INTERLEUKIN 1 (IL-1) AND TUMOR NECROSIS FACTOR (TNF) ACTIVATION OF VASCULAR ENDOTHELIUM: EFFECTS ON PLASMINOGEN ACTIVATOR INHIBITOR (PAI-1) AND TISSUE-TYPE PLASMINOGEN ACTIVATOR (tPA)

1987 ◽  
Author(s):  
R R Schleef ◽  
M P Bevilacqua ◽  
M Sawdey ◽  
M A Gimbrone ◽  
D J Loskutoff
Keyword(s):  
Plasminogen Activator ◽  
Phorbol Myristate Acetate ◽  
Local Development ◽  
Cdna Probe ◽  
Fold Increase ◽  
Tissue Type ◽  
Maximal Response ◽  
Ionophore A23187 ◽  
Myristate Acetate ◽  
Pai 1

The regulation of the fibrinolytic system of cultured human umbilical vein endothelial cells (ECs) by two distinct monokines (IL-1 and TNF) was investigated. Conditioned media (CM) was collected from ECs cultured for 24 h in the presence of the monokines and analyzed in quantitative immunological assays for PAI-1 activity and tPA antigen. Both monokines induced a dose-dependent increase in extracellular PAI-1 activity, with a concomitant decrease in tPA antigen. Maximal effects were achieved with either 10 U/ml IL-1 or 200 U/ml of TNF, and resulted in a 4 fold increase in PAI-1 and a 50% decrease in tPA. The kinetics of the effects of both monokines on EC PAI-1 and tPA were similar, with maximal response detected at 24 h. Cell-associated PAI-1 also increased in response to these monokines. For example, a 24 h exposure of EC to TNF (250 U/ml) or IL-1 (5 U/ml) caused a 5-fold increase in cell-associated PAI-1. Northern blot analysis using a PAI-1 cDNA probe indicated that the monokines increased the levels of two RNA species, corresponding to PAI mRNAs of approximately 3.0 and 2.2 kb in length. To determine if the increase in cel 1-associated PAI-1 reflects a storage pool of rapidly releasable PAI-1, ECs were pretreated with IL-1 for 24 h, washed and the PAI-1 activity in CM measured after 5, 15 and 60 min treatment with known secretagogues (i.e., phorbol myristate acetate, calcium ionophore A23187). Although IL-1 treated ECs released PAI-1 at a rate which was 5-fold higher than controls, this rate was not increased further by treatment with phorbol myristate acetate or ionophore. The fact that both monokines act in a similar manner strengthens the hypothesis that the local development of immune and inflammatory processes could reduce endothelial fibrinolytic activity, thus leading to the pathologic formation of intravascular thrombi.

Postoperative Hemostasis and Fibrinolysis in Patients Undergoing Cardiopulmonary Bypass with or without Aprotinin Therapy

1994 ◽  
Vol 72 (03) ◽  
pp. 438-443 ◽  
Author(s):  
He Lu ◽  
Charles Du Buit ◽  
Jeannette Soria ◽  
Bernard Touchot ◽  
Bernard Chollet ◽  
...  
Keyword(s):  
Placebo Group ◽  
Cardiopulmonary Bypass ◽  
Blood Loss ◽  
Plasminogen Activator ◽  
Fold Increase ◽  
Tissue Type ◽  
Postoperative Blood Loss ◽  
High Dose ◽  
Double Blind Study ◽  
Pai 1

SummaryIntra- and postoperative blood loss during open heart surgery is reduced by approximately 50% when aprotinin, a potent inhibitor for plasmin and kallikrein, is administered during surgery. But whether aprotinin increases the risk of thrombotic complications remains controversial. The aim of this study was to evaluate the effects of aprotinin administration on coagulation and fibrinolysis during and after cardiopulmonary bypass (CPB). Thirty patients undergoing CPB were randomly assigned to two comparable groups for a double-blind study (16 patients receiving high-dose aprotinin, 14 patients receiving placebo). Patients’ plasma levels of ATM (thrombin-induced modified antithrombin III), FbDP (fibrin degradation products, D-Dimers), t-PA (tissue-type plasminogen activator) and PAI-1 (plasminogen activator inhibitor type 1) were measured at regular intervals. In both groups, ATM level increased during surgery (from less than 30 to 90-110 ng/ml) and returned to normal 24 h after surgery and remained unchanged thereafter. Aprotinin reduced this increase in ATM levels (p = 0.02 at 30 min after the start of CPB). The FbDP generated during surgery was greatly reduced in the aprotinin group (945 ng/ml) in comparison with the placebo group (1889 ng/ml, p = 0.004). After surgery, FbDP levels decreased in both groups with nadirs at 2nd day (placebo group: 940 ng/ml and aprotinin group: 865 ng/ml) indicating a hypo-fibrinolytic period. Then, the FbDP level in both groups started to increase up to the 9th day, in an identical manner. This postoperative hypofibrinolysis is related to the changes of t-PA and PAI-1 levels: immediately after surgery there was a 2’fold increase in t-PA level and a 4-5 fold increase in PAI-1 level in the two groups. During the following 24 h, t-PA levels decreased in both groups. In contrast, PAI-1 levels in the placebo group during the same time increased sharply to a maximum level (175.7 ng/ml). This further increase did not occur in the aprotinin group although it remained at a high level (79.2 ng/ml). The difference in the increase of PAI-1 between the 2 groups (value at 24 h minus preoperative value: Dl-Tl) was significantly different (p = 0.04). Then t-PA continued to decrease and PAI-1 began to decrease steadily. Total blood loss was significantly reduced by aprotinin therapy (3.06 ml/kg versus 5.86 ml/kg). The present study confirms the inhibitory effects of aprotinin on both fibrinolytic activity and blood coagulation activation during CPB, and reveals an hypofibrinolytic period that lasts 48 h after surgery in both aprotinin and placebo groups. This inhibition of fibrinolysis is apparently associated with high PAI-1 level. The data of this study also show that 2 days after aprotinin therapy, there is no prolonged effect of aprotinin on fibrinolysis. In addition, the lower level of PAI-1 in the aprotinin group after surgery might result from a protection of endothelial cells by aprotinin, suggesting an unexpected benefit of aprotinin therapy.

