Increase In PAI-1 Research Articles

Ramipril Prior to Thrombolysis Attenuates the Early Increase of PAI-1 in Patients with Acute Myocardial Infarction

In a placebo-controlled, double-blinded, randomized study we evaluated the effect of ramipril prior to thrombolysis on the course of PAI-1 plasma levels and on the frequency of postinfarct ischemic events in patients with acute myocardial infarction. Fifty-one out of 99 patients received 2.5 mg ramipril orally prior to thrombolysis and 12 h later. The blood samples for determination of PAI-1 plasma levels were collected on admission and 2, 4, 8, 12 and 24 h after thrombolysis. Postinfarct ischemic events were registered until coronary angiography was performed and defined as recurrent chest pain and/or evidence of ischemic signs on the ECG (ST-depression or ST-segment elevation of 1 mm in one or more inferior or anterior leads). Coronary angiography was performed within 7 days after the onset of myocardial infarction. Patients were classified into two groups: those without reperfusion of the infarct-related artery (TIMI grade 0 or 1) and those with reperfusion of the infarct-related artery (TIMI grade 2.3). On admission, PAI-1 plasma levels were similar in both groups (ramipril: 47.1 [4.8] ng/ml; placebo: 49.1 [4.8] ng/ml). The PAI-1AUC was 77.2 [6.7] ng/ml/h in the ramipril group and 95.4 [6.2] ng/ml/h in the placebo group (p = 0.013). Significant differences between groups were observed at 4, 8 and 12 h after thrombolysis (4 h: 85.5 (11.3) vs. 116 (12.3) ng/ml, p < 0.01; 8 h: 79.1 (11.2) vs. 100.9 (9.3) ng/ml, p < 0.01; 12 hrs: 71.3 (9.5) vs. 87.4 (7.7) ng/ml, p < 0.05). The relative frequency of postinfarct ischemic events was significantly lower in the ramipril group (2.5% versus 7.1%, p = 0.001). Additionally, we observed a significant higher rate of TIMI grade 2 and 3 of the infarct-related artery in patients receiving oral ramipril compared to the placebo group (73% versus 54%; p = 0.035). Our study demonstrates a favorable effect of ramipril on the fibrinolytic system after thrombolysis associated with a lower rate of postinfarct ischemic events within the first days after myocardial infarction. Therefore, the application of ramipril prior to thrombolysis appears to be a reasonable concomitant treatment which may reduce early infarct-related complications.

Transcriptional regulation of plasminogen activator inhibitor type 1 gene by insulin: insights into the signaling pathway.

Impairment of the fibrinolytic system, caused primarily by increases in the plasma levels of plasminogen activator inhibitor (PAI) type 1, are frequently found in diabetes and the insulin-resistance syndrome. Among the factors responsible for the increases of PAI-1, insulin has recently attracted attention. In this study, we analyzed the effects of insulin on PAI-1 biosynthesis in HepG2 cells, paying particular attention to the signaling network evoked by this hormone. Experiments performed in CHO cells overexpressing the insulin receptor indicate that insulin increases PAI-1 gene transcription through interaction with its receptor. By using inhibitors of the different signaling pathways evoked by insulin-receptor binding, it has been shown that the biosynthesis of PAI-1 is due to phosphatidylinositol (PI) 3-kinase activation, followed by protein kinase C and ultimately by mitogen-activated protein (MAP) kinase activation and extracellular signal-regulated kinase 2 phosphorylation. We also showed that this pathway is Ras-independent. Transfection of HepG2 cells with several truncations of the PAI-1 promoter coupled to a CAT gene allowed us to recognize two major response elements located in the regions between -804 and -708 and between -211 and -54. Electrophoretic mobility shift assay identified three binding sites for insulin-induced factors, all colocalized with putative Sp1 binding sites. Using supershifting antibodies, the binding of Sp1 could only be confirmed at the binding site located just upstream from the transcription start site of the PAI-1 promoter. A construct comprising four tandem repeat copies of the -93/-62 region of the PAI-1 promoter linked to CAT was transcriptionally activated in HepG2 cells by insulin. These results outline the central role of MAP kinase activation in the regulation of PAI-1 induced by insulin.

Activation of coagulation and fibrinolysis during dengue virus infection.

