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.     

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.     

Uremic medium increases cytokine-induced PAI-1 secretion by cultured endothelial cells

The overall fibrinolytic activity is depressed in patients with chronic renal failure where a prothrombotic state is described, thereby enhancing the risk of vascular occlusive events. The mechanism responsible for fibrinolysis derangement has not yet been elucidated. To evaluate the effect of the uremic environment on the fibrinolytic activity of endothelial cells, we studied plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) production by human umbilical vein endothelial cells (HUVEC) in culture, exposed either to uremic or normal sera, before and after cytokine stimulation. Twenty uremics were studied: 11 were on conservative dietary treatment and nine were on maintenance hemodialysis. Eight healthy subjects served as controls. Before cytokine stimulation, no difference in the HUVEC supernatant concentration of t-PA and PAI-1 was found among the groups studied. After stimulation with interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha, the HUVEC supernatant levels of PAI-1 in the uremics were higher than in the controls, whereas the supernatant levels of t-PA did not differ. Our data provide evidence that uremic serum, in concert with IL-1 or TNF-alpha, can enhance PAI-1 secretion by endothelial cells, thereby depressing the fibrinolytic system. This impaired endothelial fibrinolytic response to hypercoagulation could favor vascular events, which are the major cause of morbidity and mortality in patients with chronic uremia.     

Production of urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor-1 (PAI-1) in human brain tumours

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-environment contributes (ascribes) to tumour cell invasion.     

Effects of platelet-activating factor (PAF) receptor blockage on mucous glycoprotein secretion in cultured chinchilla middle ear epithelium

The relationship between liver steatosis, evaluated by ultrasonography, and various plasma haemostatic factors was examined in 64 apparently healthy males, aged 38 years. Plasma levels of factor VII clotting activity (F-VIIc), plasminogen activator inhibitor-1 (PAI-1) activity and antigen, tissue-type plasminogen activator (t-PA) activity significantly differed in men with liver steatosis (n = 31) as compared with those without steatosis (n = 33). No significant differences were found in t-PA antigen and F-VII antigen. The men with liver steatosis also had significantly higher body mass index (BMI), plasma triglyceride and 2 h post-load insulin concentrations. While the differences in plasma haemostatic factors were substantially unchanged after adjustment for BMI, they totally disappeared when further allowance was made for plasma triglyceride and 2 h insulin concentrations. In conclusion, these results indicate that liver steatosis correlates specifically with increased PAI-1, F-VIIc and decreased t-PA levels, and suggest that such a relation is largely mediated by concomitant alterations in plasma triglyceride and insulin concentrations.     

Ceramide accumulation during oxidant renal tubular injury: mechanisms and potential consequences

Ceramide is an important signaling molecule that is typically generated via sphingomyelinase (SMase)-mediated sphingomyelin (SM) hydrolysis. Although diverse forms of renal injury elicit ceramide accumulation, the molecular determinants of this change and its contribution to tissue damage are poorly defined. The present study uses iron (Fe/hydroxyquinoline)-mediated injury of cultured human proximal tubular (HK-2) cells to gain additional insights into these issues. A 4-h Fe exposure doubled ceramide levels in the absence of cell death. This was independent of de novo synthesis, since ceramide synthase inhibition (with fumonisin B1) had no effect. Oxidant stress directly suppressed, rather than stimulated, SMase activity by: (1) decreasing SMase levels; (2) depleting SMase-stimulating glutathione; and (3) increasing SM resistance to SMase attack. Fe suppressed cell sphingosine levels (3 to 4 times ceramide/sphingosine ratio increments), suggesting a possible ceramidase block. Fe did not directly affect HK-2 ceramidase levels. However, arachidonic acid (C20:4) accumulation, a consequence of oxidant-induced phospholipase A2 (PLA2) activation, markedly suppressed ceramidase and stimulated SMase activity. Exogenous C20:4, as well as PLA2 (in doses simulating Fe-induced deacylation) recapitulated Fe's ceramide-generating effect. Because C20:4 is directly cytotoxic, it was hypothesized that ceramide might offset some of C20:4's adverse effects. Supporting this possibility were the following: (1) C20:4 exacerbated Fe toxicity; (2) this was abrogated by ceramide treatment; and (3) ceramide blunted Fe-mediated cell death. Conclusions: (1) ceramide accumulation during acute cell injury can be an adaptive response to PLA2 activation/C20:4 generation; (2) C20:4-induced ceramidase inhibition, coupled with SMase stimulation, may trigger this result; and (3) these ceramide increments may exert a "biostat" function, helping to offset C20:4/PLA2- and "catalytic" iron-mediated tubular cell death.     

