Tumor necrosis factor alpha increases antifibrinolytic activity of cultured human mesangial cells.

Tumor necrosis factor alpha (TNF alpha) is likely to exert a major influence in the pathogenesis of glomerulopathies. Besides its proinflammatory properties. TNF alpha interacts with cell growth and synthesis of components of the fibrinolytic system. In this study, we report the effects of recombinant human TNF alpha on the synthesis of tissue-type plasminogen activator (t-PA) and its inhibitor (PAI-1) by human mesangial cells in culture. We first demonstrate that TNF alpha binds specifically to a single class of high affinity receptors (Kd 5.10(-11) M; 1500 receptors/cell). TNF alpha has an antimitogenic effect on human mesangial cells since it decreased DNA synthesis, measured by 3H-thymidine incorporation, in a dose-dependent manner. Release of cytosolic LDH and incorporated 51Cr was not increased by 100 ng/ml TNF alpha as compared with control, indicating that this monokine is not cytotoxic for cultured human mesangial cells. Zymographic analysis and reverse fibrin autography disclosed a 120 kD t-PA-PAI-1 complex and a 50 kD free form of PAI-1 in the supernatants of both unstimulated and TNF-stimulated cells; PAI-1 was released in excess and free t-PA was not observed. TNF alpha (0 to 100 ng/ml) had no effect on t-PA synthesis, but enhanced PAI-1 release in a time- and dose-dependent manner (97% increase of PAI-1 synthesis after a 24 hour incubation). This effect was abolished by cycloheximide, suggesting that protein synthesis was required. Northern blot analysis showed that TNF alpha increased the steady-state PAI-1 mRNA levels in a time-dependent manner, with a maximal effect at two hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a plasminogen activator and its inhibitor in human mesangial cells

In the course of some pathological and experimental nephropathies, intraglomerular fibrin deposits develop, possibly as a consequence of inefficient fibrinolysis. In vitro human glomeruli exhibit fibrinolytic activity due to the synthesis of plasminogen activators (PAs) such as, tissue-type PA (t-PA) and urokinase-type PA (u-PA). Immunofluorescence studies have previously shown that t-PA is localized in the capillary tufts and u-PA in the visceral epithelial cells. We have now investigated the fibrinolytic activity of cultured human mesangial cells. Inhibitory activity towards u-PA or t-PA but not plasmin was found in both conditioned medium and cellular extracts. Analysis of the conditioned medium by zymography revealed a single band of PA-activity (Mr: 110 to 120 kDa). Immunoneutralization with anti-t-PA and anti-plasminogen activator inhibitor (PAI-1) IgG but not anti-u-PA or anti-PAI-2 removed this band. Reverse fibrin autography demonstrated the presence of PAI-1 in both cellular extracts and in conditioned medium. Western Blot analysis showed that two bands (50 kD and 120 kD) were recognized by the anti-PAI-1 antibody. By ELISA t-PA and PAI-1 antigens were found to increase progressively with time in the culture medium but not in cellular extracts. Both t-PA and PAI-1, but not u-PA and PAI-2, were also detected by immunofluorescence studies. Thus human glomerular mesangial cells synthesize and secrete t-PA and PAI-1 in vitro. PAI-1 is produced in excess, therefore t-PA is only found in the form of a complex with PAI-1.     

Secretion of plasminogen activator and its inhibitor by glomerular epithelial cells

