and is accompanied by complete loss of activity. The interaction of alpha 2-antiplasmin with plasminogen or fragments derived from plasminogen by elastase digestion has been studied by circular dichroism analysis in the near ultravoilet region. The results indicate that the fragment of plasminogen constituting the three NH2-terminal triple-loop structures contains at least two lysine-binding sites: one with high affinity and one with low affinity. One of these sites, probably the high-affinity site, is involved in the interaction with alpha 2-antiplasmin. This site seems also to be exposed in the intact plasminogen molecule. The formation of the stable complex between plasmin (EC 3.4.21.7) and alpha 2-antiplasmin is also accompanied by conformational changes.

On the specific interaction between the lysine-binding sites in plasmin and complementary sites in alpha2-antiplasmin and in fibrinogen.

Plasminogen and plasminogen derivatives which contain lysine-binding sites were found to decrease the reaction rate between plasmin and alpha2-antiplasmin by competing with plasmin for the complementary site(s) in alpha2-antiplasmin. The dissocwation constant Kd for the interaction between intact plasminogen (Glu-plasminogen) and alpha2-antiplasmin is 4.0 microM but those for Lys-plasminogen or TLCK-plasmin are about 10-fold lower indicating a stronger interaction. The lysine-binding site(s) which is situated in triple-loops 1--3 in the plasmin A-chain is mainly responsible for the interaction with alpha2-antiplasmin. The interaction between Glu-plasminogen and alpha2-antiplasmin furthermore enhances the activation of Glu-plasminogen by urokinase to a comparable extent as 6-aminohexanoic acid, suggesting that similar conformational changes occur in the proenzyme after complex formation. Fibrinogen, fibrinogen digested with plasmin, purified fragment E and purified fragment D interfere with the reaction between plasmin and alpha2-antiplasmin by competing with alpha2-antiplasmin for the lysine-binding site(s) in the plasmin A-chain. The Kd obtained for these interactions varied between 0.2 microM and 1.4 microM; fragment E being the most effective. Thus the fibrinogen molecule contains several complementary sites to the lysine-binding sites located both in its NH2-terminal and COOH-terminal regions; these sites are to a large extent.

The role of His(143) for the pH-dependent stability of plasminogen activator inhibitor-1.

Using site-directed mutagenesis, His(143) on the alpha-helix F of PAI-1 was substituted by Lys, Asp, Phe and Thr, respectively. The generated single-site changed plasminogen activator inhibitor-1 (PAI-1) mutants were expressed in Escherichia coli and purified by heparin-Sepharose and anhydrotrypsin agarose chromatographies. When compared with wild-type (wtPAI-1), the PAI-1 mutants His143Asp and His143Phe had shorter half-lives at pH 7.5 (1.1 and 1.4 h, respectively, vs. 2 h for wtPAI-1). They also exhibited less pH dependency of their stability, with half-lives at pH 5.5 of 2.5 and 2.2 h, respectively, vs. 18 h for wtPAI-1. However, the PAI-1 mutants His143Lys and His143Thr had similar properties as wtPAI-1 in this respect. In conclusion, our results suggest that His(143) in one way or another might be involved in the pH-dependent stability of PAI-1. However, it seems that the protonation of this particular residue is of less importance. The PAI-1 mutants His143Asp and His143Phe only displayed about 20% of the specific activity obtained for wtPAI-1, because they, to a large extent, act as substrates for tissue-type plasminogen activator.

Stability of plasminogen activator inhibitor-1: role of tyrosine221.

Using site-directed mutagenesis, changes of Tyr221 in plasminogen activator inhibitor-1 (PAI-1) have provided mutants with normal activity, but with increased stability. At physiological conditions, the transition of the PAI-1 mutants Tyr221His and Tyr221Ser to the latent form was significantly prolonged (half-lives 14.8 and 4.1 h, respectively) as compared to wild-type PAI-1 (2.0 h). Their half-lives, especially for the Tyr221Ser mutant, were even more prolonged in the presence of vitronectin (23.8 and 53.7 h, respectively). While wild-type PAI-1 was more stable at lower pH, the PAI-1 mutants Tyr221His and Tyr221Ser had stability optima at about pH 6.5, but displayed shorter half-lives at pH 5.5.

Plasminogen activator inhibitor 1 (PAI) is bound to vitronectin in plasma.

Functionally active plasminogen activator inhibitor 1 (PAI) is bound to a discrete binding protein in plasma [(1988) Thromb. Haemost. 59, 392-395]. The binding protein has now been partially purified using conventional chromatographic techniques. After addition of active PAI its complex with the binding protein was purified by chromatography on insolubilized monoclonal antibodies towards PAI. Dodecylsulphate (polyacrylamide gel electrophoresis revealed two main compounds with molecular masses of 50 and 75 kDa respectively. NH2-terminal amino acid sequence analysis and immunoblotting analysis suggested that the two compounds were PAI (50 kDa) and vitronectin (75 kDa). We conclude that the PAI-binding protein is identical to vitronectin.