Contribution of plasminogen activators and their inhibitors to the survival prognosis of patients with Dukes' stage B and C colorectal cancer

Despite the advances in pre-, peri- and post-operative medical care of colorectal carcinoma patients, the prognosis has improved only marginally over recent decades. Thus, additional prognostic indicators would be of great clinical value to select patients for adjuvant therapy. In previous studies we found that colorectal carcinomas have a marked increase of the urokinase-type of plasminogen activator (u-PA), and the inhibitors PAI-1 and PAI-2, whereas the tissue-type plasminogen activator (t-PA) is found to be decreased in comparison with adjacent normal mucosa. In the present study we evaluated the prognostic value of several plasminogen activation parameters, determined in both normal and carcinomatous tissue from colorectal resection specimens, for overall survival of 136 Dukes' stage B and C colorectal cancer patients, in relation to major clinicopathological parameters. Uni- and multivariate analyses indicated that a high PAI-2 antigen level in carcinoma, a low t-PA activity and antigen level and a high u-PA/t-PA antigen ratio in adjacent normal mucosa are significantly associated with a poor overall survival. A high ratio of u-PA antigen in the carcinomas and t-PA antigen in normal mucosa, i.e. u-PA(C)/t-PA(N), was found to be predictive of a poor overall survival as well. All these parameters were found to be prognostically independent of the clinicopathological parameters. Multivariate analysis of combinations of these prognostically significant plasminogen activation parameters revealed that they are important independent prognostic indicators and have in fact a better prognostic value than their separate components. Based on these combined parameters, subgroups of patients with Dukes' stage B and C colorectal cancer could be identified as having either a high or a low risk regarding overall survival. In conclusion, these findings emphasize the relevance of the intestinal plasminogen activation system for survival prognosis of patients with colorectal cancer and, in the future, might constitute a patient selection criterion for adjuvant therapy.     

Evidence that ceramide selectively inhibits protein kinase C-alpha translocation and modulates bradykinin activation of phospholipase D

Sphingomyelinase (SMase) treatment (0.1 unit/ml for up to 30 min) of mouse epidermal (HEL-37) or human skin fibroblast (SF 3155) cells preincubated with [3H]serine to label the sphingomyelin pool caused the accumulation of labeled ceramide but not sphingosine or ceramide 1-phosphate. Incubation of HEL-37 cells with dioctanoylglycerol (diC8) or SF 3155 cells with bradykinin caused translocation of calcium/phosphatidylserine-dependent protein kinase C (PKC) activity to particulate material. In both cell lines the translocation was blocked by SMase treatment of the cells or by incubation with the cell-permeable ceramide analogue N-acetylsphingosine (C2-Cer). Western blot analysis indicated that treatment of HEL-37 cells with diC8 or SF 3155 cells with bradykinin resulted in the translocation of both PKC-alpha and PKC-espilon to particulate material. Treatment with SMase or C2-Cer specifically blocked the translocation of PKC-alpha but not that of PKC-epsilon. Pretreatment of cells with SMase or C2-Cer also inhibited the activation of phospholipase D activity induced by either diC8 (HEL-37 cells) or bradykinin (SF 3155 cells). The data provide strong evidence that ceramide can negatively regulate the translocation of PKC-alpha but not PKC-epsilon and further suggest that PKC-alpha may be involved in regulating phospholipase D activity.     