Dengue virus infection can induce mild dengue fever (DF) or severe dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) in human. The pathogenesis of hemorrhage in dengue virus infection is not fully understood. Since hemostasis depends on the balance between coagulation and fibrinolysis, alternation of some coagulation parameters (platelet count and activated partial thromoboplastin time, APTT) as well as fibrinolytic parameters (tissue plasminogen activator, tPA and plasminogen activator inhibitor-1, PAI-1) were compared in 8 DHF/DSS and 17 DF patients. Patients showed thrombocytopenia, APTT prolongation, and tPA increase in the acute stage of disease, indicating activation of coagulation and fibrinolysis. The activation of coagulation and fibrinolysis in DHF/DSS patients was much more severe than DF patients. In the convalescent stage, a rise of PAI-1 level and platelet count with concomitant decline of tPA level and APTT returned to normal in both DHF/DSS and DF patients. Therefore, the activation of coagulation and fibrinolysis during the acute stage of dengue virus infection is offset by the increase of platelet and PAI-1 during convalescent stage. Taken together, these results suggest that the degree of coagulation and fibrinolysis activation induced by dengue virus infection is associated with the disease severity.

Induction of the Plasminogen Activator Inhibitor-1 Gene Expression by Mild Hypoxia Via a Hypoxia Response Element Binding the Hypoxia-Inducible Factor-1 in Rat Hepatocytes

AbstractPlasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of both tissue-type and urokinase-type plasminogen activators. The balance between plasminogen activators and PAI-1 plays an important role in several physiological and pathophysiological processes such as atherosclerosis or thrombosis. Because these conditions are associated with hypoxia, it was the aim of the present study to investigate the influence of low O2tension on the expression of PAI-1 mRNA and protein using primary cultured rat hepatocytes as a model system. We found that PAI-1 mRNA and protein were induced by mild hypoxia (8% O2). The hypoxia-dependent PAI-1 mRNA induction was transcriptionally regulated because it was inhibited by actinomycin D (ActD). Luciferase (LUC) reporter gene constructs driven by about 800 bp of the 5′-flanking region of the rat PAI-1 gene were transiently transfected into primary rat hepatocytes; mild hypoxia caused a 3-fold induction, which was mediated by the PAI-1 promoter region -175/-158 containing 2 putative hypoxia response elements (HRE) binding the hypoxia-inducible factor (HIF-1). Mutation of the HRE-1 (-175/-168) or HRE-2 (-165/-158) also abolished the induction by mild hypoxia. Cotransfection of a HIF-1α vector and the PAI-1–LUC constructs, as well as gel shift assays, showed that the HRE-2 of the PAI-1 promoter was most critical for induction by hypoxia and HIF-1 binding. Thus, PAI-1 induction by mild hypoxia via a HIF-1 binding HRE in the rat PAI-1 promoter appears to be the mechanism causing the increase in PAI-1 in many clinical conditions associated with O2deficiency.

The course of fibrinolytic proteins in children with malignant bone tumours

To evaluate the role of fibrinolytic and proteolytic proteins in children and adolescents suffering from Ewing sarcoma or osteosarcoma with respect to postoperative complications and late outcome, a prospective two-arm two-centre study was conducted. Plasminogen, plasminogen activator inhibitor (PAI)-1, tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) were investigated in the pre-surgical period and in the postoperative follow-up period in children suffering from Ewing sarcoma (ES; n = 36) or osteosarcoma (OS; n = 39). In addition, the factor V mutation (FV) Q506, protein C, protein S, antithrombin and lipoprotein (a) were determined. All children received LMWH (EnoxaparinR) 1 mg/kg s.c. once daily over a period of 6 weeks to 3 months. Besides a short-lasting increase of PAI-1 in patients with OS on day 1 and in children with Es on day 14, a small and significant but clinically irrelevant difference was found on days 7-10 for plasminogen, t-PA and u-PA. No thromboembolic complications occurred in patients treated with LMWH and having a prothrombotic genetic risk factor. Within one year of surgery 7 out of 36 patients with ES and 5 out of 39 children with OS showed a relapse of their disease. Prior to the first local tumour therapy, 5 out of 7 children with ES and relapse had elevated u-PA concentrations compared with 2 out of 5 children in the OS group. No such differences were found for PAI-1- or t-PA antigen. The role of u-PA as a possible follow-up marker for a poorer outcome in children with ES should be evaluated in a prospective multicentre study.

