Human renal microvascular endothelial cells as a potential target in the development of the hemolytic uremic syndrome as related to fibrinolysis factor expression, in vitro

Renal glomerular microvascular endothelial cell damage is characteristic of Shiga toxin-associated hemolytic uremic syndrome (HUS). An impaired renal fibrinolysis may be responsible for renal microvascular fibrin accumulation during the course of HUS disease. This study examined the effect of Shiga toxin, bacterial lipopolysaccharide (LPS, endotoxin), and tumor necrosis factor (TNF) on the expression of fibrinolysis factors by human renal glomerular microvascular endothelial cells (HRMEC) in vitro. The results were compared to a previously better-characterized endothelial cell type, human umbilical vein endothelial cells (HUVEC). In HUVEC, the ratio of fibrinolysis antigens was antifibrinolytic, consisting of 55-fold more plasminogen activator inhibitor type 1 (PAI-1) than tissue-type plasminogen activator (tPA). Treatment of HUVEC with LPS or TNF accentuated this ratio by decreasing tPA and increasing PAI-1 expression. In contrast, HRMEC produced urokinase-type plasminogen activator (uPA) in a 24-fold excess to PAI-1 and were thereby profibrinolytic with regard to fibrinolysis antigen expression. LPS and TNF further decreased PAI-1 antigen expression by HRMEC. These results argue against a role for LPS or TNF in decreasing renal fibrinolysis at the level of fibrinolysis factor expression by renal endothelial cells. Nevertheless, HUVEC and HRMEC were responsive to the same LPS analogs in the same order of potency. Shiga toxin decreased fibrinolysis factor expression to a greater extent in HRMEC than in HUVEC. Since HRMEC fibrinolysis antigen expression was profibrinolytic, the Shiga toxin-mediated decrease in renal endothelial uPA synthesis may predispose renal microvasculature to thrombosis and may have implications for the development of HUS.     

Hyperinsulinemia and hypofibrinolysis: effects of short-term optimized glycemic control with continuous insulin infusion in type II diabetic patients

A defect in the fibrinolytic system results from an increase in type 1 plasminogen activator inhibitor (PAI-1) in diabetes. It can be considered an independent risk factor for the development of cardiovascular disease. In obese and type II diabetic patients, plasma PAI-1 level correlates with fasting insulinemia. However, during the euglycemic clamp, acute hyperinsulinemia does not increase PAI-1 production. The present study was undertaken to investigate the effect of optimized glycemic control by continuous subcutaneous insulin infusion (CSII) on the hypofibrinolytic state for 14 days in 16 type II diabetic patients with poor metabolic control despite maximal oral antidiabetic treatment. Plasma PAI-1 activity levels decreased from 13.38 +/- 2.85 IU/mL to 6.77 +/- 1.81 IU/mL (P = .002) during CSII, along with a concurrent improvement in insulin sensitivity (index obtained by basal glycemia-nadir glycemia/basal glycemia) during the insulin sensitivity test (0.121 +/- 0.03 v 0.057 +/- 0.02, P = .02). These results suggest that insulin resistance rather than hyperinsulinism may be involved in the hypofibrinolytic state in type II diabetic patients. The positive correlation between the changes in triglycerides and in PAI-1 activity (r = .589, P = .026) strongly suggests a role for triglycerides in the impairment of fibrinolysis, which could be a link between insulin resistance and hypofibrinolysis.     

Interleukin-1-mediated regulation of plasminogen activation in pregnant mare serum gonadotropin-primed rat granulosa cells is independent of prostaglandin production