Thrombin stimulates expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in cultured human vascular smooth muscle cells

The effect of thrombin on the fibrinolytic potential of human vascular smooth muscle cells (SMC) in culture was studied. SMC of different origin responded to thrombin treatment with a dose and time dependent increase in tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) levels in both cell lysates and conditioned media with maximum effects achieved at 10-20 IU/ml thrombin. PAI-1 antigen levels also increased in the extracellular matrix of thrombin treated SMC. PAI-2 levels in cell lysates of such SMC were not affected by thrombin. The effect was restricted to active thrombin, since DFP-thrombin and thrombin treated with hirudin showed no increasing effect on t-PA and PAI-1 levels in SMC. Enzymatically active thrombin also caused a four-fold increase in specific PAI-1 mRNA and a three-fold increase in t-PA mRNA. Furthermore we demonstrated the presence of high and low affinity binding sites for thrombin on the surface of SMC with a KD = 4.3 x 10(-10)M and 9.0 x 10(4) sites per cell and a KD = 0.6 x 10(-8) M and 5.8 x 10(5) sites per cell respectively. Thrombin could come in contact with SMC in case of vascular injury or following gap formation between endothelial cells. Our data support the idea that besides its known proliferative effect for SMC, thrombin could also modulate their fibrinolytic system.     

Characterization of tTA and its functional domain in tetracycline repressor-mediated gene repression system

The tissue concentrations of urokinase-type plasminogen activator (u-PA), urokinase-type plasminogen activator receptor (u-PAR), plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were investigated by an ELISA technique in normal and malignant samples of the prostate from 24 patients undergoing radical prostatectomy for organ-confined prostate cancer. The median concentration of u-PA was significantly higher in cancerous than in normal prostate tissue (p = 0.006). No significant increase of u-PAR, PAI-1 and t-PA was found in cancer tissue in comparison with the benign samples (p > 0.05). Assessment of the relationship between fibrinolytic proteins and DNA ploidy revealed an increased u-PA, u-PAR and PAI-1 in diploid prostate cancer as compared with the normal controls. However, in aneuploid cancer u-PA remained high but u-PAR and PAI-1 were decreased. This led to a higher local concentration of u-PA in aneuploid samples than in normal prostate and in diploid prostate cancer. No alteration of median t-PA was found in benign prostate or in diploid or aneuploid prostate cancer. The altered expression of u-PA, u-PAR and PAI-1 in diploid and aneuploid prostate cancer suggests a possible role of fibrinolytic proteins in the different biologic behavior of tumors, and may be one explanation for the higher metastatic potential of aneuploid tumors.     

Immunoelectron microscopic localization of plasminogen activator inhibitor type 1 (PAI-1) in smooth muscle cells from morphologically normal and atherosclerotic human arteries

Vascular wall fibrinolytic system proteins are believed to play a pivotal role in atherogenesis. Tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) influence persistence of luminal thrombi and proteolysis of extracellular matrix, respectively. The major physiologic inhibitor of t-PA and u-PA is plasminogen activator inhibitor type 1 (PAI-1). All three of these fibrinolytic system proteins have been detected in vascular endothelial cells, smooth muscle cells, and macrophages by light microscopic immunohistochemistry. This study was undertaken to delineate, by immunoelectron microscopy, the loci of PAI-1 in smooth muscle cells from intact morphologically normal and atherosclerotic human arteries as well as in isolated and cultured smooth muscle cells from arteries. In intact vessels, PAI-1 immunoreactivity was associated with contractile filaments in cells in both normal and atherosclerotic tissues. Lipid-laden smooth muscle cells in atherosclerotic vessels were mainly of the synthetic phenotype and displayed lesser amounts of PAI-1 associated with rough endoplasmic reticulum and contractile filaments. Isolated smooth muscle cells exhibited either a contractile or synthetic phenotype. In the cells with a contractile phenotype, PAI-1 was associated with the contractile elements, whereas in the cells with a synthetic phenotype, the PAI-1 was associated predominantly with elements of the endoplasmic reticulum. Because PAI-1 is associated predominantly with contractile filaments in smooth muscle cells, the net amount of immunodetectable PAI-1 appears to be greater in contractile compared with synthetic phenotype cells.     