The effects of thrombin, interleukin-1 (IL-1), tumor necrosis factor (TNF) and gamma-interferon (gamma-IFN) on the release of plasminogen activator (PA) and inhibitor (PAI) were studied using cultivated human glomerular epithelial cells (GECs). Species of PAs and PAI secreted from the GECs were urokinase-type PA (u-PA) and tissue-type PA (t-PA), while the major species was a single chain u-PA in the amount of 28.6 +/- 2.34 ng/10(5) cells for 24 hours (N = 4, mean +/- SD), and PAI-1. The addition of increased concentrations of thrombin (0.1 to 31.6 U/ml) into confluent cultures enhanced the GECs to release u-PA, t-PA and PAI-1 in a dose- and time-dependent manner. The incubation of the GECs with 10 U/ml thrombin resulted in about a fourfold increase in the concentration of u-PA, threefold in t-PA and twofold in PAI-1. All thrombin effects, however, were suppressed by the simultaneous addition of cycloheximide, indicating that the enhancing effects of thrombin were due to an increase in the production of PAs and PAI-1, via protein synthesis. These thrombin effects appeared to be dependent upon the enzymatically active site of thrombin because DFP-thrombin had no effect. In the conditioned medium which was under continuous thrombin stimulation for 24 hours, no u-PA activity was detectable, even after the plasmin treatment, because a single chain u-PA was degraded by the thrombin. The stimulation of cultured GECs with thrombin only for the first three hours in 24 hour cultivation showed an apparent increase in the antigenic amount of u-PA. IL-1 enhanced the release of t-PA and PAI-1, and TNF did that of u-PA and t-PA, while gamma-IFN showed no significant effects. These findings indicate that the GECs participate in the regulation of extracapillary fibrinolysis in the glomerular microenvironment, as being modulated by thrombin and two cytokines, IL-1 and TNF.     

Simvastatin increases fibrinolytic activity in human peritoneal mesothelial cells independent of cholesterol lowering

BACKGROUND: The continuous physical and chemical irritation of the peritoneum in peritoneal dialysis patients can result in a nonbacterial serositis with increased fibrin deposition, thus promoting peritoneal fibrosis and adhesion development. By expressing the fibrinolytic enzyme tissue-type plasminogen activator (t-PA) and its specific inhibitor, plasminogen activator inhibitor-1 (PAI-1), human peritoneal mesothelial cells (HMC) play an important role in regulating peritoneal fibrinolysis. METHODS: Cultured HMC were used to examine the effect of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, simvastatin, on the expression of t-PA and PAI-1. Antigen concentrations in the cell supernatants were measured by ELISA and Northern blot analysis was conducted for mRNA expression. RESULTS: Simvastatin time- and concentration-dependently increased t-PA and decreased PAI-1 synthesis, reaching maximal effects after 48 hours, when simvastatin (1 micromol/L) increased t-PA levels 5.1 +/- 0.1-fold and suppressed PAI-1 levels 2.6 +/- 0.2-fold. This was accompanied by a twofold increase in mesothelial cell-associated t-PA activity. Qualitatively similar results were obtained in cultured human endothelial cells, but the effects were less pronounced and required higher simvastatin concentrations. Northern blot analysis revealed that the action of simvastatin on t-PA and PAI-1 expression in HMC can be explained by parallel changes in t-PA and PAI-1 mRNA. The effects of simvastatin were prevented in the presence of mevalonate and geranylgeraniol, suggesting that the effect of simvastatin on t-PA and PAI-1 synthesis is mediated through geranylgeranyl-modified intermediates. Experiments using specific inhibitors of geranylgeranylated Rho GTPases excluded a role of members of this family of small GTP-binding proteins in simvastatin action in HMC. The effects of simvastatin on t-PA and PAI-1 expression as well as on cell shape were completely mimicked by cytochalasin D, a disrupter of cellular actin filaments, but not by colchicine, a disrupter of microtubules. CONCLUSIONS: In conclusion, the cholesterol-lowering drug simvastatin is an effective stimulator of local peritoneal fibrinolytic activity, as it increases t-PA and decreases PAI-1 production in mesothelial cells by a mechanism involving geranylgeranyl-modified intermediates and actin skeleton perturbation. These results provide a new rationale to prevent peritoneal fibrin deposition and adhesion development in peritoneal dialysis patients.     