OBJECTIVES: This study examines the effects of interleukin-1 (IL-1) on plasminogen activator (PA) activity and prostaglandin (PG) E production in pregnant mare serum gonadotropin (PMSG)-primed granulosa cells and the potential involvement of PGE in the regulation of ovarian plasminogen activation. METHODS: Granulosa cells were obtained from PMSG-primed rat (27-day-old) ovaries and cultured in serum-free conditions for 48 hours in the absence or presence of IL-1 beta (10 ng/mL) with and without transforming growth factor-beta 1 (10 ng/mL). Cellular PA activity was measured through the conversion of plasminogen to plasmin and assay of the plasmin-mediated cleavage of [14C]-labeled globin to acid-soluble products. RESULTS: Exposure of PMSG-primed granulosa cells to IL-1 resulted in a 30% reduction (P < .05) in PA activity. Addition of hCG (1 IU/mL) to the granulosa cell cultures resulted in a 2.3-fold increase in PA activity, an effect significantly attenuated by co-administration of IL-1. The IL-1-mediated inhibition occurred concurrent with a 6.6-fold increase in the ability of the corresponding conditioned media to inhibit exogenous urokinase activity. This latter inhibitory capacity was the result of a significant increase in plasminogen activator inhibitor type 1 (PAI-1), given its abolition by a polyclonal anti-rat PAI-1 immunoglobulin G. The IL-1-mediated effects on PA/PAI-1 were accompanied by a sevenfold increase in PGE content of the spent culture medium. This response was dose dependent. The IL-1 effects on plasminogen activation and PG production were abolished by the IL-1 receptor antagonist, suggesting specific IL-1 receptor-mediated responses. Indomethacin, an inhibitor of PG biosynthesis, prevented the IL-1-induced increase in PGE accumulation but failed to affect the response of the PA system. Transforming growth factor-beta 1, a known regulator of IL-1 action, significantly attenuated the IL-1-stimulated PGE production but did not interfere with the ability of IL-1 to affect the PA system. CONCLUSIONS: The present observations suggest a pleiotropic response of PMSG-primed granulosa cells to IL-1, characterized by the induction of PAI-1 concurrent with but independent of PG production. These findings corroborate and extend earlier observations suggesting that IL-1 affects PA activity and PGE production in immature rat ovaries. Moreover, these observations support our contention that IL-1 may play a major regulatory role in the cellular events leading to ovulation and early corpus luteum formation.     

Coagulation and fibrinolysis parameters in normal pregnancy and in gestational diabetes

The changes in coagulation and fibrinolysis parameters during pregnancy, delivery and 3 days after delivery were evaluated in normotensive and gestational diabetes pregnant women. Normal pregnant women (n = 60) and pregnant women with gestational diabetes (n = 15) formed the study population. Coagulation and fibrinolysis parameters were estimated using commercial tests. Antithrombin III, thrombin-antithrombin III complexes, heparin cofactor II, protein C, protein S, tissue plasminogen activator, (t-PA) D-dimer and plasminogen activator inhibitor (PAI-1 and PAI-2) activities in normal and gestational diabetes pregnancies were determined. Thrombin-antithrombin III complexes increased and coagulation inhibitors decreased in gestational diabetes. Plasminogen activator inhibitors remained unchanged and t-PA levels increased in gestational diabetes.     

Modulation by endothelin-1 of tissue plasminogen activator and plasminogen activator inhibitor-1 release from cultured human vascular endothelial cells: interaction of endothelin-1 with cytokines

The interaction of endothelin-1 (ET-1) with either interleukin-1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF alpha) on the release of tissue plasminogen activator antigen (t-PA:Ag) and plasminogen activator inhibitor-1 antigen (PAI-1:Ag) was investigated in a culture system of vascular endothelial cells derived from human umbilical vein. The t-PA:Ag release was significantly decreased by either IL-1 beta or TNF alpha; ET-1 intensified the suppressive effect of the cytokines. In contrast, PAI-1:Ag release was significantly increased by either IL-1 beta or TNF alpha; ET-1 significantly reduced the stimulatory effect of the cytokines. The data suggest that endothelial cell-mediated fibrinolysis may be modulated by ET-1.     

Recombinant lys-plasminogen given before, but not after, recombinant tissue-type plasminogen activator markedly improves coronary thrombolysis in dogs: relationship of thrombolytic efficacy with parameters of fibrinolysis

Recombinant tissue-type plasminogen activator (rt-PA) administration rapidly restores blood flow in thrombosed coronary arteries, but coronary arteries often reocclude after initial thrombolysis. This occurs because of the short half-life of rt-PA and rapid increase in plasminogen activator inhibitor (PAI-1) and alpha2-antiplasmin levels in plasma. We hypothesized that administration of lys-plasminogen, which binds to fibrin with 10 times greater affinity and results in a loose fibrin structure (as compared with native glu-plasminogen), before rt-PA would enhance the thrombolytic efficacy of rt-PA and modulate parameters of fibrinolysis. To examine this hypothesis, dogs with electrically induced stable thrombus in the left anterior descending coronary artery (LAD) were treated with saline (group A, n = 9) or lys-plasminogen (group B, 2 mg/kg, n = 5), followed 10 min later by rt-PA (1 mg/kg in 20 min). Four other dogs with occlusive LAD thrombus were first given rt-PA, followed by lys-plasminogen (2 mg/kg) 50 min later (group C). Lys-plasminogen given before rt-PA restored flow in all dogs in 14 +/- 4 min (vs. 22 +/- 9 min in group A, p < 0.05), continuing > 2 h (vs. 41 +/- 15 min in group A, p < 0.02). Lys-plasminogen given after rt-PA did not potentiate the effect of rt-PA. Plasma t-PA antigen concentrations were highest in group B dogs at 2 h after rt-PA infusion. PAI-1 and alpha2-antiplasmin plasma levels were suppressed in all dogs receiving lys-plasminogen whether it was given before or after rt-PA. Therefore, lys-plasminogen given before rt-PA markedly potentiates the effect of rt-PA and alters the parameters of fibrinolysis. In contrast, lys-plasminogen given after rt-PA does not influence the thrombolytic effect of rt-PA, whereas it suppresses PAI-1 and alpha2-antiplasmin levels in plasma. This study also suggests that binding of plasminogen to the clot is more important than the plasma levels of PAI-1 and alpha2-antiplasmin.     

