Decreased Plasminogen Activator Research Articles

A proatherogenic role for cGMP-dependent protein kinase in vascular smooth muscle cells.

Nitric oxide (NO) exerts both antiatherogenic and proatherogenic effects, but the cellular and molecular mechanisms that contribute to modulation of atherosclerosis by NO are not understood completely. The cGMP-dependent protein kinase I (cGKI) is a potential mediator of NO signaling in vascular smooth muscle cells (SMCs). Postnatal ablation of cGKI selectively in the SMCs of mice reduced atherosclerotic lesion area, demonstrating that smooth muscle cGKI promotes atherogenesis. Cell-fate mapping indicated that cGKI is involved in the development of SMC-derived plaque cells. Activation of endogenous cGKI in primary aortic SMCs resulted in cells with increased levels of proliferation; increased levels of vascular cell adhesion molecule-1, peroxisome proliferator-activated receptor gamma, and phosphatidylinositol 3-kinase/Akt signaling; and decreased plasminogen activator inhibitor 1 mRNA, which all are potentially proatherogenic properties. Taken together, these results highlight the pathophysiologic significance of vascular SMCs in atherogenesis and identify a key role for cGKI in the development of atherogenic SMCs in vitro and in vivo. We suggest that activation of smooth muscle cGKI contributes to the proatherogenic effect of NO and that inhibition of cGKI might be a therapeutic option for treating atherosclerosis in humans.

Decreased plasminogen activator inhibitor-1 secretion in hypoxic corneal epithelial cells is associated with increased urokinase plasminogen activator activity

The aim of this study was to determine the effects of hypoxia on mRNA levels, cell-associated and -secreted protein concentration, activity, and protein complex formation of urokinase-type plasminogen activator, its receptor, and plasminogen activator inhibitor type-1 in corneal epithelium. Non-transformed human corneal epithelial cells were cultured in 20% oxygen (normoxic conditions) or 2% oxygen (hypoxic conditions) for 1, 3, 5, or 7 days. Relative changes in mRNA levels of plasminogen activator, receptor, and plasminogen activator inhibitor-1 were determined using a cDNA expression array, chemiluminescence, and densitometry. Protein concentrations were determined using enzyme linked immunosorbent assays. Activity assays were also used. Protein complex formation was assayed using cell surface biotinylation, immunoprecipitation, and Western blot analysis. Hypoxic corneal epithelial cells demonstrated no significant differences in plasminogen activator or receptor mRNA. Cell-associated plasminogen activator and membrane-associated receptor protein levels were unchanged. In contrast decreases in mRNA and secreted plasminogen activator inhibitor-1 protein were observed in hypoxic cells. Concurrently, increased cell-associated plasminogen activator activity was observed in hypoxic cells. The formation of plasminogen activator/receptor/plasminogen activator inhibitor-1 complex at the cell surface was not inhibited by hypoxia. However, in hypoxic cells less plasminogen activator inhibitor-1 was associated with receptor. It is concluded that in corneal epithelium cultured in 2% oxygen plasminogen activator inhibitor-1 may be an important regulatory factor of the plasminogen activator system resulting in increased urokinase plasminogen activator activity.

Atheroprotective Mechanisms Activated by Fluid Shear Stress in Endothelial Cells.

Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, while laminar flow and high shear stress are atheroprotective. Well characterized atheroprotective mechanisms include inhibition of thrombosis (increased tissue-type plasminogen activator and decreased plasminogen activator inhibitor-1), inhibition of endothelial cell apoptosis, limitation of permeability (uptake of low-density lipoprotein), prevention of white blood cell binding and transmigration (no expression of adhesion molecules such as intercellular adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1] and no release of monocyte chemotactic protein-1) and increased bioavailability of nitric oxide (because of increased expression of endothelial nitric oxide synthase and manganese superoxide dismutase). Our lab has investigated flow-mediated inhibition of inflammatory cytokine action. In particular, we have shown that flow prevents tumor necrosis factor-alpha (TNF-alpha) mediated signal transduction. TNF regulates inflammatory gene expression (e.g., ICAM-1 and VCAM-1) in endothelial cells, in part, by stimulating mitogen activated protein (MAP) kinases that phosphorylate transcription factors. We hypothesized that fluid shear stress inhibits TNF inflammatory effects on endothelial cells by inhibiting TNF mediated activation of the c-Jun N-terminal kinase. To test this hypothesis, we determined the effects of steady laminar flow on TNF-stimulated activity of c-Jun N-terminal kinase. The results show that flow inhibits c-Jun N-terminal kinase activation through multiple mechanisms, including stimulation of counter-regulatory MAP kinases (extracellular signal regulated kinases [ERK]1/2 and ERK5) and inhibition of apoptosis signal-regulated kinase. In summary, the atheroprotective effects of steady laminar flow on the endothelium involve multiple synergistic mechanisms. These multiple mechanisms offer attractive targets for new drug therapies aimed at limiting atherosclerosis development and progression. (c) 2002 Prous Science. All rights reserved.

External pneumatic compression and fibrinolysis in abdominal surgery

Introduction: External pneumatic compression (EPC) devices prevent lower extremity deep venous thrombosis by increasing venous flow and thereby reducing stasis. Early studies suggested that they also enhance systemic fibrinolytic activity and thus prevent thrombus formation; more recent studies have been conflicting. The hypothesis of this study was that EPC devices enhance systemic fibrinolysis or reduce postoperative fibrinolytic impairment in patients undergoing abdominal surgical procedures. Methods: Each of 48 patients (98% male; mean age, 67 years) undergoing major intra-abdominal surgical procedures (36 bowel procedures, 12 aortic reconstructions) was prospectively randomized to one of three treatments for deep venous thrombosis prophylaxis: subcutaneous heparin injections (HEP group), use of a thigh-length sequential EPC device (EPC group), or both (HEP + EPC group). Antecubital venous samples were collected for measurement of systemic fibrinolytic activity on the day before surgery, after induction of anesthesia but before prophylaxis was initiated, and on postoperative days 1, 3, and 5. Fibrinolysis was assessed through measurement of the activities of the rate limiting fibrinolytic activator, tissue plasminogen activator, and its inhibitor plasminogen activator inhibitor-1 with amidolytic methods. Results: On the day before surgery, plasminogen activator inhibitor-1 activity was elevated in all groups in comparison with that in age-matched and sex-matched controls (20.3 ± 0.6 AU/mL). In the HEP group, plasminogen activator inhibitor-1 activity was further elevated above the value for the day before surgery on postoperative day 1 (28.5 ± 4.3 AU/mL; P =.04) and postoperative day 3 (25.1 ± 1.9 AU/mL; P =.07). No significant decrease in plasminogen activator inhibitor-1 activity occurred in either group treated with EPC devices in comparison with the HEP group at any time. There were no changes in tissue plasminogen activator activity postoperatively in the HEP group and no significant increases in either EPC group at any point. Conclusions: Reduced systemic fibrinolytic activity (“fibrinolytic shutdown”) occurred in these patients after abdominal surgery; it was manifested as increased plasminogen activator inhibitor-1 activity. EPC devices did not enhance systemic fibrinolysis or prevent postoperative shutdown either by decreasing plasminogen activator inhibitor-1 activity or by increasing tissue plasminogen activator activity. These data suggest that EPC devices do not prevent deep venous thrombosis by fibrinolytic enhancement; effective prophylaxis is achieved only when the devices are used in a manner that reduces lower extremity venous stasis. (J Vasc Surg 2000;32:537-43.)