Effect of dextran on plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1(PAI-1) during surgery

Dextran is known to increase the plasminogen activation rate in vitro and to decrease the α₂-antiplasmin activity.
We decided to explore the effect of dextran on plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1(PAI-1) during surgical trauma.
Thirty-one patients undergoing elective surgery were given 500 ml of 6% dextran 70. Another nine patients serving as controls were given 500 ml of a glucose-electrolyte solution. The activities of t-PA and PAI-1during surgery were determined, as was the concentration of t-PA antigen.
PAI-1activity was decreased by 19% after infusion of 250 ml of dextran. After 500 ml, the activity was reduced by 22% (both P <0.05). The activity of t-PA was increased by 43% and 29% (both P <0.05) and the antigenic amount of t-PA was increased by 18% and 15% (both P <0.05) after infusion of 250 ml and 500 ml of dextran, respectively. No changes in these variables were observed in the control patients.
It is concluded that infusion of dextran promotes fibrinolysis by enhancing plasminogen activation in patients subjected to trauma. Since elevated levels of PAI-1prior to surgery are known to predispose to deep vein thrombosis, which may form already during the operation, the effect of dextran on PAI-1described here may explain its clot preventing properties.     

多囊卵巢综合征患者血纤溶能力的研究

目的了解多囊卵巢综合征(PCOS)患者的血纤溶能力及服用二甲双胍后血纤溶能力的变化。方法(1)对照组20例、PCOS患者30例,分别取血检测组织纤溶酶原激活物(t-PA)、纤溶酶原激活抑制物(PAI-1)的活性;(2)PCOS高胰岛素血症者10例,分别给予二甲双胍750~1 500 mg/d治疗,比较治疗前、后t-PA、PAI-1活性。结果PCOS组的血胰岛素(INS)、游离睾酮(F-T)t、-PA、PAI-1分别为(24.42±12.30)mU/L(、2.70±1.50)ng/L、(0.17±0.06)KU/L(、0.88±0.05)AU/ml,对照组分别为(10.04±6.12)mU/L(、1.70±1.00)ng/L(、0.28±0.04)KU/L、(0.70±0.09)AU/ml。10例PCOS患者经二甲双胍治疗后INS、F-Tt、-PA、PAI-1分别为(12.20±7.78)mU/L、(1.70±1.00)ng/L、(0.26±0.08)KU/L、(0.70±0.35)AU/ml。结论PCOS患者的血纤溶能力低于正常者,而服用二甲双胍后血纤溶能力明显改善。     

Enhanced fibrinolytic activity during the course of hemodialysis.

In order to clarify the effect of hemodialysis (HD) on the fibrinolytic system, fibrinolytic activity was evaluated in 27 patients undergoing regular hemodialysis treatment (RDT) using new parameters including plasma alpha 2-plasmin inhibitor (alpha 2 PI), alpha 2-plasmin inhibitor-plasmin complex (alpha 2 PIC), cross-linked fibrin degradation products (XL-FDP), tissue plasminogen activator (t-PA) activity, t-PA antigen and plasminogen activator inhibitor-1 (PAI-1) antigen. Predialysis baseline levels of plasminogen and alpha 2PI activity in RDT patients were significantly lower and those of alpha 2PIC were significantly higher than normal control values. During a single HD session, alpha 2PIC exhibited a continuous, significant increase reaching about 180% of initial values by the end of HD. alpha 2PI activity was significantly decreased at the end of the HD, though there were no significant changes in plasminogen activity during HD. Predialysis baseline levels of XL-FDP in RDT patients were significantly higher than normal control values. No significant changes in XL-FDP were observed during HD. Both t-PA activity and t-PA antigen significantly increased during HD, and PAI-1 antigen significantly decreased during HD. Von Willebrand factor (vWF) antigen in plasma, which is regarded as reflecting a release reaction by vascular endothelial cells to certain stimuli, also significantly increased during HD. However, neither vWF antigen nor t-PA antigen was increased by heparin administration alone. The changes in alpha 2PI and alpha 2PIC levels suggest that fibrinolytic activity is slightly higher in RDT patients and is even higher during HD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasminogen activator inhibitor 1 in renal fibrin deposits of human nephropathies