PAI-1 stability: the role of histidine residues.

The role of the 13 histidine residues in plasminogen activator inhibitor 1 (PAI-1) for the stability of the molecule was studied by replacing these residues by threonine, using site-directed mutagenesis. The generated mutants were expressed in Escherichia coli, purified and characterized. All variants had a normal activity and formed stable complexes with tissue-type plasminogen activator. Most of these PAI-1 variants displayed a similar pH-dependency in stability as wild-type PAI-1, with increased half-lives at lower pH. However, the variant His364Thr had a half-life of about 50 min at 37 degrees C and had almost completely lost its pH-dependency. Therefore, our data suggest that His(364), in the COOH-terminal end of the molecule might be responsible for the pH-dependent stability of PAI-1.

Increased expression of alpha-enolase in c-jun transformed rat fibroblasts without increased activation of plasminogen.

Two-dimensional gel electrophoresis was used to identify polypeptides differentially expressed between normal and c-jun transformed rat fibroblasts. The level of a 49 kDa polypeptide was 3-fold elevated in c-jun transformed cells. Sequence analysis by ion trap mass spectrometry identified the polypeptide as rat alpha-enolase. Enolase functions as a cell surface receptor for plasminogen, suggesting that upregulation may increase plasminogen activation and cell surface proteolysis important for tumor growth. However, no difference was observed between normal and transformed cells in formation of plasmin, suggesting that upregulation of alpha-enolase may contribute to an increased metabolic capacity, but not to increased plasminogen activation.

ERK signalling in metastatic human MDA-MB-231 breast carcinoma cells is adapted to obtain high urokinase expression and rapid cell proliferation.

Increased urokinase plasminogen activator (u-PA) production is associated with tumor invasion and metastasis in several malignancies, including breast cancer. The mechanisms underlying constitutive u-PA expression are not well understood. We examined the relationship between the signal strength of the ERK pathway and the level of u-PA expression in the metastatic human breast cancer cell line MDA-MB-231. Treatment with the MEK1 inhibitor PD98059 resulted in decreased ERK1/2 phosphorylation and decreased u-PA mRNA and protein expression. Inhibition of ERK1/2 activity also led to decreased cell proliferation and to decreased cyclin D1 expression. Less than 5% of total ERK1/2 was phosphorylated in exponentially growing MDA-MB-231 cells, and ERK1/2 activity could be stimulated by okadaic acid. Okadaic acid did not stimulate u-PA expression, but induced strong expression of the cdk-inhibitor p21Cip1. These findings suggest that ERK1/2 signaling is tuned to a level which results in high u-PA expression and rapid cell proliferation.

Inflammatory and hemostatic markers in relation to cardiovascular prognosis in patients with stable angina pectoris. Results from the APSIS study. The Angina Prognosis Study in Stockholm.

Increased inflammatory activity and platelet activation have been associated with an increased risk of cardiovascular (CV) events in epidemiological studies, but their prognostic importance in patients with stable angina pectoris is less well established. The Angina Prognosis Study in Stockholm (APSIS), comprised 809 patients (2766 patient years) with stable angina pectoris on double-blind treatment with verapamil or metoprolol. Plasma levels of fibrinogen and orosomucoid (an acute phase reactant), white blood cell counts (WBC), platelet counts and the urinary excretion of beta-thromboglobulin (reflecting platelet secretion), were related to the risk of CV death (n=36), non-fatal myocardial infarction (MI) (n=30) or revascularization (n=99) in a subgroup of 782 patients. Verapamil and metoprolol had only minor effects on the inflammatory variables. In multivariate Cox regression analyses (adjusted for previous MI, hypertension, diabetes mellitus and smoking), fibrinogen and WBC were independent predictors of CV death or non-fatal MI, as well as the risk of revascularization. Orosomucoid did not carry any independent information. Platelet counts and urinary beta-thromboglobulin were not significantly related to CV prognosis. The treatment given did not significantly influence the prognostic impact of either fibrinogen or WBC. Fibrinogen and WBC were independent predictors of CV death or non-fatal MI as well as disease progression leading to revascularization in patients with stable angina pectoris. As fibrinogen is also an acute-phase reactant, the present findings indicate that inflammatory activity is involved in disease progression in stable angina pectoris.

Plasminogen activator inhibitor 1 (PAI-1) in plasma: its role in thrombotic disease.