Insulin sensitivity, insulin action, and fibrinolysis activity in nondiabetic and diabetic obese subjects

Because inconsistencies occur with regard to the relative contribution of insulin to the hypofibrinolysis characteristic of obesity and diabetes, we explored the relationship between insulin and fibrinolysis, assessing both insulin sensitivity and insulin action. Seventeen markedly obese subjects (body mass index [BMI], 34.0+/-1.6 kg/m2; 12 nondiabetic and five diabetic) were studied using the three-step euglycemic-hyperinsulinemic clamp technique. Since the circadian rhythm of the fibrinolytic system may obscure a true effect of insulin, variations in fibrinolysis parameters observed during the glucose clamp were compared with those occurring spontaneously because of the circadian rhythm. Compared with six normal-weight subjects (BMI, 21.0+/-0.9 kg/m2), all obese subjects exhibited basal hyperinsulinism (fasting plasma insulin, 16.0+/-1.4 v 9.8+/-1.3 microU/microL, P < .001; fasting plasma C-peptide, 1.4+/-0.2 v 0.5+/-0.2 ng/mL, P < .001), hypofibrinolysis (euglobulin lysis time [ELT], 378+/-29 v 222+/-31 minutes, P=.01; tissue plasminogen activator [tPA] antigen, 7.8+/-0.9 v 4.2+/-0.5 ng/mL, P=.04; plasminogen activator inhibitor type 1 [PAI-1] activity, 22.2+/-2.5 v3.9+/-0.6 AU/mL, P=.004), and marked insulin resistance (M value, ie, the maximal glucose disposal rate, 9.1+/-0.6 v 18.6+/-0.8 mg/(kg x min), P < .001). The M value correlated inversely with tPA antigen (r=-.46, P=.05). During insulin infusion, values for fibrinolysis parameters decreased, but were not different compared with variations due to the circadian rhythm. In conclusion, our findings together with previously reported data reinforce the idea that chronic hyperinsulinism is linked to hypofibrinolysis, but insulin does not seem to acutely regulate the fibrinolysis system.     

Attenuation by gemfibrozil of expression of plasminogen activator inhibitor type 1 induced by insulin and its precursors

BACKGROUND: Insulin and its precursors found in increased plasma concentrations in non-insulin-dependent diabetes mellitus (NIDDM) augment synthesis of plasminogen activator inhibitor type 1 (PAI-1) in Hep G2 cells in vitro and in rabbit liver in vivo. Reduced endogenous fibrinolysis secondary to increased PAI-1 activity may exacerbate atherogenesis. Recently, the reduction of the coronary heart disease incidence in the Helsinki Heart Study has implicated favorable modulation of endogenous fibrinolysis by gemfibrozil. METHODS AND RESULTS: In Hep G2 cells, 500 (700) mumol/L gemfibrozil decreased basal secretion of PAI-1 by 26% (43%) (P = .012 and P = .021, respectively) and attenuated insulin-induced (10 nmol/L) augmentation of PAI-1 in conditioned media by 61% (109%) (P = .010) within 24 hours. Inhibition was dependent on the duration of exposure (0 to 48 hours) and on the concentration of gemfibrozil (0 to 700 mumol/L) but not on the concentration of insulin (0.1 to 100 nmol/L). Gemfibrozil attenuated the augmentation of PAI-1 secretion induced by proinsulin (> 100%), by des(31,32)proinsulin (75%), and by des(64,65) proinsulin (77%) as well (10 nmol/L each). The specificity of these effects was confirmed by the unaltered levels of newly synthesized protein (metabolic labeling) and of total protein (both in conditioned media and cell lysates). Secretion of fibrinogen by Hep G2 cells was not affected by gemfibrozil. Changes in PAI-1 protein levels reflected modulation of PAI-1 gene expression as manifested by changes in levels of 3.2-kb PAI-1 mRNA (Northern blots). CONCLUSIONS: Gemfibrozil attenuated the augmentation of synthesis and secretion of PAI-1 induced by insulin and its precursors directly and specifically. Accordingly, gemfibrozil may exert favorable therapeutic effects normalizing impaired fibrinolysis in patients with hyperinsulinemia such as NIDDM.     