The persistency of fibrin deposits in the kidney during renal diseases could reflect either a defective release of plasminogen activators (PA) or a local excess of PAI. In order to investigate this question, we studied human renal biopsies by immunofluorescence technique with specific antibodies for fibrin, tissue-type plasminogen activator (t-PA), urokinase (u-PA), PAI-1 and PAI-2. By this technique t-PA could be detected in the glomerular flocculus and the endothelium of small arteries of the normal control kidneys. We failed to detect significant fluorescence with other antibodies in normal kidneys. Conversely, in cases of vascular nephropathy with thrombosis the positive fluorescence obtained with anti-fibrin antibodies at the site of thrombosis was associated with a positive fluorescence with anti-PAI-1 and to a lesser extent with anti-t-PA antibodies. u-PA and PAI-2 were not detected in these lesions. Similarly in the most severe forms of crescentic glomerulonephritis, extracapillary fibrin deposits were associated with PAI-1. In one case u-PA was also detected. This is in agreement with our previous findings that glomerular epithelial cells release both PAI-1 and the inactive form of u-PA (pro u-PA). Thus, our results support the hypothesis that PAI-1, which is able to inhibit both t-PA and u-PA, may play a major role in the persistency of fibrin deposits in the human kidneys during pathological conditions.     

Tissue-type plasminogen activator activity in HIV-associated HUS

Background: In children, haemolytic uraemic syndrome (HUS) is associated with high plasma plasminogen activator inhibitor type 1 (PAI-1), which may contribute to the persistence of renal glomerular and arteriolar thrombi. HUS has been described in HIV-infected patients, but the pathophysiology of HUS in these patients is poorly understood. The aim of the study was to investigate plasma fibrinolytic activity in 18 patients with HIV-associated HUS. Methods: We measured tissue type plasminogen activator (t-PA) and PAI-1 activities in the plasma of 18 HIV-infected patients with biopsy-proven HUS (HIV+/HUS+0 and 48 HIV-infected patients without HUS (HIV+/HUS-). Results: Patients with HUS had a significantly higher serum creatinine, a lower platelet count and an increased incidence of cytomegalovirus (CMV) infection (72% of patients HIV+/HUS+, vs 25% of patients HIV+/HUS-). Unexpectedly, plasma PAI-1 activity was similar in both groups. However, t-PA activity was significantly higher in HUS cases (11.5 vs 4.5 U/ml, P=0.001). Patients with CMV infection, with or without HUS, had significantly increased t-PA level (P=0.01). Multivariate analysis identified high t-PA (RR=9.21) and CMV infection (RR=3.36) as risk factors for HUS. Conclusion: This study provides evidence that HIV-infected patients with HUS have high plasma t-PA activity. PAI-1 plasma activity is not significantly increased, as opposed to non-HIV-associated HUS. Thus, in the setting of HIV infection, HUS cannot be attributed to decreased fibrinolytic activity.     

Production of Urokinase-Type Plasminogen Activator (u-PA) and Plasminogen Activator Inhibitor-1 (PAI-1) in Human Brain Tumours