Impaired fibrinolytic function, mainly due to an elevation of the plasma PAI-1 concentration, is a common finding in patients with thrombotic disease. Unfortunately, regarding patients with idiopathic deep vein thrombosis (DVT), no reliable prospective or genetic studies have been published. Concerning postoperative DVT, preoperatively increased PAI-1 level seems to predict a postoperative DVT in patients subjected to hip surgery. Several longitudinal cohort studies of patients with manifest coronary heart disease (CHD) have linked elevated plasma PAI-1 or tPA antigen concentrations to future cardiovascular events, particularly myocardial infarction. Before PAI-1 can be regarded as a risk factor in the conventional epidemiological sense, its relationship to myocardial infarction must be demonstrated in prospective studies of healthy populations. Regulation of the plasma concentration of PAI-1 is complex and at present not well understood. Multiple interactions with disturbances of both carbohydrate and lipoprotein metabolism are evident. Many studies have been carried out in cultured cells, but data can hardly be transformed into human pathophysiology. It seems that both environmental and genetic factors are of importance for the plasma PAI-1 concentration. Recently, knowledge of the importance of genetic factors involved in the regulation of plasma PAI-1 concentration has become available. Interestingly, preliminary data suggest that the 4G/5G polymorphism located within the PAI-1 promoter is connected to CHD. More data on larger patient groups are needed and will certainly shed new light on the importance of impaired fibrinolytic function in the etiology of CHD in the near future.

Determination of soluble fibrin in plasma by a rapid and quantitative spectrophotometric assay.

A rapid, sensitive and quantitative spectrophotometric assay of soluble fibrin in plasma samples has been developed. The method is based on the principle that fibrin stimulates the activation of plasminogen by tissue plasminogen activator (t-PA). A sample containing fibrin is incubated with t-PA, plasminogen and a plasmin-sensitive chromogenic substrate. An increase in absorbance which is dependent on the fibrin concentration in the sample is obtained. In plasma samples an initial lag-phase, due to the presence of alpha 2-antiplasmin is observed. However, the change in A405 per square minute during the later part of the reaction was found to be proportional to the fibrin content and to generate linear standard curve intercepts at the origin. The detection limit of bathroxobin-digested fibrinogen added to plasma was about 5 nmol/L. Recovery experiments at 20 and 75 nmol/L levels in plasma samples from healthy individuals were excellent. The "within-run" variation (CV) was determined as 3.7%. The formation of fibrin after addition of minute amounts of thrombin to plasma could be monitored with the method. Plasma from 8 healthy individuals was found to contain about 9.2 +/- 1.9 nmol/L fibrin. Plasma samples from 46 patients with a suspected haemostatic disturbance had higher levels of soluble fibrin (53 +/- 62 nmol/L). Seven of the 46 samples had concentrations above 150 nmol/L.

The significance of hypofibrinolysis for the risk of recurrence of venous thromboembolism. Duration of Anticoagulation (DURAC) Trial Study Group.

An impaired fibrinolytic function has been described in several case-control studies of patients with venous thromboembolism (VTE). In the present study the correlations between some fibrinolytic compounds and future recurrent VTE were investigated. Blood samples for analysis of tissue-type plasminogen activator (t-PA) antigen before and after 10 min of venous occlusion (V.O.) and plasminogen activator inhibitor type 1 (PAI-1) activity were taken at 6 months after the first episode of VTE or the first recurrent VTE in 784 and 207 patients, respectively, who were anticoagulated for 1.5 or 6 months (first VTE) and 6 months or indefinitely (first recurrence). During a follow-up of 3-6 years from the event which qualified for inclusion there have been 177 recurrences. All initial and recurrent events were verified with objective diagnostic methods. Using cut off points of 10.0 ng/ml for t-PA antigen before V.O. and 30 AU/ml for PAI-1 in samples taken at rest, there were more patients above those levels in the groups with than without further recurrence (t-PA antigen, 50% versus 36%, p = 0.001; PAI-1, 18% versus 12%, p = 0.045). In the 495 patients, who received oral anticoagulation for 6 months, t-PA antigen at rest discriminated better, with 59% versus 34% of patients above 10 ng/ml in the groups with and without recurrence, respectively (p < 0.001). The t-PA antigen levels after V.O. and the fibrinolytic capacity (t-PA antigen after V.O. minus t-PA antigen before V.O.) were distributed similarly in patients with and without new recurrences. There was a statistically significant positive correlation between age and t-PA antigen (p < 0.001), and by analysis of covariance the difference between the groups with and without further recurrence regarding t-PA antigen disappeared. In conclusion, increased levels of PAI-1 and t-PA antigen in VTE-patients correlate with development of recurrent VTE within the next 3-6 years, but the value of these components in predicting future events for the individual patients is limited.