The role of gap junctional intercellular communication (GJIC) disorders in experimental and human carcinogenesis

BACKGROUND: To evaluate the effect of plasminogen activator inhibitor type 1 (PAI-1) levels on the clearance of total tissue plasminogen activator (TPA) antigen, we studied the clearance of active TPA and TPA/PAI-1 complex in subjects with low (181+/-109 pmol/L; n=7) and high (1166+/-322 pmol/L; n=4) baseline active PAI-1. METHODS AND RESULTS: A 5-microg/kg bolus of TPA was infused over a 15-second period followed by measurement of TPA activity, TPA antigen, TPA/PAI-1, TPA/C1 inhibitor, PAI-1 activity, and PAI-1 antigen over a 4-hour period. alpha-Phase clearance of total TPA antigen was faster in subjects with low PAI-1 (t(1/2) of 3.5+/-0.7 minutes) versus high PAI-1 (t(1/2) of 5.3+/-0.9 minutes) (P=.006). Clearance of all factors was best fit by a two-compartment pharmacokinetic model based on a computer-simulated human circulatory system. The average hepatic clearance fraction in the two-compartment model was greater for active TPA (89+/-10%, t(1/2) of 2.4+/-0.3 minutes) than for TPA/PAI-1 complex (48+/-17%, t(1/2) of 5.0+/-1.8 minutes) (P=.0006). CONCLUSIONS: Plasma clearance of active TPA was faster than clearance of TPA/PAI-1 complex. High levels of active PAI-1 converted more TPA into TPA/PAI-1 complex, effectively slowing the clearance of total TPA antigen and explaining in part why high levels of PAI-1 activity are associated with increases in total TPA antigen.     

Effect of weight change and metformin on fibrinolysis and the von Willebrand factor in obese nondiabetic subjects: the BIGPRO1 Study. Biguanides and the Prevention of the Risk of Obesity

OBJECTIVE: Insulin resistance is associated with hypofibrinolysis. Metformin has been shown to improve insulin sensitivity and fibrinolysis. Its action on fibrinolysis and the von Willebrand factor was evaluated in the Biguanides and the Prevention of the Risk of Obesity (BIGPRO)1 trial in nondiabetic men (n = 151) and women (n = 306) aged between 34 and 65 years with a central fat distribution and a mean BMI of 32.5 kg/m2. RESEARCH DESIGN AND METHODS: The subjects were randomly allocated to a 1-year treatment with metformin (850 mg b.i.d.) or placebo, in addition to diet and exercise recommendations. RESULTS: Plasminogen activator inhibitor 1 (PAI-1) activity and antigen decreased significantly but similarly by 30 and 40%, respectively, in both the placebo and the metformin groups. This decrease occurred mainly in subjects who lost weight. Metformin did not have any significant additional effect on PAI-1. In contrast to the results for PAI-1, there was a significantly greater decrease in tissue-type plasminogen activator (tPA) antigen in the metformin than in the placebo group (mean+/-SD: -1.1+/-3.1 vs. 0.2+/-3.2 ng/ml, P < 0.02). The von Willebrand factor (vWF) also decreased significantly more in the metformin group (-0.17+/-0.42 vs. -0.05+/-0.38 U/I, P < 0.02). CONCLUSIONS: Weight loss was the main factor associated with the decrease in PAI-1, in accordance with the recent demonstration of production of PAI-1 by adipocytes. Metformin had a significant effect on two factors, tPA antigen and vWF, mainly secreted by the endothelial cells, which suggests an effect of the drug on the production or the metabolism of these two hemostatic proteins.     