Vitronectin deficiency is associated with increased wound fibrinolysis and decreased microvascular angiogenesis in mice

Background: Vitronectin has several putative functions including regulating hemostasis, cell adhesion, and cell migration. However, the targeted deletion of vitronectin in mice results in normal development and normal coagulation parameters. To determine whether vitronectin may be necessary for nondevelopmental processes, we examined the response to tissue injury in vitronectin-null mice. Methods: We examined wound healing in control and vitronectin-null mice by healing rate, zymography, reverse zymography, and Western blots. Results: We found that dermal wound healing was slightly delayed in mice lacking vitronectin. More importantly, we found extensive areas of delayed hemorrhage near the sprouting tips of microvessels between days 7 and 14, which temporally coincided with increased urokinase-type plasminogen activator and tissue-type plasminogen activator activity by zymography. Though Western blots confirmed the presence of plasminogen activator inhibitor-1 protein throughout wound repair and reverse zymograms showed decreased plasminogen activator inhibitor-1 activity between days 7 and 14. Conclusions: Loss of vitronectin in mice was associated with changes in the fibrinolytic balance, and this may have led to focal sites of delayed hemorrhage. The mechanism that resulted in decreased angiogenesis and the formation of larger blood vessels in response to tissue injury remains unknown. This study suggests that vitronectin may have several distinct functions that are not required for normal development but are manifested in response to tissue injury. (Surgery 2000;127:696-704.)

Activation of Guanine Nucleotide-binding Proteins and Induction of Endothelial Tissue-type Plasminogen Activator Gene Transcription by Alcohol

The mechanism by which moderate alcohol ingestion lowers the risk of cardiovascular disease is unknown but may be due, in part, to the ability of alcohol to increase the level of tissue-type plasminogen activator (t-PA). Human endothelial cells were treated with low concentrations of ethanol (0.25-25 mM, 0-24 h), which are associated with moderate alcohol consumption. Although treatment with ethanol alone did not affect t-PA gene transcription or mRNA expression, it augmented isoproterenol (ISO)-stimulated t-PA gene transcription and mRNA levels by 3.4- and 2.8-fold, respectively, and decreased plasminogen activator inhibitor-1 mRNA levels by 65%. These effects of ethanol correlated with 2.5- and 6.9-fold increases in ISO-stimulated cyclic AMP levels and 4x-cyclic AMP response element heterologous promoter activity, respectively. To determine whether alcohol-induced changes in agonist-stimulated cyclic AMP levels were because of modulation of guanine nucleotide-binding proteins (G proteins), we assessed the effects of ethanol on Galphas and Galphai2. Although ethanol did not affect the expression of Galphas or Galphai2, it increased ISO-stimulated Galphas GTPase and GTP binding activity by 2.2- and 2.9-fold and decreased UK14304-stimulated Galphai2 GTPase and GTP binding activity by 38 and 80%. These results indicate that treatment with relatively low concentrations of ethanol enhances agonist-stimulated cyclic AMP-dependent t-PA gene transcription in vascular endothelial cells through differential modulation of G protein.

Pravastatin therapy in hyperlipidemia: effects on thrombus formation and the systemic hemostatic profile

OBJECTIVESThe study sought to determine the effects of lipid-lowering with pravastatin on the systemic fibrinolytic profile and on thrombus formation under dynamic flow conditions.BACKGROUNDLowering cholesterol (C) decreases clinical events in coronary artery disease (CAD) patients, but an analysis of the effects of lipid-lowering on the entire hemostatic and thrombotic profile has not been conducted.METHODSWe prospectively studied 93 stable patients with untreated low-density lipoprotein cholesterol (LDL-C) >145 mg/dl. The CAD patients received pravastatin, and non-CAD patients were randomized to pravastatin versus placebo (double-blind). Thrombus formation upon an injured vascular surface was assessed in a substudy of 40 patients with a previously validated ex vivo perfusion chamber system. Systemic hemostatic markers and thrombus formation were evaluated at baseline, three and six months.RESULTSPlacebo produced no changes in either the lipid profile, any of the hemostatic markers, or the ex vivo thrombus formation. Both pravastatin groups (CAD and non-CAD) showed decreased LDL-C by 30% within 6 weeks (188 to 126 mg/dl, p < 0.001 vs. baseline), and decreased plasminogen activator inhibitor-1 at 3- and 6-month follow-up compared to baseline (15% to 18% decrease at 3 months and 21% to 23% at 6 months). For the tissue plasminogen activator antigen, CAD and non-CAD groups showed significant decreases at 6 months compared to baseline (10% and 13%, respectively). No significant changes were observed with treatment in d-dimer, fibrinopeptide A, prothrombin fragment F1.2, factor VIIa, von Willebrand factor, or C-reactive protein. Fibrinogen levels were significantly increased at 6 months compared to baseline, though still below the upper normal limit. In the perfusion chamber substudy, there was a decrease in thrombus area in non-CAD patients treated with pravastatin at both 3 and 6 months compared to baseline (by 21% and 34%, respectively). The CAD patients showed decreases in thrombus formation by 13% at 3 months, and by 16% at 6 months. The change in LDL-C- correlated modestly with the change in thrombus formation (r = 0.49; p < 0.01).CONCLUSIONSPravastatin therapy significantly decreased thrombus formation and improved the fibrinolytic profile in patients with and without CAD. These early effects may, in part, explain the benefit rendered in primary and secondary prevention of CAD.