Relation between long-term steroid treatment after heart transplantation, hypofibrinolysis and myocardial microthrombi generation

Background Thrombotic complications and transplant coronary artery disease are among the main causes of morbidity and mortality after heart transplantation. A thrombophilic state has been described in transplant recipients, and correlated to immunosuppressive therapy with Cyclosporine A or Azathioprine, whereas the prothrombotic effects of steroids, even though always given, have never been duly considered. A reduced fibrinolytic capacity due to high levels of PAI-1, the most important inhibitor of plasminogen activators, was suggested to play a role in the development of cardiovascular diseases and transplant coronary artery disease. A severe hypofibrinolytic state secondary to PAI-1 increase has been found in patients with Cushing’s disease, and in hypercorticism secondary to long-term steroid treatment after renal transplantation. Methods We evaluated plasma clotting and fibrinolytic behaviors in 2 groups of heart transplant patients treated with (26 cases) or without (23 cases) steroids together with Cyclosporine A and Azathioprine. Twenty-five healthy subjects were studied as normal controls. The following tests were assayed at least 1 year after transplantation: fibrinogen, factor VIII coagulant activity, von Willebrand factor antigen, Euglobulin Lysis Time, tissue plasminogen activator antigen and activity, PAI-1 antigen and activity. In addition, the presence of cardiac microthrombi was evaluated on 2 endomyocardial biopsy specimens obtained in each patient both on Day 7 after heart transplantation (first control) and usually 1 year or more later (last control). Results Plasma levels of fibrinogen, factor VIII and von Willebrand factor were significantly higher in both groups of patients than in normal controls. Fibrinolytic activity was significantly reduced in transplant patients treated with steroids, compared with steroid-free patients and normal controls. In steroid-treated heart transplant recipients, the hypofibrinolytic state was due to a significant and pathological increase in PAI-1 antigen and activity levels. The fibrinolytic impairment was more evident in patients transplanted for ischemic heart disease and treated with steroids than in patients with previous dilated cardiomyopathy and treated either with or without steroids. Myocardial microthrombi were found in 2/49 cases at the first biopsy control, and in 12/49 cases at the last biopsy control after transplantation. This different prevalence was statistically significant (χ 2 = 8.33, p = .003). Plasma PAI-1 activity was significantly higher and, as a consequence, Euglobulin Lysis Time was more prolonged in microthrombi-positive patients than in microthrombi-negative ones. Among the 12 transplant recipients who developed cardiac microthrombi, 7 patients were treated with steroids and showed higher PAI-1 levels and more reduced fibrinolytic activity than the 5 steroid-free patients. Conclusions Our data confirm the prothrombotic state induced by long-term steroid treatment, characterized by an increase in PAI-1 levels and secondary impairment of fibrinolytic capacity. In heart transplant patients, steroid-related hypofibrinolysis might constitute a further risk factor for transplant coronary artery disease.

Perioperative fibronolytic activity in cholecystectomized patients The postoperative fibrinolytic shutdown is only a temporary event

Objective: to estimate the influence of successive steps of abdominal surgery and of postoperative course on the components of fibrinolytic system. Design: a prospective open study. Setting: the patients were treated in medical research institute and the laboratory tests were done in postgraduate teaching hospital. Interventions: open cholecystectomy, performed in general anesthesia. Main outcome measures: the plasminogen concentration in plasma was determined by amidolytic method. The concentrations of t-PA antigen, PAI-1 antigen, and PAP complex antigen were measured by enzyme-linked immunosorbent assays [ELISA]. Results: the induction of anaesthesia was followed by the decline in plasminogen concentration and this decline was observed until the beginning of intraperitoneal part of operation. The decrease of PAI-1 concentration was encountered also at this period of time but was significant only during surgical opening of abdominal wall. Both variables increased then gradually till the end of investigations, i.e. 7th postoperative day. t-PA and PAP complex increased during the intraperitoneal part of cholecystectomy but their concentrations differed postoperatively. t-PA level declined but the concentration of PAP complex increased until the end of investigations, except of the slight, transient decline on the 1st postoperative day. Conclusion: the early drop of plasminogen concentration cannot be explained by its conversion to plasmin. It is hypothesized that the binding of this zymogen to proteins, endothelial cells, and to subendothelial matrix may be regarded as a cause of its early decline in plasma. We suppose that the surgical maneuvers inside the peritoneal cavity during cholecystectomy resulted in the release of t-PA from endothelial cells of systemic circulation and it is speculated that this release is mediated by cytokines. t-PA level decreased gradually after the end of operation, while PAP complex rose till the end of the 1st postoperative week after a slight, transient decrease on the 1st postoperative day. These latter findings can be explained by the local activation of fibrinolysis in the surgical wound: t-PA remains bound to fibrin inside this wound while plasmin, generated on the fibrin matrix, is released into the circulation as PAP complex. The gradual increase of PAI-1 together with an enhanced production of plasminogen, observed after cholecystectomy up to the 7th postoperative day, can be explained by a delayed systemic response to surgery.