Visceral fat accumulation is an important determinant of PAI-1 levels in young, nonobese men and women: modulation by cross-sex hormone administration

Increased plasminogen activator inhibitor type-1 (PAI-1) levels, leading to impaired fibrinolysis, are associated with increased visceral fat in middle-aged and obese subjects. It is unknown, however, whether this association is independent of other disturbances clustered in the insulin resistance syndrome. We analyzed this association in young, nonobese transsexual men and women before and after administration of cross-sex steroids, which potentially influence many elements of the insulin resistance syndrome, including PAI-1 levels and visceral fat accumulation. We assessed the visceral fat area (by MRI); total body fat; insulin sensitivity (with a glucose clamp technique); and plasma levels of PAI-1, insulin, and triglycerides in young (<37 years old), nonobese (body mass index <28 kg/m2), healthy men (n=18) and women (n=15) before and after 12 months of cross-sex hormone administration. Men were treated with ethinyl estradiol 100 microgram/d plus cyproterone acetate 100 mg/d, and women were treated with testosterone esters 250 mg IM every 2 weeks. At baseline, only visceral fat area was significantly correlated with plasma PAI-1 levels in both men (r=0.57, P=0.03) and women (r=0.59, P=0.03). In multivariate linear regression analysis, this association was independent of total body fat, insulin sensitivity, and plasma levels of triglycerides and insulin. After 12 months of cross-sex hormone administration, the plasma PAI-1 levels were no longer correlated with visceral fat (which had increased). We conclude that in young, nonobese men and women, visceral fat area is an important determinant of plasma PAI-1 levels. After cross-sex hormone administration, this association was no longer demonstrable.     

Troglitazone improves defects in insulin action, insulin secretion, ovarian steroidogenesis, and fibrinolysis in women with polycystic ovary syndrome

Women with polycystic ovary syndrome (PCOS) are characterized by defects in insulin action, insulin secretion, ovarian steroidogenesis, and fibrinolysis. We administered the insulin-sensitizing agent troglitazone to 13 obese women with PCOS and impaired glucose tolerance to determine whether attenuation of hyperinsulinemia ameliorates these defects. All subjects had oligomenorrhea, hirsutism, polycystic ovaries, and hyperandrogenemia. Before and after treatment with troglitazone (400 mg daily for 12 weeks), all had 1) a GnRH agonist (leuprolide) test, 2) a 75-g oral glucose tolerance test, 3) a frequently sampled iv glucose tolerance test to determine the insulin sensitivity index and the acute insulin response to glucose, 4) an oscillatory glucose infusion to assess the ability of the beta-cell to entrain to glucose as quantitated by the normalized spectral power for the insulin secretion rate, and 5) measures of fibrinolytic capacity [plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator]. There was no change in body mass index (39.9 +/- 1.4 vs. 40.2 +/- 1.4 kg/m2) or body fat distribution after treatment. Both the fasting (91 +/- 3 vs. 103 +/- 3 mg/dL; P < 0.001) and 2 h (146 +/- 8 vs. 171 +/- 6 mg/dL; P < 0.02) plasma glucose concentrations during the oral glucose tolerance test declined significantly. There was a concordant reduction in glycosylated hemoglobin to 5.7 +/- 0.1 from a pretreatment level of 6.1 +/- 0.1% (P < 0.03). Insulin sensitivity increased from 0.58 +/- 0.14 to 0.95 +/- 0.26 10(-5) min-1/pmol.L (P < 0.01) after treatment as did the disposition index (745 +/- 135 vs. 381 +/- 96; P < 0.05). The ability of the beta-cell to appropriately detect and respond to an oscillatory glucose infusion improved significantly after troglitazone treatment; the normalized spectral power for the insulin secretion rate increased to 5.9 +/- 1.1 from 4.3 +/- 0.8 (P < 0.05). Basal levels of total testosterone (109.3 +/- 15.2 vs. 79.4 +/- 9.8 ng/dL; P < 0.05) and free testosterone (33.3 +/- 4.0 vs. 21.2 +/- 2.6 pg/mL; P < 0.01) declined significantly after troglitazone treatment. Leuprolide-stimulated levels of 17-hydroxyprogesterone, androstenedione, and total testosterone were significantly lower posttreatment compared to pretreatment. The reduction in androgen levels occurred independently of any changes in gonadotropin levels. A decreased functional activity of PAI-1 in blood (from 12.7 +/- 2.8 to 6.3 +/- 1.4 AU/mL P < 0.05) was associated with a decreased concentration of PAI-1 protein (from 64.9 +/- 9.1 to 44.8 +/- 6.1 ng/mL; P < 0.05). No change in the functional activity of tissue plasminogen activator (from 5.3 +/- 0.4 to 5.1 +/- 0.5 IU/mL) was observed despite a decrease in its concentration (from 9.6 +/- 0.9 to 8.2 +/- 0.7 ng/mL; P < 0.05). The marked reduction in PAI-1 could be expected to improve the fibrinolytic response to thrombosis in these subjects. We conclude that administration of troglitazone to women with PCOS and impaired glucose tolerance ameliorates the metabolic and hormonal derangements characteristic of the syndrome. Troglitazone holds potential as a useful primary or adjunctive treatment for women with PCOS.     