Summary We investigated the role of plasminogen activators (PAs) and their inhibitor (plasminogen activator inhibitor-1, PAI-1) in human brain tumours. The amounts of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1), and the activity of u-PA and t-PA were determined by enzyme-linked immunosorbent assay (ELISA), and u-PA and PAI-1 were immunolocalized using monoclonal antibodies in human brain tumours and normal brain tissues. The tissues were surgically removed from 64 patients; normal brain tissue (5 cases), low-grade glioma (4 cases), high-grade glioma (17 cases), metastatic tumour (9 cases), meningioma (benign 12 cases, malignant 6 cases), acoustic schwannoma (11 cases). u-PA activity and u-PA and PAI-1 antigen levels were significantly elevated in malignant brain tumours (malignant meningiomas, high-grade gliomas, and metastatic tumours) and acoustic schwannomas but very low in benign meningiomas, low-grade gliomas and normal brain. There was no difference in t-PA antigen levels among normal and malignant tissues, however levels of t-PA activity were markedly decreased in metastastic tumours. All malignant brain tumour tissues showed positive immunostaining for u-PA and PAI-1, however, some tumour cells showed negative intensity while others showed strong intensity for these antibodies. This contrasts to the homogeneous staining pattern found in acoustic schwannoma. These findings indicate that malignancy in human brain tumours is associated with elevated levels of u-PA and PAI-1 and that an imbalance between these proteins in a micro-enviroment contributes (ascribes) to tumour cell invasion.     

Hyaluronan levels during laparoscopic versus open colonic resections

Background Plasma hyaluronan binds to fibrinogen, affecting intravascular fibrin polymerization and fibrin clot formation. It has been hypothesized that alterations in fibrin clot formation influence the risk of thromboembolism in those undergoing surgery. The aim of this study is to quantify the intravascular components, especially plasma hyaluronan levels, in laparoscopic and conventional colorectal resections that contribute to thromboembolism formation. Methods Prospective cohort analysis of consecutive patients which were participating in the prospective randomized multi-center trial Lapkon II comparing the long-term effects of laparoscopic and conventional resection for colon cancer. Plasma samples were obtained from 15 patients at the beginning and the end of laparoscopic or conventional colorectal resections. Concentrations and activities of tissue plasminogen activator(t-PA), plasminogen activator inhibitor type 1(PAI-1), t-PA/PAI complex, fibrinogen, d-dimers and hyaluronan were determined by using commercial enzyme-linked immunosorbent assay (ELISA) kits. Results No differences in age, sex and type of resection between the laparoscopic and conventional-surgery groups were observed. Laparoscopic procedures lasted longer (p < 0.05). Concentration and activities of t-PA, PAI-1, t-PA/PAI complex, fibrinogen and d-dimers did not vary between the two groups. Plasma hyaluronan decreased from 28.6 to 17.9 IU/ml (p < 0.05) during laparoscopic compared to conventional procedures. Plasma hyaluronan levels were significantly different at the end of operation between the two groups (p < 0.05) . Conclusions Plasma hyaluronan levels were decreased in patients undergoing laparoscopic colorectal resections, compared to those undergoing conventional procedures. Therefore, interactions between plasma hyaluronan and fibrinogen may be lower, with a sequential decrease in fibrin polymerization, and a possibly reduced risk of deep venous thrombosis.     

t-PA promotes glomerular plasmin generation and matrix degradation in experimental glomerulonephritis

BACKGROUND: In addition to its well-known role in degrading fibrin, recent evidence suggests that plasmin degrades matrix proteins and activates prometalloproteinases. Plasmin is generated from plasminogen by tissue plasminogen activator (t-PA). We hypothesized that t-PA treatment increases plasmin generation in nephritic glomeruli and degrades pathological matrix leading to a therapeutic reduction in matrix accumulation. METHODS: Anti-Thy-1 nephritis was induced by injection of OX-7 antibody. Rats were given twice daily intravenous injections of saline (disease control group) or human recombinant t-PA (rt-PA; 1 mg/kg body weight) on days 3 through 5. Proteinuria, glomerular matrix protein staining, and glomerular mRNA levels for transforming growth factor-beta 1 (TGF-beta 1), fibronectin, and plasminogen activator inhibitor type 1 (PAI-1) were evaluated at day 6. Localization of rt-PA, plasmin generation by glomeruli in vitro, and glomerular production and content of active TGF-beta1 were also investigated. RESULTS: Compared with disease control animals, proteinuria and staining score for periodic acid-Schiff (2.75 +/- 0.17 vs. 1.41 +/- 0.09), fibronectin-EDA+ (19 +/- 2 vs. 14 +/- 1), laminin (35 +/- 2 vs. 25 +/- 2), type I collagen (33 +/- 1 vs. 21 +/- 3), and type IV collagen (27 +/- 2 vs. 23 +/- 1) were significantly reduced in treated rats (P < 0.01). Glomerular TGF-beta 1, fibronectin, and PAI-1 mRNA levels were unchanged. rt-PA colocalized with fibrin along glomerular capillary walls and in the mesangium. Nephritic glomeruli in vitro had decreased plasmin activity, which was elevated by an in vivo presacrifice injection of rt-PA. Glomerular production and content of active TGF-beta 1 were unchanged by the rt-PA injection. CONCLUSIONS:: These results show that injected rt-PA binds to fibrin in nephritic glomeruli, thus increasing plasmin generation and promoting pathological matrix degradation without activating latent TGF-beta. Agents that increase plasmin generation, such as t-PA, may have potential as antifibrotic therapies.     