Effect of the administration of recombinant hirudin and/or tissue‐plasminogen activator (t‐PA) on endotoxin‐induced disseminated intravascular coagulation model in rabbits

We evaluated the effect of r-hirudin and/or tissue-plasminogen activator (t-PA) in a model of DIC in rabbits induced by i.v. infusion of 100 micrograms/kg/h/6 h endotoxin. Rabbits were treated with saline (endotoxin control group), r-hirudin at 0.3 mg/kg/h/6 h, t-PA at 0.3 mg/kg for 90 min and r-hirudin plus t-PA at the doses described above. The best results were achieved when r-hirudin and t-PA were infused together. This treatment reduced the consumption of platelets and protein C and attenuated the increase of PAI-1 more efficiently than r-hirudin or t-PA alone. r-Hirudin plus t-PA also resulted in the lowest formation of fibrin deposits in the kidneys. Finally, mortality at 24 h dropped from 70% in the endotoxin control group to 40%, 10% and 0% in the t-PA, r-hirudin and r-hirudin plus t-PA groups respectively. None of the t-PA-infused rabbits which had died by 24 h showed macroscopic signs of haemorrhage. r-Hirudin alone was better than t-PA alone, as was shown by fibrin deposits and mortality. We conclude that r-hirudin and t-PA given simultaneously were more efficient than either given alone in this model of DIC. Effective thrombin inhibition, which could influence other pathophysiological mechanisms apart from coagulation, together with the improvement in fibrinolysis, would explain these results.

Effect of the administration of recombinant hirudin and/or tissue-plasminogen activator (t-PA) on endotoxin-induced disseminated intravascular coagulation model in rabbits

We evaluated the effect of r-hirudin and/or tissue-plasminogen activator (t-PA) in a model of DIC in rabbits induced by i.v. infusion of 100 μg/kg/h/6 h endotoxin. Rabbits were treated with saline (endotoxin control group), r-hirudin at 0.3 mg/kg/h/6 h, t-PA at 0.3 mg/kg for 90 min and r-hirudin plus t-PA at the doses described above. The best results were achieved when r-hirudin and t-PA were infused together. This treatment reduced the consumption of platelets and protein C and attenuated the increase of PAI-1 more efficiently than r-hirudin or t-PA alone. r-Hirudin plus t-PA also resulted in the lowest formation of fibrin deposits in the kidneys. Finally, mortality at 24 h dropped from 70% in the endotoxin control group to 40%, 10% and 0% in the t-PA, r-hirudin and r-hirudin plus t-PA groups respectively. None of the t-PA-infused rabbits which had died by 24 h showed macroscopic signs of haemorrhage. r-Hirudin alone was better than t-PA alone, as was shown by fibrin deposits and mortality. We conclude that r-hirudin and t-PA given simultaneously were more efficient than either given alone in this model of DIC. Effective thrombin inhibition, which could influence other pathophysiological mechanisms apart from coagulation, together with the improvement in fibrinolysis, would explain these results.

Severe heat stroke associated with high plasma levels of plasminogen activator inhibitor 1

In a 38-year-old man, severe heat stroke caused disseminated intravascular coagulation (DIC) associated with significantly elevated plasma plasminogen activator inhibitor 1 levels. Investigation of the effects of hyperthermia on coagulation and fibrinolysis showed, in apparent conflict with previous reports, a time lag between the initial hypercoagulable and hyperfibrinolytic response (within 24 hr) and hypofibrinolysis shown by a disproportionate increase of PAI-1 (after 24 h), which possibly occurs in correspondence with the recovery of vascular endothelial integrity. The patient was discharged without sequelae although computed tomography (CT) scans indicated the likelihood of venous infarction or posterior inferior cerebellar artery area infarction secondary to DIC.