Fibrinolytic response in subjects with hypertriglyceridemia and low HDL cholesterol

The combination of hypertriglyceridemia and low high-density lipoprotein cholesterol (HDL-C) appears to be an excessively high risk factor for coronary artery disease (CAD). In the Helsinki study, both coronary events and mortality were decreased by gemfibrozil, especially in subjects with low HDL-C and high triglycerides (TG). On the other hand, it is known that high levels of TG can be associated with high levels of circulating plasminogen activator inhibitor (PAI), which is also a possible risk factor for CAD. The aim of the present study was to see: 1) whether the combination of low HDL-C and high TG is associated with a more impaired fibrinolytic response than in either isolated condition, and 2) whether gemfibrozil administration can improve fibrinolysis in patients with both high TG and low HDL-C. Twelve non-obese, non-diabetic subjects (eight men, four women; mean age 55 +/- 13 yrs) with low HDL-C (< 35 mg/dL men; < 45 mg/dL women) and high TG (mean 253.6 +/- 42.6 mg/dL) entered the study (Group A). Additionally fourteen comparable subjects with normal HDL-C were also investigated (Group B), plus 12 comparable subjects with isolated low HDL-C (Group C). Ten healthy people served as the control group. The following plasma fibrinolytic parameters were measured: tissue plasminogen activator antigen, PAI antigen and activity, euglobulin fibrinolytic activity (EFA) on fibrin plates, plasminogen and alpha-2-antiplasmin activities. All except the latter two values were also measured after venous occlusion (vo). In baseline conditions, patients in Groups A and B had higher EFA values before vo and higher PAI-1 antigen and alpha-2-antiplasmin levels after vo than those of controls or the subjects in Group C. The relationship between PAI antigen and PAI activity and TG was not confirmed in our population (n = 48). We also saw no interference due to HDL-C, while there was a significant relationship between EFA before vo and both TG and cholesterol. After gemfibrozil treatment (600 mg bid for 12 weeks), the lipid profiles of subjects with high TG and low HDL-C were significantly improved. There was also a slight reduction of PAI activity after vo, while the PAI-1 antigen had decreased significantly from baseline after vo (56.3 +/- 13 ng/mL before vo; 48.4 +/- 21 ng/mL after vo; P = 0.04). The higher risk of CAD in patients with low HDL-C and high TG might be in part related to impairment of fibrinolysis, which occurs in patients with isolated high TG. The close relationship existing between both TG and cholesterol levels and fibrinolytic activity confirm the key role of this latter process in the development of CAD.     

Role of plasminogen activator inhibitor-1 in promoting fibrin deposition in rabbits infused with ancrod or thrombin

The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P < .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.     

Atheromatous plaque macrophages produce plasminogen activator inhibitor type-1 and stimulate its production by endothelial cells and vascular smooth muscle cells

The capacity of macrophages to influence directly and indirectly fibrinolytic processes in atherosclerosis was studied using macrophages isolated from atherosclerotic plaques of patients undergoing surgical repair of distal aortic and femoral arteries. These cells were characterized by their morphology, adherence, esterase positivity, and expression of CD14 antigen. Production of plasminogen activator inhibitor type-1 (PAI-1) by plaque macrophages (6.7 +/- 2.7 ng/10(5) cells/24 hours [mean +/- SEM]) was significantly greater than PAI-1 production by blood monocytes isolated simultaneously from the same patients (1.8 +/- 1.5 ng/10(5) cells/24 hours). Production of tissue type plasminogen activator and urokinase type was not augmented compared to blood monocytes. Conditioned medium from cultured plaque macrophages significantly increased production of PAI-1 by endothelial cells (85 +/- 11% above basal) and vascular smooth muscle cells (25 +/- 10%) in vitro. This response was significantly greater than the response to monocyte-conditioned medium (endothelial cells 38 +/- 11%, vascular smooth muscle cells 2.5 +/- 2.0%). Stimulation of endothelial cell PAI-1 production by macrophage-conditioned medium was partially inhibitable by a monoclonal antibody to transforming growth factor-beta. Tissue type plasminogen activator production by endothelial cells and vascular smooth muscle cells was not affected by plaque macrophage- or monocyte-conditioned medium. Urokinase type plasminogen activator production by endothelial cells and vascular smooth muscle cells was undetectable in control medium and was augmented to similar levels in response to plaque macrophage- and monocyte-conditioned media. These results demonstrate upregulation of PAI-1 production by macrophages in atheromatous plaques and the capacity of soluble products from plaque macrophages to upregulate PAI-1 production by endothelial cells and vascular smooth muscle cells in vitro. These data suggest that macrophages in atherosclerotic plaques may inhibit thrombolysis both directly and indirectly by effects of their soluble products on endothelial cells and vascular smooth muscle cells.     