The coagulation and fibrinolytic responses of baboons after in vivo thrombin generation--effect of interleukin 6

Disseminated intravascular coagulation (DIC) may lead to severe thrombotic or hemorrhagic complications. The present work was undertaken to study the effect of interleukin 6 (IL-6) on variations of key coagulation and fibrinolytic parameters in plasma in a baboon model of experimental DIC induced by injection of factor Xa and phospholipids at dosages leading to partial (48%) or complete fibrinogen depletion. Transient increases of D-dimer, fibrinopeptide A, thrombin-antithrombin and the activated partial thromboplastin time were observed. Each parameter had a particular (time and Xa/phospholipid dose dependent) pattern of changes. The principal effect of IL-6 was a more rapid restoration of fibrinogen concentrations and of overall coagulation tests. Injection of factor Xa/phospholipids led also to a rapid increase of tissue-type plasminogen activator (t-PA) the extent of which was dependent on Xa/phospholipid dose. Pretreatment with IL-6 induced a threefold increase of basal t-PA and a corresponding increase of the t-PA response. Plasminogen activator inhibitor type 1 (PAI-1) concentrations did not change after low dose Xa/phospholipids, but increased eightfold after high dose Xa/phospholipids, IL-6 pretreatment induced within 8 h a twentyfold increase of PAI-1 but no further increase was observed after injection of factor Xa/phospholipids. Thus, in vivo thrombin generation leads to dynamic modifications of the coagulation and fibrinolytic systems. The principal effect of IL-6 is a more rapid normalization of overall coagulation tests, due to normalization of fibrinogen, and an increased t-PA release response which is partially counteracted by increased PAI-1 concentrations.     

Production of tissue-type plasminogen activator (t-PA) and type-1 plasminogen activator inhibitor (PAI-1) in mildly cirrhotic rat liver

We have studied the production of tissue-type plasminogen activator (t-PA) and type-1 plasminogen activator inhibitor (PAI-1) in liver of normal rats and in rats with mild cirrhosis induced by carbon tetrachloride inhalation, to demonstrate the production of these fibrinolytic components and their pathophysiologic role in the liver in vivo. Immunohistochemical study of paraffin-embedded liver sections and fibrin autography of frozen sections showed that the normal rat liver produces very little t-PA or PAI-1. On the contrary, striking t-PA activity and both t-PA and PAI-1 antigens were observed in the cirrhotic liver. Both t-PA and PAI-1 in plasma were also markedly increased in the cirrhotic rats. Because the hepatocyte can internalize t-PA or PA/PAI-1 complexes from circulation, Northern blot analysis of the total liver RNA was performed to demonstrate the endogenous synthesis of t-PA and PAI in the liver. Although the normal liver hardly expresses either t-PA or PAI-1 mRNA, striking t-PA and PAI-1 mRNA expression was observed in the liver of rats with mild cirrhosis. These data demonstrate that t-PA and PAI-1 production is strongly upregulated in the liver in rats with mild cirrhosis. These fibrinolytic components, whose production is closely associated with liver failure, may play important roles in the regulation of hepatocyte proliferation and liver regeneration in vivo.     

Release of luteinizing hormone-releasing hormone from enzymatically dispersed rat hypothalamic explants is pulsatile

Impaired whole blood fibrinolytic activity (FA), measured by the dilute clot lysis time (DCLT), is associated with first episodes of ischaemic heart disease (IHD) in the Northwick Park Heart Study in men, especially under 55 years, and in women. In a community-based study to investigate possible determinants of the DCLT, and therefore to assess which fibrinolytic components might be predictors of first IHD events, we measured fibrinolytic variables in a sub-sample of 150 healthy adults (73 males, 77 females) randomly selected from a single general practice. Most of the variance in DCLT (68% in men, 63% in women) was explained by tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) activities. In multiple regression analysis there was a significant difference in the strength of the association of t-PA activity with DCLT in men compared to women (test for interaction p = 0.05), the association of t-PA activity with DCLT being significant in males but not in females. Plasma PAI-1 activity was strongly associated with DCLT in both sexes. There was no independent association of DCLT with plasma fibrinogen, t-PA antigen, other fibrinolytic inhibitors, body mass index, serum lipids or C-reactive protein. Plasma PAI-1 activity in females and both t-PA and PAI-1 activities in males are the main determinants of whole blood FA measured by DCLT. It is therefore likely that these modulators of the plasma fibrinolytic system are associated with the onset of first clinical episodes of IHD. Elevated levels of t-PA antigen were positively associated with DCLT after adjustment for age and sex and therefore indicate impaired rather than enhanced FA. Further studies of the association of FA with risk of IHD should include not only "global" measures but also assessment of t-PA and PAI-1 activities, particularly as our results suggest that their associations with IHD may differ in men and women.     