Abnormal Expression of Type 1 Plasminogen Activator Inhibitor and Tissue Factor in Severe Preeclampsia

Preeclampsia is a multisystemic obstetric disease of unknown etiology that is commonly associated with fibrin deposition, occlusive lesions in placental vasculature, and intrauterine fetal growth retardation. We previously reported that type 1 plasminogen activator inhibitor (PAI-1) levels are significantly increased in plasma and placenta from pregnant women with preeclampsia compared to normal pregnant women. In the present report we localize the expression of placental PAI-1 in greater detail and compare it with that of tissue factor (TF), a procoagulant molecule, and vitronectin (Vn), a PAI-1 cofactor. We also examine the expression of two cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1), in order to begin to define the underlying mechanisms responsible for the elevated levels of PAI-1 and fibrin deposits observed in placenta from preeclampsia. We demonstrate a significant increase in PAI-1, TF and TNFalpha antigen and PAI-1 and TF mRNA in placentas from preeclamptic patients. PAI-1 mRNA was increased not only in syncytiotrophoblast and infarction areas, but also in fibroblasts and in some endothelial cells of fetal vessels in placentas from preeclamptic patients. However, there was no colocalization between PAI-1, TF, Vn and TNFalpha in placental villi. The elevated TNFalpha in the placenta may induce PAI-1 and TF, and thus promote the thrombotic alterations associated with preeclampsia.

Characterization and fibrinolytic properties of mesothelial cells isolated from peritoneal lavage

Human peritoneal mesothelial cells were harvested from patients undergoing open or laparoscopic surgery for non-septic conditions using three different approaches: (1) from a peritoneal biopsy, (2) from peritoneal fluid, and (3) from lavage fluid collected from peritoneal cavity. When these different methods were compared, cells derived from peritoneal fluid or lavage were more likely to result in established cultures than those obtained from biopsies. The cells displayed morphological, immunohistochemical and ultrastructural characteristics of mesothelial cells. The cultured mesothelial cells produced tissue type plasminogen activator (t-PA), urokinase plasminogen activator (uPA), and plasminogen activator inhibitor type-1 and type-2 (PAI-1 and PAI-2) during unstimulated conditions. Treatment with the proinflammatory mediators LPS and TNF-alpha resulted in an overall decreased fibrinolytic capacity with a decrease in the release of t-PA and an increase in plasminogen activator inhibitors PAI-1 and PAI-2. TNF-alpha had a more profound effect than LPS, especially on the release of t-PA. This may be an important mechanism by which inflammatory mediators disrupt the fibrin degradation. In conclusion, peritoneal lavage is a convenient and reproducible source of mesothelial cells for culture.

Attenuation of Thrombolysis-induced Increase of Plasminogen Activator Inhibitor-1 by Intravenous Enalaprilat

We examined the effect of intravenous enalaprilat on the course of PAI-1 plasma levels in 23 patients with acute myocardial infarction undergoing thrombolytic therapy. All patients received 100 mg aspirin, 1000 IU/h heparin, thrombolysis with 100 mg rt-PA within 90 min, and betablockers. Eleven out of 23 patients received 5 mg enalaprilat intravenously prior to thrombolysis. Blood samples for determination of PAI-1 plasma levels were collected on admission, 2, 4, 6, 12, and 24 h after thrombolysis. PAI-1 plasma levels in patients receiving enalaprilat were similar to those of the control patients before thrombolysis (5 ng/ml, 95% confidence interval: 2-10 vs. 7 ng/ml, 95% confidence interval: 2-10; p = 0.5). The PAI-1AUC was 9 ng/ml/h (95% confidence interval: 5-10) in the enalaprilat group and 19 ng/ml/h (95% confidence interval: 13-26) in the control group (p = 0.0006). The maximum difference was observed 6 h after thrombolysis (enalaprilat: 13 ng/ml, 95% confidence interval: 5-25, control: 42 ng/ml, 95% confidence interval: 18-55; p = 0.003). Our study clearly demonstrates that application of intravenous enalaprilat prior to thrombolysis attenuates the thrombolysis-related increase of PAI-1. This finding may suggest a possible therapeutic approach to influence the fibrinolytic system in patients with acute myocardial infarction after thrombolysis.