Stimulation of PAI-1 in rabbit anti-GBM glomerulonephritis

SUMMARY: It has previously been shown in human disease and animal models of glomerulonephritis (GN) that fibrin deposition is associated with a net reduction of glomerular fibrinolytic activity as a result of reduced expression of plasminogen activators and increased expression of plasminogen activator inhibitor type 1 (PAI-1). Conditioned media (CM) prepared from cultured glomeruli of normal rabbits and rabbits 24 (Day 1) and 96 (Day 4) h after induction of anti-GBM GN were compared for their effects on the synthesis of fibrinolytic molecules in human endothelial cells (EC). Only CM from Day 4 GN rabbits showed PAI-1 protein stimulatory activity of up to 148% ( P <0.05; n = 3) above that of untreated EC. This was also seen at the mRNA level. Glomerulonephritis Day 4 CM showed significantly higher amounts of tumour necrosis factor (TNF) and thrombin and transforming growth factor-β (TGF-β) bioactivity in comparison to glomerular CM from normal rabbits. After high performance liquid chromatography (HPLC) of Day 4 GN CM, PAI-1 stimulatory activity was found to correlate with the presence of interleukin 1 (IL-1), TNF and TGF-β. These results suggest a correlation between severity of anti-GBM GN in a rabbit model, increased PAI-1 synthesis and increased expression of TNF and TGF-β. This may potentiate glomerular fibrin and extracellular matrix deposition in anti-GBM GN, leading to glomerular crescent formation and eventual renal failure.     

Intraglomerular coagulation and fibrinolysis in human primary glomerular diseases]

It is a well known fact that intraglomerular coagulation plays an important role in the development of human primary glomerular diseases. However, the precise mechanism of intraglomerular coagulation, and intraglomerular coagulability and/or fibrinolytic activity remains obscure. The present study was aimed to elucidate the role of the intraglomerular coagulation and fibrinolysis in human primary glomerular diseases. Subjects enrolled in this study were 27 patients with minimal change nephrotic syndrome (MCNS), 14 patients with focal glomerular sclerosis (FGS), 36 patients with membranous nephropathy (MN), 161 patients with mesangial proliferative glomerulonephritis (mesPGN), 9 patients with membranoproliferative glomerulonephritis (MPGN), and 40 healthy volunteers as controls. Normal human renal cortex as controls of isolated intraglomerular plasminogen activator activity (PAA) was obtained at the time of nephrectomy from the normal pole of kidneys removed because of an opposite pole tumor. Urinary urokinase (UK), fibrinopeptide A (FPA) and fibrinopeptide B beta 15-42 (B beta 15-42) antigens were measured by RIA. Urinary tissue plasminogen activator (t-PA) antigen was measured by ELISA. Urinary fibrin/fibrinogen degradation products (FDP) were measured by latex agglutination method. Moreover, PAA was measured by 125I-fibrin films. The following results were obtained: 1) In primary glomerular diseases, levels of urinary UK and t-PA were significantly lower than those in healthy volunteers, 2) Urinary UK and t-PA showed gradual decrease along with the development of mesangial proliferation, 3) Urinary UK and t-PA were significantly correlated with both the urinary FPA and B beta 15-42, 4) In mesPGN and FGS, PAA was significantly lower than that in normal controls, 5) PAA was significantly correlated with urinary UK, t-PA, FPA and B beta 15-42, 6) Urinary UK and t-PA in the patients with urinary FDP were significantly lower than those in patients without urinary FDP, 7) Urinary UK, t-PA and PAA were significantly lower in patients with intraglomerular fibrin deposition than those in patients without fibrin depositions. These findings suggest that the decrease of urinary UK and t-PA levels and the diminution of isolated intraglomerular plasminogen activator activity contribute to the progression of primary glomerular diseases.     