Inhibition of plasminogen activator inhibitor-1 by 11-keto-9(E),12(E)-octadecadienoic acid, a novel fatty acid produced by Trichoderma sp.

We have recently found a novel fatty acid, 11-keto-9(E),12(E)-octadecadienoic acid (KOD), that enhances fibrinolytic activity of endothelial cells. The mechanism of action of KOD has been investigated. KOD increased 2-fold the plasmin activity of bovine aortic endothelial cells at 250 microM. The stimulation was dependent on plasminogen and was inhibited by anti-urokinase, whereas KOD did not enhance the urokinase-catalyzed plasminogen activation and the resulting plasmin activity in a cell-free system. Neither the production of urokinase nor the conversion of pro-urokinase to the active, two-chain form was elevated by KOD, but it decreased plasminogen activator inhibitor-1 (PAI-1) activity of cells and inactivated PAI-1 irreversibly in a purified system. These results demonstrated that the KOD enhancement of endothelial fibrinolytic activity was mediated, at least in part, by inactivation of PAI-1.

The Influence of Labor and Placental Separation on Hemostasis in Term Pregnancy

We studied the hemostatic effects in 68 term preg nant subjects with normal pregnancy (n = 39), gestational diabetes mellitus (n = 21), pregnancy-induced hypertension (n = 8), during labor, after placental separation, and at 24 hours postpartum. During labor, a hypercoagulable state with en hanced fibrinolysis and platelet activation along with elevated plasminogen activator inhibitor-1 and plasminogen activator inhibitor-2 were seen. In all cases following placental separa tion, enhanced fibrinolysis persisted with further elevated tissue plasminogen activator antigen and D-dimer levels, increased thrombin generation and platelet activation with decreasing plasminogen activator inhibitor-1 levels. By 24 hours postpar tum, thrombin generation (thrombin-antithrombin complex) decreased to a nonpregnant level except for pregnancy induced hypertension, but enhanced prothrombin activation (F1+2) was evident in normal, gestational diabetes mellitus and pregnancy- induced hypertension. Reduced total protein S and antithrom bin III activity with normal protein C and elevated fibrinogen levels seen during labor were not affected by delivery. How ever, although factor VII showed a decreasing trend at 24 hours postpartum it remained elevated above the normal nonpregnant level. At 24 hours postpartum, a hypercoagulable state still persisted in normal, gestational diabetes mellitus and preg nancy-induced hypertension term pregnancy accompanied by enhanced fibrinolysis and elevated plasminogen substrate for fibrinolysis, suggesting a critical dynamic equilibrium between a thrombotic and hemorrhagic state.

Significance of plasminogen activator inhibitor 2 as a prognostic marker in primary lung cancer: association of decreased plasminogen activator inhibitor 2 with lymph node metastasis.