Comparative analysis of plasminogen activator inhibitor-1 expression in different types of atherosclerotic lesions in coronary arteries from human heart explants.

Clinical manifestations of coronary heart disease result primarily from the progressive development of atherosclerotic plaques and subsequent thrombus formation: processes which may be accelerated by an enhanced expression of plasminogen activator inhibitor (PAI-1) in the vessel wall. In the present study, content and expression of PAI-1 were comparatively analyzed in human coronary arteries in relation to the presence and severity of atherosclerotic lesions. Segments of coronary arteries obtained from heart explants (n = 15) were classified by the presence and types of atherosclerotic lesions. Antigen and activity levels of PAI-1 were determined in protein extracts of intimal and medial layers. In situ hybridization and immunohistochemical analyses were performed on serial sections of representative tissue specimens. Total PAI-1 antigen consistently increased from macroscopically normal areas (MNAs) to early lesions (ELs) and to maximal levels in fibrous (FPs) and calcified (CPs) plaques. No PAI activity was detected, although PAI-1 in its free form was present in all vascular specimens. Both free PAI-1 and PAI-1 complexed with plasminogen activators were significantly increased in extracts of advanced lesions. However, there was a 2-3 fold molar excess of free versus complexed PAI-1 in FPs and CPs. These findings suggest the presence of relevant amounts of PAI-1 in its substrate rather than in its inhibitor conformation in areas of advanced lesions. Compared with MNAs, PAI-1 mRNA was strongly expressed within the thickened intima of ELs. The highest PAI-1 expression was observed in FPs and CPs, being mainly localized in areas surrounding the necrotic cores in co-localization with infiltrating macrophages. PAI-1 content is consistently increased in relation to the severity of the lesions in atherosclerotic coronary arteries. The concomitant elevation of PAI-1 mRNA suggests that the PAI-1 increase in regulated by local synthesis in the areas of atherosclerotic lesions.

Plasminogen activator inhibitor type 1 and tissue inhibitor of metalloproteinases-2 increase after arterial injury in rats.

Vascular injury induced by angioplasty causes smooth muscle cells to migrate, proliferate, and form a neointima. The neointima is further enlarged by the accumulation of matrix molecules synthesized by smooth muscle cells. Smooth muscle cell migration and matrix accumulation are associated with an increase in the expression of matrix-degrading enzymes and might be regulated by the balance of protease and anti-protease activity. We have studied the inhibitors of two major classes of matrix-degrading enzymes, the plasminogen activators and the matrix metalloproteinases (MMPs) to understand better the regulation of proteolytic activity following balloon catheter injury in the rat carotid artery. At various times after injury, protease inhibitor expression was analyzed by Northern blotting, reverse zymography, immunohistochemistry, and Western blotting. During the first month after injury, we found that the expression of two proteinase inhibitors (plasminogen activator inhibitor type 1 [PAI-1] and tissue inhibitor of metalloproteinases-2 [TIMP-2]) was modulated. PAI-1 mRNA expression reached a maximum 6 hours after injury before tapering off to baseline levels by 3 days. PAI-1 activity, as measured by reverse zymography, followed the same temporal profile. PAI-1, localized by immunohistochemistry, was expressed at low levels in the media of control arteries and was increased after injury primarily in the medial smooth muscle cells. TIMP-2 mRNA levels began to increase 24 hours after injury and reached a maximum at day 7. TIMP-2 activity, measured by reverse zymography, peaked at day 3 after injury. TIMP-2 protein was increased in the intima compared with the media and adventitia at day 7 after injury. The increase of PAI-1 and TIMP-2 after injury supports the hypothesis that changes in the proteolytic balance play an important role in smooth muscle cell migration after arterial injury.