Urethrolysis after surgical cure of stress urinary incontinence using colpopexy: surgical technics

Vitronectin (VN) is an obligatory cofactor for the inhibition of thrombin by plasminogen activator inhibitor 1 (PAI-1). It accelerates the rate of association between thrombin and PAI-1 more than two orders of magnitude. In contrast, VN does not accelerate the association between tissue-type plasminogen activator (t-PA) and PAI-1. Previously, we reported that the anti-PAI-1 monoclonal antibody (MoAb) CLB-2C8 binds to a short stretch of amino acids of PAI-1, located between residues 128 and 145, and prevents PAI-1 binding to VN. Furthermore, MoAb CLB-2C8 fully blocks the inhibitory activity of PAI-1 towards t-PA, emphasizing the importance of this area for the interaction with t-PA. Here, we show that this area is also required for the interaction between thrombin and PAI-1, since MoAb CLB-2C8 fully prevents inhibition of thrombin by PAI-1. In spite of similar structural requirements for the interaction between t-PA, PAI-1 and VN and between thrombin, PAI-1 and VN, the intermediate reaction products are clearly distinct. By employing surface plasmon resonance (SPR), using the BIAcore equipment, and by immunoprecipitation we demonstrate that, in the presence of VN, t-PA and PAI-1 form exclusively equimolar binary t-PA/PAI-1 complexes. Thrombin, PAI-1 and VN generate equimolar, binary thrombin/PAI-1 complexes and in addition equimolar, ternary complexes and multimers.     

Plasminogen activator inhibitor 1 contains a cryptic high affinity receptor binding site that is exposed upon complex formation with tissue-type plasminogen activator

The low density lipoprotein receptor-related protein (LRP), a multi-functional endocytic receptor, mediates the cellular internalization of tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator and their complexes with plasminogen activator inhibitor type 1 (PAI-1). LRP preferentially binds the complexed forms, exemplified by equilibrium dissociation constants (KD) that are at least an order of magnitude lower than those of the free components. To understand the molecular interactions, underlying the preference of the receptor for complexes rather than for the free components, we have performed a detailed analysis of the affinity and kinetics of the binding of PAI-1 and t-PA:PAI-1 complexes to the receptor, using surface plasmon resonance. To assess the involvement of the heparin-binding domain of PAI-1 for the interaction with LRP, we determined the equilibrium dissociation constants for the binding to LRP of a panel of PAI-1 mutants with single- and multiple amino-acid substitutions of the basic residues that constitute the heparin binding site of PAI-1 (K65, K69, R76, K80 and K88). The binding of these PAI-1 mutants was partially reduced with a 2 to 4 fold increase in KD values for single (K80, K88) and combined (K80, 88) substitution mutant proteins respectively. LRP binding of complexes, composed of t-PA with either wild type PAI-1 or any one of the single PAI-1 mutants indicated a major role of lysine 69 (K69) for the binding of t-PA:PAI-1 complexes to LRP (KD values of 6.1, 3.7. 75.4, 5.4, 12.5 and 8.1 nM for wild type, K65A, K69A, R76A, K80A and K88A complexes, respectively). Since the KD for the binding of free t-PA to LRP is 158 nM, we conclude that the PAI-1 moiety harbors the major determinant for t-PA:PAI-1 complex binding to LRP. The in vitro binding studies were extended by binding and clearance studies with COS-1 cells. Degradation of both 125I-t-PA:PAI-1 K69A and 125I-t-PA:PAI-1 K69A K80A K88A complexes after 2 h of incubation was reduced compared to the degradation of 125I-t-PA:PAI-1 complexes. We conclude that PAI-1 contains a cryptic binding site (lysine 69) for LRP, that is specifically expressed upon t-PA:PAI-1 complex formation.     