The expression of urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR) and plasminogen activator inhibitor (PAI) 1 and 2 was examined in 105 cases of primary lung cancer tissue using immunohistochemical staining and reverse transcriptase polymerase chain reaction (RT-PCR) techniques. The expression of u-PA, u-PAR and PAI-1 was detected in approximately 80% of primary lung cancers, whereas detectable PAI-2 expression was observed only in half of the overall cases. We assessed the relationships between the expression pattern and clinicopathological findings and found that a diminished expression level of PAI-2 was significantly correlated with lymph node metastasis and a poor prognosis. These results indicate that PAI-2 may play a critical role in the regulation of extracellular matrix degradation during tumour cell invasion and metastasis, and the expression of PAI-2 may be useful as a marker for evaluating the prognosis of lung cancer.

Familial dysplasminogenemia with central retinal vein and cilioretinal artery occlusion

PURPOSE: To report a 49-year-old woman with unilateral central retinal vein occlusion and ipsilateral cilioretinal artery occlusion who showed familial dysplasminogenemia associated with elevated lipoprotein(a). METHOD: Case report. RESULTS: Extensive hemostatic and coagulation studies for causes of thrombosis, as well as family studies, disclosed decreased plasminogen activity without reduction of plasminogen antigen in the patient, her two siblings, and her two children. The patient also showed elevated lipoprotein(a). CONCLUSION: The combination of decreased plasminogen activity and elevated lipoprotein(a) should be considered as a possible cause of retinal vein and artery occlusion.

Vitamin D metabolites regulate matrix vesicle metalloproteinase content in a cell maturation-dependent manner.

Matrix vesicles are extracellular organelles produced by cells that mineralize their matrix. They contain enzymes that are associated with calcification and are regulated by vitamin D metabolites in a cell maturation-dependent manner. Matrix vesicles also contain metalloproteinases that degrade proteoglycans, macromolecules known to inhibit calcification in vitro, as well as plasminogen activator, a proteinase postulated to play a role in activation of latent TGF-beta. In the present study, we examined whether matrix vesicle metalloproteinase and plasminogen activator are regulated by 1, 25(OH)2D3 and 24,25(OH)2D3. Matrix vesicles and plasma membranes were isolated from fourth passage cultures of resting zone chondrocytes that had been incubated with 10(-10)-10(-7) M24, 25(OH)2D3 or growth zone chondrocytes incubated with 10(-11)-10(-8) M 1,25(OH)2D3, and their alkaline phosphatase, active and total neutral metalloproteinase, and plasminogen activator activities determined. 24,25(OH)2D3 increased alkaline phosphatase by 35-60%, decreased active and total metalloproteinase by 75%, and increased plasminogen activator by fivefold in matrix vesicles from resting zone chondrocyte cultures. No effect of vitamin D treatment was observed in plasma membranes isolated from these cultures. In contrast, 1,25(OH)2D3 increased alkaline phosphatase by 35-60%, but increased active and total metalloproteinase three- to fivefold and decreased plasminogen activator by as much as 75% in matrix vesicles isolated from growth zone chondrocyte cultures. Vitamin D treatment had no effect on plasma membrane alkaline phosphatase or metalloproteinase, but decreased plasminogen activator activity. The results demonstrate that neutral metalloproteinase and plasminogen activator activity in matrix vesicles are regulated by vitamin D metabolites in a cell maturation-specific manner. In addition, they support the hypothesis that 1,25(OH)2D3 regulation of matrix vesicle function facilitates calcification by increasing alkaline phosphatase and phospholipase A2 specific activities as well as metalloproteinases which degrade proteoglycans.

The effect of oestrogen dose and progestogen type on haemostatic changes in women taking low dose oral contraceptives.