Effect of Strenuous Exercise on Fibrinogen and Fibrinolysis in Healthy Elderly Men and Women

The elevated incidence of thrombotic disease in elderly people may be associated with an increase in PAI-1 and fibrinogen with ageing. Cross-sectional studies report an inverse relation of PAI-1 and fibrinogen with physical activity, but training studies show inconsistent results. In a controlled intervention study among elderly subjects (aged 60-80 years) we observed a moderate decrease in PAI-1 antigen (4%, -2.1 +/- 2.4 ng/ml), a significant increase in t-PA activity (11%, 0.07 +/- 0.04 IU/ml) and an unexpected significant increase in fibrinogen (6%, 0.18 +/- 0.07 g/l) in subjects following a 6-month intensive training program as compared to controls. Reduction in PAI-1 antigen was significantly associated with a decrease in triglycerides (beta = 10.3 ng/ml per 1 mM, p <0.01) and insulin (beta = 2.37 ng/ml per 1 mU/l, p = 0.07). Increase in fibrinogen coincided with a rise in C-reactive protein (p <0.001). These data suggest that regular intensive activity may increase fibrinolytic activity in a moderate way, but also may cause chronically elevated plasma levels of acute phase proteins in elderly persons.

Clinical Impact of the Plasminogen Activation System in Tumor Invasion and Metastasis: Prognostic Relevance and Target for Therapy

Extravasation and intravasation of solid malignant tumors is controlled by attachment of tumor cells to components of the basement membrane and the extracellular matrix, by local proteolysis and tumor cell migration. Strong clinical and experimental evidence has accumulated that the tumor-associated serine protease plasmin, its activator uPA (urokinase-type plasminogen activator), the receptor uPA-R (CD87), and the inhibitors PAI-1 and PAI-2 are linked to cancer invasion and metastasis. In cancer, increase of uPA, uPA-R, and/or PAI-1 is associated with tumor progression and with shortened disease-free and/or overall survival in patients afflicted with malignant solid tumors. uPA and/or its inhibitor PAI-1 appear to be one of the strongest prognostic markers so far described. Strong prognostic value to predict disease recurrence and overall survival has been documented for patients with cancer of the breast, ovary, cervix, endometrium, stomach, colon, lung, bladder, kidney, brain, and soft-tissue. Due to the strong correlation between elevated uPA and/or PAI-1 values in primary cancer tissues and the tumor invasion/ metastasis capacity of cancer cells, proteolytic factors have been selected as targets for therapy. Various very different approaches to interfere with the expression or reactivity of uPA or CD87 at the gene or protein level were successfully tested including antisense oligonucleotides, antibodies, enzyme inhibitors, and recombinant or synthetic uPA and uPA-R analogues.

Restoration of Thrombolytic Potential in Plasminogen-Deficient Mice by Bolus Administration of Plasminogen

Homozygous plasminogen-deficient (Plg-/-) mice had a significantly reduced thrombolytic capacity toward intravenously injected 125I-fibrin labeled plasma clots prepared from Plg-/- murine plasma (9% +/- 3% lysis after 8 hours; (mean +/- SEM, n = 6), as compared with 82% +/- 8% in wild-type mice; P < .0001). Bolus injection of 1 mg purified murine plasminogen in 10- to 17-week-old Plg-/- mice increased the plasminogen antigen and activity levels at 8 hours to normal levels (130 +/- 5 micrograms/mL). Plasminogen administration was associated with significant restoration of thrombolytic potential (64% +/- 7% spontaneous clot lysis; P < .0001 versus lysis without plasminogen injection). Bolus injection of 1 mg plasminogen in homozygous tissue-type plasminogen activator-deficient (t-PA-/-) mice doubled the plasminogen antigen and activity levels after 8 hours and increased 125I-fibrin clot lysis at 8 hours from 13% +/- 3% to 34% +/- 5% (P = .008). Fibrinogen, t-PA antigen and alpha 2-antiplasmin activity levels after 8 hours were not significantly different in the groups with or without plasminogen injection. Injection of plasminogen induced a variable increase (on average 7- to 10-fold) of PAI-1, but no correlation with the extent of spontaneous clot lysis was observed. Histopathologic examination at the end of the experiments revealed that fibrin deposition in the liver of Plg-/- mice was slightly reduced 8 hours after bolus plasminogen injection (P = .007) and markedly reduced after 24 hours (P < .0001). Plasminogen antigen levels in liver extracts were comparable with those found in wild-type mice at 8 hours (130 +/- 20 versus 110 +/- 15 ng/mg protein) and decreased to 25 +/- 3.2 ng/mg protein at 24 hours. Thus, restoration of normal plasminogen levels in Plg-/- mice normalized the thrombolytic potential toward experimentally induced pulmonary emboli, and resulted in removal of endogenous fibrin deposits within 24 hours.