The relationship between cardiovascular complications of estrogen therapy and fibrinolysis in patients with prostatic cancer.

To determine the relationship between cardiovascular complications of estrogen therapy and fibrinolysis, fibrinolysis parameters plasminogen, urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1), were assessed in 12 prostatic cancer patients before and 6 weeks after the onset of estrogen therapy. The levels of plasminogen, u-PA, and PAI-1 in the patients treated with the estrogen therapy were significantly higher than those in the patients before the therapy. The t-PA level in the patients during the therapy was significantly lower than that before the treatment. Cardiovascular complications were found in two patients (16.7%) during estrogen therapy. In the two patients, marked elevation of PAI-1 and decreased level of t-PA were observed during the therapy. These results indicate that cardiovascular complications of estrogen therapy in patients with prostatic cancer may be related to hypofibrinolysis resulting from changes of PAI-1 and t-PA.     

Systemic effects of tissue plasminogen activator-associated fibrinolysis and its relation to thrombin generation in orthotopic liver transplantation

Orthotopic liver transplantation is frequently associated with hyperfibrinolysis, the origin and clinical relevance of which is largely unknown. In 20 orthotopic liver transplantations, we studied the occurrence and systemic effects of hyperfibrinolysis. Severe fibrinolysis was defined to be present when the euglobulin-clot lysis time and the whole-blood-clot lysis time, as measured by thrombelastography, were shorter than 60 and 90 min, respectively, at some time during the operation. Based on these criteria, 7 patients had minimal fibrinolysis (group I), and 13 patients had severe fibrinolysis (group II). In group II a gradual increase of tissue-type plasminogen activator (t-PA) activity was seen during the anhepatic stage, followed by an "explosive" increase immediately after graft reperfusion (P = 0.0004, compared with group I), and a reduction of plasminogen activator inhibitor (PAI) activity. Plasma degradation products of fibrinogen and fibrin increased parallel to t-PA activity, and levels were significantly higher at 45 min after graft reperfusion in group II compared with group I (P less than 0.04). Thrombin-antithrombin III complexes showed an identical steady increase in both groups, indicating that increased t-PA activity was not related to thrombin formation. A combination of increased endothelial release and reduced hepatic clearance may have caused the increased t-PA activity. The t-PA-associated destruction of fibrinogen and fibrin after graft reperfusion is consistent with the clinical signs of severe oozing often seen in this period. These observations may have important clinical implications for the treatment of bleeding in patients undergoing orthotopic liver transplantation.     