To determine the effect of oestrogen dose and progestogen type on the coagulation and fibrinolytic systems of a group of normal healthy women taking three different oral contraceptive combinations. Plasma levels of factor VII, X, antithrombin III, protein C, fibrinogen, tissue plasminogen activator activity, plasminogen activator inhibitor I antigen and fibrin (D-dimer) degradation products were measured at pretreatment, 6, 14, 22 weeks of treatment and at 6 weeks post-treatment in a group of 67 women taking either 30 micrograms ethinyloestradiol/150 micrograms desogestrel (n = 21), 20 micrograms ethinyloestradiol/150 micrograms desogestrel (n = 24), 30 micrograms ethinyloestradiol/75 micrograms gestodene (n = 22). Sixty-seven healthy normal women, 18 to 34 years, smoking fewer than 15 cigarettes per day. The subjects were within 10% of their normal body weight and had no history of thromboembolic disease. Coombe Women's Hospital and St James's Hospital, Dublin, Ireland. Factor VII and X levels were significantly raised on treatment with both the 30 micrograms ethinyloestradiol/desogestrel and gestodene combinations. Higher levels of factor VII activity were observed in the 30 micrograms ethinyloestradiol/desogestrel combination compared with the gestodene combination. Factor VII and X were not significantly affected by the 20 micrograms ethinyloestradiol combination. Increased plasminogen, fibrinogen and D-dimer levels and decreased plasminogen activator inhibitor I antigen levels were observed during the treatment phases in all three groups. Antithrombin III and protein C activity did not change during treatment with any of the oral contraceptives studied. Low dose oral contraceptives cause an activation of the coagulation system which is balanced by an activation of the fibrinolytic system. Reducing the dose of ethinyloestradiol from 30 micrograms to 20 micrograms reduces the effect on factor VII and X. This effect can be modified by the progestogen. The lesser effect of the 20 micrograms combination may make this a safer option for some women than pills containing a higher dose of oestrogen.

Fibrinolytic system and reproductive process with special reference to fibrinolytic failure in pre-eclampsia.

Here we summarize the recent progress in research on the role of the fibrinolytic system in reproduction, with a special emphasis on the role of the plasminogen activator inhibitors in fetal development. Trophoblasts produce fibrinolytic proteins that can promote normal implantation and regulate blood flow to the fetus and placenta throughout pregnancy. Normal pregnancy is associated with a hypofibrinolytic state that is fundamentally caused by an increase in plasminogen activator inhibitors types 1 and 2. In pre-eclampsia, a fibrinolytic failure, resulting from an increase in plasma and placental concentrations of plasminogen activator inhibitor-1, was observed. The localized elevated concentrations of placenta plasminogen activator inhibitor-1 protein and mRNA observed in pre-eclamptic patients would be expected to foster the deposition of fibrin and thus play a role in the complications associated with this disease. The decreased plasminogen activator inhibitor-2 concentrations in placenta and plasma from intrauterine fetal growth retardation pregnancies and the positive correlation between plasma/placenta plasminogen activator inhibitor-2 concentration and birthweight suggest that this inhibitor could be considered an adequate marker of placental function.

Low proteins C and S and activation of fibrinolysis in treated essential thrombocythemia.

The aim of this study was to investigate whether abnormalities in the fibrinolytic system and in the naturally occurring anticoagulant proteins could contribute to the thrombotic risk in essential thrombocythemia. Euglobulin lysis time, fibrin plate lysis area, tissue plasminogen activator antigen, and activity and plasminogen activator inhibitor antigen were measured before and after venous occlusion in a group of 16 patients with essential thrombocythemia and in 16 healthy age and sex matched controls. In addition, resting levels of antithrombin III, D-dimer, prothrombin fragment 1 + 2, and protein C and S were assessed. The results were related to the presence or absence of a thrombotic history. The results demonstrated that the patients had a significantly elevated fibrin plate lysis area and significantly decreased plasminogen activator antigen, both at baseline and after venous occlusion. They also had significantly decreased levels of plasma protein C and total protein S. There was a modest, non-significant elevation in the plasma concentration of D-Dimer and F 1 + 2. Those patients with a history of thrombosis had significantly lower protein C levels compared with individuals without a thrombotic history. We conclude that patients with essential thrombocythemia have evidence of activated fibrinolysis in the resting state and after stimulation. This, and the decreased levels of protein C and total protein S, may be secondary to chronic clinically occult thrombosis occurring in myeloproliferative disorders.