Effect of a recombinant dimeric tumor necrosis factor receptor on inflammatory responses to intravenous endotoxin in normal humans

To determine the role of tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-induced inflammation, 12 healthy subjects received an intravenous injection with LPS (2 ng/kg) preceded by infusion of either a recombinant human dimeric TNF receptor type II-IgG fusion protein (TNFR:Fc; 6 mg/m2; n = 6) or vehicle (n = 6) from -30 minutes to directly before LPS injection. LPS elicited a transient increase in plasma TNF activity, peaking after 1.5 hours (219 +/- 42 pg/mL; P < .05). Infusion of TNFR:Fc completely neutralized endogenous TNF activity. LPS administration was associated with an early activation of fibrinolysis (plasma concentrations of tissue-type plasminogen activator, plasminogen activator activity, and plasmin-alpha2-antiplasmin complexes), followed by inhibition (plasma plasminogen activator inhibitor type I), changes that were completely prevented by TNFR:Fc. By contrast, TNFR:Fc did not influence LPS-induced activation of coagulation (plasma levels of prothrombin fragment F1 + 2 and thrombin-antithrombin III complexes). TNFR:Fc strongly inhibited endothelial cell activation (plasma levels of soluble E-selectin), modestly reduced neutrophil responses (neutrophilia and plasma concentrations of elastase-alpha1-antitrypsin complexes and lactoferrin), but did not affect the release of secretory phospholipase A2 or lipopolysaccharide-binding protein (P > .05). Infusion of TNFR:Fc only (without LPS) in another 6 normal subjects did not induce any inflammatory response. These data indicate that TNF is involved in only some inflammatory responses to intravenous LPS in humans.     

The mast cell as site of tissue-type plasminogen activator expression and fibrinolysis

Recent data suggest that mast cells (MC) and their products (heparin, proteases) are involved in the regulation of coagulation and fibrino(geno)lysis. The key enzyme of fibrinolysis, plasmin, derives from its inactive progenitor, plasminogen, through catalytic action of plasminogen activators (PAs). In most cell systems, however, PAs are neutralized by plasminogen activator inhibitors (PAIs). We report that human tissue MC as well as the MC line HMC-1 constitutively produce, express, and release tissue-type plasminogen activator (tPA) without producing inhibitory PAIs. As assessed by Northern blotting, highly enriched lung MC (>98% pure) as well as HMC-1 expressed tPA mRNA, but did not express mRNA for PAI-1, PAI-2, or PAI-3. The tPA protein was detectable in MC-conditioned medium by Western blotting and immunoassay, and the MC agonist stem cell factor (c-Kit ligand) was found to promote the release of tPA from MC. In addition, MC-conditioned medium induced fibrin-independent plasmin generation as well as clot lysis in vitro. These observations raise the possibility that MC play an important role in endogenous fibrinolysis.     

Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in tissue and serum of head and neck squamous cell carcinoma patients

The aim of this study was to determine urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) concentrations in tumour and adjacent normal tissue samples from 58 patients, and in serum samples from 40 of 58 patients with squamous cell carcinoma of the head and neck obtained at diagnosis and after completion of therapy. uPA and PAI-1 serum concentrations were also measured in 28 healthy volunteers who served as controls. Measurements were made using enzyme-linked immunosorbent assay (ELISA) techniques. For both uPA and PAI-1, significantly elevated concentrations were measured in tumour tissue as compared with normal tissue (uPA: 8.89 versus 0.41 ng/mg total protein (mgp), P < 0.0001; PAI-1: 23.9 versus 1.47 ng/mgp, P < 0.0001). A statistically significant difference in uPA concentrations was found between normal laryngeal and nonlaryngeal tissue (0.52 versus 0.3 ng/mgp, P = 0.008), and in PAI-1 concentrations between T1 + 2 and T3 + 4 stage of disease (17.32 versus 35.63 ng/mgp, P = 0.04). The uPA concentrations positively correlated with those of PAI-1 measured in both tumour (Rs = 0.62, P < 0.0001) and normal tissue (Rs = 0.30, P = 0.02). In serum samples, lower concentrations of PAI-1 were measured in the control group than in patients with cancer (412.0 versus 680.5 ng/ml serum (mls), P = 0.0006). The time of collection of the serum sample did not influence uPA and PAI-1 concentrations, and no association was observed between their concentrations and any clinical and histopathological prognostic factors tested. Our results indicate that both uPA and PAI-1 may play a specific role in the process of invasion and metastasis, and might also be of prognostic value in squamous cell carcinoma of the head and neck.     