Postoperative changes in plasma tissue-type plasminogen activator and type I plasminogen activator inhibitor

To clarify the changes which occur postoperatively in intravascular fibrinolysis, plasma levels of tissue-type plasminogen activator (t-PA) antigen, the total plasminogen activator inhibitor type-1 (PAI-1) antigen, and the t-PA-PAI-1 complexes were assayed in this study. Blood samples were taken the morning before surgery, then at 0, 12, 24, 36, 60, 108, and 156h postoperatively in ten patients who underwent radical surgery for thoracic esophageal cancer. The plasma levels of the t-PA and total PAI-1 antigens, and the t-PA-PAI-1 complexes were then measured by enzyme immunoassay. The plasma t-PA and total PAI-1 levels increased significantly in the immediate postoperative period, the percent increase of the latter being much greater than that of the former. Moreover, the calculated free t-PA antigen level was decreased throughout the postoperative period, suggesting postoperative hypofibrinolysis. The platelet count and neutrophil elastase level were significantly correlated with the free t-PA antigen level atr = 0.630,P < 0.001, andr = -0.447,P < 0.01, respectively. The results of this study indicated that postoperative hypofibrinolysis caused by the increased synthesis of PAI-1 may enhance postoperative hypercoagulability, and this may lead to the development of organ damage. Thus, the concentration of the PAI-1 antigen may be a potentially important index for the prediction of postoperative illness.     

Stimulation of PAI-1 in rabbit anti-GBM glomerulonephritis

It has previously been shown in human disease and animal models of glomerulonephritis (GN) that fibrin deposition is associated with a net reduction of glomerular fibrinolytic activity as a result of reduced expression of plasminogen activators and increased expression of plasminogen activator inhibitor type 1 (PAI-1). Conditioned media (CM) prepared from cultured glomeruli of normal rabbits and rabbits 24 (Day 1) and 96 (Day 4) h after induction of anti-GBM GN were compared for their effects on the synthesis of fibrinolytic molecules in human endothelial cells (EC). Only CM from Day 4 GN rabbits showed PAI-1 protein stimulatory activity of up to 148% ( P <0.05; n =3) above that of untreated EC. This was also seen at the mRNA level. Glomerulonephritis Day 4 CM showed significantly higher amounts of tumour necrosis factor (TNF) and thrombin and transforming growth factor-β (TGF-β) bioactivity in comparison to glomerular CM from normal rabbits. After high performance liquid chromatography (HPLC) of Day 4 GN CM, PAI-1 stimulatory activity was found to correlate with the presence of interleukin 1 (IL-1), TNF and TGF-β. These results suggest a correlation between severity of anti-GBM GN in a rabbit model, increased PAI-1 synthesis and increased expression of TNF and TGF-β. This may potentiate glomerular fibrin and extracellular matrix deposition in anti-GBM GN, leading to glomerular crescent formation and eventual renal failure.     

Secretory activity of the coronary artery endothelial cells in conditions of the peritoneal dialysis

IntroductionEndothelial dysfunction is frequent in patients treated with peritoneal dialysis and may lead to cardiac complications. We evaluated the effect of effluent dialysates and serum on the function of coronary artery endothelial cells (CAEC).MethodsHuman CAEC in in vitro culture were exposed to serum and dialysates from 24 patients treated with continuous ambulatory peritoneal dialysis (CAPD) and secretion of interleukin-6 (IL6), von Willebrand factor (vWF), tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were measured. Modulation of the secretory activity of CAEC by Sulodexide, mixture of glycosaminoglycans: heparin sulfate and dermatan sulfate, was studied.ResultsSerum from CAPD patients stimulated synthesis of IL6 (+93%), vWF (+18%), and PAI-1 (+20%) and did not change t-PA secretion in CAEC. Dialysates stimulated secretion of IL6 (+89%), vWF (+29%), and PAI-1 (+31%) and did not change t-PA synthesis. Dialysates collected in 12 patients after 6 months more strongly stimulated synthesis of IL6 (+37%) and PAI-1 (+7%). Sulodexide suppressed the secretory activity of CAEC stimulated by the studied sera: IL6 (–38%), vWF (–19%), t-PA (–13%), and PAI-1 (–12%).ConclusionsSerum and the dialysate from CAPD patients induce inflammatory and prothrombotic reaction in coronary arterial endothelial cells. The general pattern of the observed effects for serum and dialysates was similar but the intensity of the effects was not identical. Sulodexide reduced these effects.