Effect of notoginsenoside R1 on the synthesis of components of the fibrinolytic system in cultured human pulmonary artery endothelial cells and human skin microvascular endothelial cells

Summary We have previously reported that Notoginsenoside R1 (NR1) increases the synthesis of tissue-type plasminogen activator (t-PA) and decreases plasminogen activator inhibitor-1 (PAI-1) activity in cultured human umbilical vein endothelial cells (HUVECs). It was the aim of this study to investigate whether the effect of NR1 on the synthesis of components of the fibrinolytic system is also operative in endothelial cells from other vascular beds. Therefore cultured human pulmonary artery endothelial cells (HPAECs) and human skin microvascular endothelial cells (HSMECs) were treated with NR1. When cultured HPAECs and HSMECs (passage 2–3) were conditioned with NR1, a dose (0.01–100 g NR1/ml for 24 h) dependent increase in t-PA and u-PA synthesis was observed, which was significant from 0.1 g NR1/ml on. t-PA antigen increased from 5.5 ± 0.2 ng/10 5 cells/24 h to 9.8 ± 0.6 ng/10 5 cells/24 h in HPAECs, and from 1.9 ± 0.1 ng/10 5 cells/24 h to 3.0 ± 0.3 ng/10 5 cells/24 h in HSMECs; u-PA antigen increased from 0.8 ± 0.1 ng/10 5 cells/24 h to 1.8 ± 0.4 ng/10 5 cells/24 h in HPAECs, and from 3.0 ± 0.1 ng/10 5 cells/24 h to 5.7 ± 0.7 ng/10 5 cells/24 h in HSMECs on addition of 100 g NR1/ml. In contrast, no change in PAI-1 antigen synthesis in both HPAECs and HSMECs was seen. As judged from fibrin autography (FA) and reverse fibrin autography (RFA), t-PA activity increased 3.5-fold whereas PAI-1 activity decreased to approximately 50% in conditioned media of HPAECs treated with 100 g/ml NR1 as compared to control cells. On Northern blot analysis of mRNA obtained from NR1-stimulated and control HPAECs and HSMECs, NR1 induced a significant increase in specific mRNA levels of t-PA and u-PA (t-PA mRNA: 180% and 160% of control value in HPAECs and HSMECs, respectively, at 100 g NR1/ml; u-PA mRNA: 140% of control value in HSMECs at 100 g NR1/ml), while PAI-1 mRNA remained unchanged. In conclusion, our data give evidence that NR1 can increase the fibrinolytic and proteolytic potential of endothelial cells from different vascular sources in vitro by increasing the production of t-PA and u-PA.

Morphological changes in carotid arteries of rabbits induced by defibrotide infusion

Defibrotide is an antithrombotic and profibrinolytic drug which modulates endothelial function. The drug increases prostacyclin and tissue plasminogen activator while it decreases plasminogen activator inhibitor synthesis by endothelial cells. In this study, in vivo effects of defibrotide on the morphology of endothelial cells and vessel wall of the healthy rabbits were investigated by light and electron microscopy. The examination of the carotid arteries of healthy rabbits after infusion of saline or defibrotide (10 mg/kg/hr) in saline solution for three hours revealed that the drug had induced dramatic morphological changes in all the test animals while no change was observed in control group. The changes observed after defibrotide administration, such as the decrease in hill and valley-like appearance of endothelial surface, and thinning of the intimal layer provides evidence for the vasorelaxant effect of the drug, while the decrease in the number of blood cells adhering to the endothelial surface confirms the antithrombotic effect of defibrotide. Finally the decrease in the number of crater-like structures may be due to the cytoprotective effect of the drug.