Novel low-molecular-weight inhibitor of PAI-1 (XR5118) promotes endogenous fibrinolysis and reduces postthrombolysis thrombus growth in rabbits

BACKGROUND: Elevated levels of plasminogen activator inhibitor 1 (PAI-1) have been associated with the occurrence of thrombotic disease, and inhibition of PAI-1 activity in vivo resulted in enhanced thrombolysis and a reduction in reocclusion. Besides monoclonal antibodies and peptides, no suitable agents that are able to block PAI-1 activity are available to date. The present study was designed to test the interaction between a nonantibody, nonpeptide, diketopiperazine-based inhibitor of PAI-1, XR5118, and PAI-1 and to assess the effect of XR5118 on PAI-1 activity in vitro and on in vivo thrombolysis and thrombus growth in an experimental thrombosis model in rabbits. METHODS AND RESULTS: The binding site of XR5118 on the PAI-1 molecule was studied by competitive binding experiments with mapped anti-PAI-1 monoclonal antibodies by use of surface plasmon resonance experiments. XR5118 selectively and competitively inhibited binding of the PAl-1-inhibiting monoclonal antibody CLB-2C8, indicating that binding of XR5118 to PAI-1 takes place at the area between amino acids 110 and 145 of the PAI-1 molecule, which is known to be involved with the binding of PAI-1 to tissue plasminogen activator (TPA). Incubation of plasma or platelet releasate with XR5118 resulted in a dose-dependent inhibition of PAI-1 activity. Systemic infusion of XR5118 induced a significant reduction in plasma PAI-1 activity levels from 23.7+/-4.9 to 10.9+/-3.4 IU/mL. Administration of XR5118 resulted in a significant, twofold increase in endogenous thrombolysis compared with the control. Thrombus growth in rabbits receiving both XR5118 and rTPA was significantly attenuated compared with rabbits receiving rTPA alone (13.5+/-2.7% versus 19.9+/-3.8%, respectively). CONCLUSIONS: XR5118 binds to PAI-1 and reduces plasma PAI-1 activity levels. Furthermore, XR5118 promotes endogenous thrombolysis and inhibits thrombus accretion and is the first nonpeptide compound with significant anti-PAI-1 activity in vivo in these models.     

Small GTP-binding proteins in the brain-corpus cardiacum-corpus allatum complex of the silkworm, Bombyx mori: involvement in the secretion of prothoracicotropic hormone

BACKGROUND: Proteolysis, modulated in part by intramural fibrinolytic system proteins and their inhibitors, appears to influence vascular smooth muscle cell (SMC) migration and proliferation and remodeling of extracellular matrix (ECM). Alterations of fibrinolysis in circulating blood and of proteolysis within vessel walls in experimental animals and patients with diabetes have been associated with accelerated vascular disease. Hyaluronan, a prominent component of ECM in normal vessels, is increased in the tunica media of macroscopically normal arterial vessels from patients with type 2 diabetes. OBJECTIVE: To determine whether hyaluronan alters the expression of the fibrinolytic system protein, plasminogen activator inhibitor type-1 (PAI-1), in human vascular SMCs, thereby potentially accelerating vascular disease in patients with type 2 diabetes. METHODS: Urokinase-type and tissue-type plasminogen activators (uPA and tPA) and PAI-1 were assayed in vascular SMC conditioned media and in cell lysates, using enzyme-linked immunosorbent assay and western blotting. RESULTS: Hyaluronan increased the 24-h release of PAI-1 into conditioned media in a concentration-dependent and time-dependent manner (1.8-fold compared with control with 1 mg/ml hyaluronan; n = 9, P < 0.01). Although the accumulation of uPA in conditioned media tended to increase also, uPA content was reduced in cell lysates (64% of control with 0.1 mg/ml hyaluronan at 24 h; n = 9, P < 0.01) without any change in PAI-1. Concentrations of tPA in conditioned media and cell lysates were unchanged. Digestion of hyaluronan with hyaluronidase (50 turbidity reducing units (TRU)/ml) or exposure of the smooth muscle cells to antihuman CD44 antibody (1 microgram/ml) that binds to the hyaluronan cell surface receptor obviated the effects of hyaluronan. CONCLUSION: Our results indicate that increases in hyaluronan increase vascular SMC expression of PAI-1, a phenomenon that may alter the balance between proteolysis and its inhibition in vessels of patients with type 2 diabetes, thereby contributing to the acceleration of macroangiopathy.