Impact of weight loss on plasminogen activator inhibitor (PAI-1), factor VII, and other hemostatic factors in moderately overweight adults

Based on previous cross-sectional findings, we hypothesized that weight loss could improve several hemostatic factors associated with cardiovascular disease. In a randomized controlled trial, moderately overweight men and women were assigned to one of four weight loss treatment groups or to a control group. Measurements of plasminogen activator inhibitor-1 (PAI-1) antigen, tissue-type plasminogen activator (t-PA) antigen, D-dimer antigen, factor VII activity, fibrinogen, and protein C antigens were made at baseline and after 6 months in 90 men and 88 women. Net treatment weight loss was 9.4 kg in men and 7.4 kg in women. There was no net change (p > 0.05) in D-dimer, fibrinogen, or protein C with weight loss. Significant (p < 0.05) decreases were observed in the combined treatment groups compared with the control group for mean PAI-1 (31% decline), t-PA antigen (24% decline), and factor VII (11% decline). Decreases in these hemostatic variables were correlated with the amount of weight lost and the degree that plasma triglycerides declined; these correlations were stronger in men than women. These findings suggest that weight loss can improve abnormalities in hemostatic factors associated with obesity.     

Modulation of plasminogen activator inhibitor type-1 biosynthesis in vitro and in vivo with oligo(nucleoside phosphorothioate)s and related constructs

Oligonucleotides with a nucleotide sequence complementary to various regions of human plasminogen activator inhibitor type-1 (PAI-1) mRNA have been studied as antisense inhibitors of expression of PAI-1 protein in cultured cells [human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells, human hybrid endothelial cells]. Hexadeca(deoxyribonucleoside phosphorothioate) 13 complementary to a fragment of a signal peptide PAI-1 mRNA was found to be most active, giving ca. 70% inhibition of PAI-1 release in a time- and dose-dependent way. The stereo-regular All-S(P) and All-R(P) diastereomers of 13 were studied and found to inhibit PAI-1 synthesis in HUVEC in a stereo-dependent manner, with the All-S(P) diastereomer considerably more active than the stereo-random construct and All-R(P) isomer. The observed stereo-dependent activity of oligonucleotide phosphorothioate constructs is presumably governed by their resistance to nucleases. The corresponding phosphodiester analogue of 13 was not active unless covalently bound at its 5'-end to a lipophilic alcohol residue (menthol, heptadecanol). The observed antisense activity of phosphodiester oligonucleotide bioconjugates in cultured human hybrid endothelial cells was paralleled by their increased stability in human plasma with respect to unconjugated oligonucleotide. The oligo(deoxyribonucleoside phosphorothioate) complementary to the same signal peptide region of rat PAI-1 mRNA was found to reduce the PAI-1 level in blood plasma of rats after intravenous administration into the tail vein. The effect was both time- and dose-dependent. The same oligonucleotide was found to protect against arterial thrombus formation in the rat (lower incidence of venous thrombosis, lower thrombus weight, and increased occlusion time in experimentally induced thrombosis). An anti-PAI-1 inhibitory activity has been independently reported for a 20-mer oligo(2'-O-methyl-ribonucleoside phosphorothioate) complementary to a 3'-untranslated region of human PAI-1 mRNA in cultured HUVEC and human aortic smooth muscle cells.     

Fibrinolytic proteins in apoptotic human umbilical vein endothelial cells

Endothelial cells express fibrinolytic proteins including: urokinase (u-PA) and tissue type (t-PA) plasminogen activators, type-1 (PAI-1) and 2 (PAI-2) plasminogen activator inhibitors, and u-PA receptor (u-PAR). Apoptotic endothelial cells detach, potentially forming both local and circulating microthrombi in vivo. In this article, apoptotic human umbilical vein endothelium was obtained by serum starvation and compared with nonapoptotic cells rescued from death with fresh medium containing serum. Antigen levels for t-PA, PAI-1, PAI-2, and u-PAR were reduced greatly in apoptosis (p< 0.05). In contrast, u-PA levels were similar in apoptotic as compared with rescued cells (p<0.05). Radioactive amino acids were used to determine absolute levels of protein synthesis and degradation in these cells. Reduced antigen levels likely were due to proteolysis as there was 98% total protein degradation and very little protein synthesis in apoptotic endothelial cells. Also, u-PA levels in apoptotic endothelial cells were not affected by the protein synthesis inhibitor cycloheximide. Endothelial cells in inflammatory sites are exposed to cytokines, which increase both apoptosis and u-PA levels. Data from this article support the idea that maintained u-PA levels in apoptotic endothelium may protect from micro-thrombosis in inflammatory sites as well as in the circulation.     

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.