Low levels of plasminogen activator inhibitor type 1 in patients with inflammatory bowel disease

Recent investigations suggest that microthrombi formation in bowel capillaries could be a determinant factor in inflammatory bowel disease (IBD) pathogenesis. To evaluate the implication of fibrinolysis in these thrombotic events, we analysed plasmatic values of physiologic activators, effectors and inhibitors of fibrinolysis. In a sample of 112 IBD patients we found decreased plasminogen activator inhibitor type 1 (PAI-1:Ag) levels, a finding which has not been reported to date: 8.8±1.1 ng/mL (mean±SEM) versus 17.8±1.1 ng/mL in controls (p<0.0001). Urokinase-type plasminogen activator (u-PA:Ag) from patients with inflammatory activity was decreased: 0.41±0.03 ng/mL in active disease versus 0.52±0.02 ng/mL in inactive disease (p=0.01) and the same applied to patients with Crohn's disease (CD) (0.38±0.03 ng/mL) with respect to ulcerative colitis (UC) patients (0.52±0.025 ng/mL), p=0.001. Levels of plasminogen, alpha 2 antiplasmin and tissue-type plasminogen activator (t-PA:Ag) showed no differences with respect to the controls. With the exception of u-PA:Ag, there were no differences between UC and CD. These results demonstrate plasmatic disturbances in the flbrinolytic system of IBD patients.

The Effects of Transdermal Estradiol and Oral Conjugated Estrogens on Haemostasis Variables

The effects of oral and transdermal administration of estrogen replacement therapy (ERT) have been fairly well investigated regarding lipoprotein and carbohydrate metabolism, while the effects of different modes of estrogen administration on the haemostatic system have been less well studied. To delineate and compare the effects of perorally administered conjugated estrogens (CE) and transdermally administered estradiol (E2) in doses needed for hormone replacement therapy (HRT) on haemostasis parameters, 23 postmenopausal women were engaged in a study with an open cross-over design. The doses compared (0.625 mg CE and 50 micrograms E2/24h) are the lowest which, with few exceptions, eliminate climacteric symptoms. Both CE and E2 increased factor VII:C, factor VII:Ag, and the prothrombin fragment1 + 2. The increase in factor VII:Ag, however, was significantly higher after treatment with CE. These changes were all towards a state of hypercoagulability. Furthermore, CE decreased plasminogen activator inhibitor (PAI) and the thrombin-antithrombin complexes (TAT), as well as antithrombin (ATIII).

Decreased Plasminogen Activator Inhibitor‐1 Activity in Newly Diagnosed Type 2 Diabetic Patients Following Dietary Modification

Increased plasminogen activator inhibitor-1 (PAI-1) activity has been reported in Type 2 (non-insulin-dependent) diabetes and is a recognized risk factor for coronary artery disease. Fourteen newly diagnosed Type 2 diabetic patients were studied before and 3 months after standard clinical dietary modification. To assess the effect of improved metabolic control on PAI-1 activity, nine Type 2 diabetic patients established on diet therapy and with previous stable glycaemic control served as controls. In the newly diagnosed patients diet therapy resulted in a significant decrease in HbA1c levels (8.3 +/- 0.5 vs 5.2 +/- 0.3% (mean +/- SEM); p < 0.001), and this was accompanied by a fall in fibrinogen (4.3 +/- 0.3 vs 3.0 +/- 0.2 g.l-1; p < 0.01) concentration, and PAI-1 (18.7 +/- 2.3 vs 12.2 +/- 0.9 arbitrary units ml-1; p < 0.02) and factor VIII (147 +/- 17 vs 115 +/- 13%; P < 0.01) activities. PAI-1 activity was correlated with triglyceride levels at the first assessment in the newly diagnosed patients (r = 0.66; p < 0.01), and this was the only independent association by multiple regression analysis when all patients (n = 23) were considered (r = 0.62; p < 0.002). However, there was no association between the changes in PAI-1 activity and the changes in HbA1c BMI, and serum triglyceride levels following treatment in the newly diagnosed patients. Serum triglyceride concentrations, HBA1c, PAI-1 activity, and the coagulation factors remained unchanged in the control group over the same treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)