Restoration of thrombolytic potential in plasminogen-deficient mice by bolus administration of plasminogen

Homozygous plasminogen-deficient (Plg-/-) mice had a significantly reduced thrombolytic capacity toward intravenously injected 125I-fibrin labeled plasma clots prepared from Plg-/- murine plasma (9% +/- 3% lysis after 8 hours; (mean +/- SEM, n = 6), as compared with 82% +/- 8% in wild-type mice; P &lt; .0001). Bolus injection of 1 mg purified murine plasminogen in 10- to 17-week-old Plg-/- mice increased the plasminogen antigen and activity levels at 8 hours to normal levels (130 +/- 5 micrograms/mL). Plasminogen administration was associated with significant restoration of thrombolytic potential (64% +/- 7% spontaneous clot lysis; P &lt; .0001 versus lysis without plasminogen injection). Bolus injection of 1 mg plasminogen in homozygous tissue- type plasminogen activator-deficient (t-PA-/-) mice doubled the plasminogen antigen and activity levels after 8 hours and increased 125I-fibrin clot lysis at 8 hours from 13% +/- 3% to 34% +/- 5% (P = .008). Fibrinogen, t-PA antigen and alpha 2-antiplasmin activity levels after 8 hours were not significantly different in the groups with or without plasminogen injection. Injection of plasminogen induced a variable increase (on average 7- to 10-fold) of PAI-1, but no correlation with the extent of spontaneous clot lysis was observed. Histopathologic examination at the end of the experiments revealed that fibrin deposition in the liver of Plg-/- mice was slightly reduced 8 hours after bolus plasminogen injection (P = .007) and markedly reduced after 24 hours (P &lt; .0001). Plasminogen antigen levels in liver extracts were comparable with those found in wild-type mice at 8 hours (130 +/- 20 versus 110 +/- 15 ng/mg protein) and decreased to 25 +/- 3.2 ng/mg protein at 24 hours. Thus, restoration of normal plasminogen levels in Plg-/- mice normalized the thrombolytic potential toward experimentally induced pulmonary emboli, and resulted in removal of endogenous fibrin deposits within 24 hours.

3 Restoration of thrombolytic potential in plasminogen-deficient mice by bolus administration of plasminogen

Homozygous plasminogen-deficient (Plg-/-) mice had a significantly reduced thrombolytic capacity toward intravenously injected 125I-fibrin labeled plasma clots prepared from Plg-/- murine plasma (9% +/- 3% lysis after 8 hours; (mean +/- SEM, n = 6), as compared with 82% +/- 8% in wild-type mice; P &lt; .0001). Bolus injection of 1 mg purified murine plasminogen in 10- to 17-week-old Plg-/- mice increased the plasminogen antigen and activity levels at 8 hours to normal levels (130 +/- 5 micrograms/mL). Plasminogen administration was associated with significant restoration of thrombolytic potential (64% +/- 7% spontaneous clot lysis; P &lt; .0001 versus lysis without plasminogen injection). Bolus injection of 1 mg plasminogen in homozygous tissue- type plasminogen activator-deficient (t-PA-/-) mice doubled the plasminogen antigen and activity levels after 8 hours and increased 125I-fibrin clot lysis at 8 hours from 13% +/- 3% to 34% +/- 5% (P = .008). Fibrinogen, t-PA antigen and alpha 2-antiplasmin activity levels after 8 hours were not significantly different in the groups with or without plasminogen injection. Injection of plasminogen induced a variable increase (on average 7- to 10-fold) of PAI-1, but no correlation with the extent of spontaneous clot lysis was observed. Histopathologic examination at the end of the experiments revealed that fibrin deposition in the liver of Plg-/- mice was slightly reduced 8 hours after bolus plasminogen injection (P = .007) and markedly reduced after 24 hours (P &lt; .0001). Plasminogen antigen levels in liver extracts were comparable with those found in wild-type mice at 8 hours (130 +/- 20 versus 110 +/- 15 ng/mg protein) and decreased to 25 +/- 3.2 ng/mg protein at 24 hours. Thus, restoration of normal plasminogen levels in Plg-/- mice normalized the thrombolytic potential toward experimentally induced pulmonary emboli, and resulted in removal of endogenous fibrin deposits within 24 hours.