Tissue-Type Plasminogen Activator after Venous Occlusion in Pregnancy and Puerperium

1993 ◽  
Vol 70 (03) ◽  
pp. 486-490 ◽  
Author(s):  
Mojca Stegnar ◽  
Andrej Zore ◽  
Živa Novak-Antolič ◽  
Neva Vovk ◽  
Egbert K O Kruithof
Keyword(s):  
Plasminogen Activator ◽  
Gestational Hypertension ◽  
Fold Increase ◽  
Normal Pregnancy ◽  
Venous Occlusion ◽  
Tissue Type ◽  
Third Trimester ◽  
The Third ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryPregnancy is associated with depressed fibrinolysis as judged from the decreased fibrinolytic response to venous occlusion. In order to elucidate if this decreased response is due to an increase in plasminogen activator inhibitors 1 and 2 (PAI-1, PAI-2), and/or to decreased release of tissue-type plasminogen activator (t-PA) antigen during venous occlusion, 36 women (18 women with normal pregnancy and 18 with gestational hypertension without proteinuria) were followed during pregnancy and puerperium. In each woman a 20 min venous occlusion was performed in the second and in the third trimester of pregnancy and 3 days after delivery. The increase in t-PA antigen after venous occlusion relative to basal value was in the second trimester of pregnancy on average 3.7 fold, in the third trimester 4.4 fold, and so not reduced compared to non-pregnant women (3.7 fold increase). After delivery the increase in t-PA antigen was significantly enhanced (8.5 fold, p <0.005). The fibrinolytic response to venous occlusion measured by euglobulin and t-PA activity was significantly decreased in the third trimester compared to non-pregnant values (both p <0.005) and returned to somewhat higher (euglobulin clot lysis) or significantly higher (t-PA activity, p <0.01) values 3 days after delivery. Decreased euglobulin and t-PA activity after venous occlusion in the third trimester coincided with significant increases in basal PAI activity, PAI-1 antigen and PAI-2 antigen (2.9, 2.5 and >30 fold increase relative to non-pregnant values, respectively, all p <0.001). No significant differences in fibrinolytic variables were observed between nor-motensive and hypertensive pregnant women. It was concluded that t-PA antigen release during venous occlusion is not decreased during pregnancy and puerperium, and that decreased fibrinolytic response measured by global methods should be attributed to increased t-PA inhibitors. Gestational hypertension without proteinuria is not characterized by changes in fibrinolytic responses different from those observed in normal pregnancy.

Thrombin Stimulates Expression of Tissue-Type Plasminogen Activator and Plasminogen Activator lnhibitor Type 1 in Cultured Human Vascular Smooth Muscle Cells

1993 ◽  
Vol 70 (03) ◽  
pp. 469-474 ◽  
Author(s):  
Johann Wojta ◽  
Marisa Gallicchio ◽  
Hans Zoellner ◽  
Peter Hufnagl ◽  
Karena Last ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Plasminogen Activator ◽  
Vascular Smooth Muscle Cells ◽  
Fold Increase ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryThe 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 K D = 4.3 × 10−10 M and 9.0 × 104 sites per cell and a KD = 0.6 × 10−8 M and 5.8 × 105 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.

scholarly journals Site-directed Targeting of Plasminogen Activator Inhibitor-1 as an Example for a Novel Approach in Rational Drug Design

2004 ◽  
Vol 279 (19) ◽  
pp. 20447-20450 ◽  
Author(s):  
Bart De Taeye ◽  
Griet Compernolle ◽  
Paul J. Declerck
Keyword(s):  
Molecular Mass ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Fold Increase ◽  
Rational Drug Design ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Rational Drug ◽  
Pai 1

As plasminogen activator inhibitor-1 (PAI-1), the physiological inhibitor of tissue-type plasminogen activator, is considered to be an important risk factor in several (patho)physiological conditions, many research activities focus on attempts to inhibit this serpin. The approach illustrated in the current study focuses on elucidating important interaction sites allowing the inhibition of PAI-1. Since monoclonal antibodies are in most cases not ideal for therapeutic use, the question of whether smaller molecules exert comparable effects is a hot issue. To answer this question, Cys residues were introduced in PAI-1 at positions previously identified as determining the epitope of a PAI-1-inhibiting antibody, MA-8H9D4, resulting in PAI-1-R300C, PAI-1-Q303C, and PAI-1-D305C. Subsequently, low molecular mass sulfhydryl-specific reagents (i.e.BODIPY® 530/550 IA (molecular mass 626 Da) and BODIPY® FL C1-IA (molecular mass 417 Da)) were allowed to react covalently with the cysteine. The functional distribution (inhibitoryversussubstrate) toward tissue-type plasminogen activator was determined for the labeled and the unlabeled samples. Labeling at position 300 leads to a 1.7- and 2.2-fold increase in SI value for BODIPY® 530/550 IA and BODIPY® FL C1-IA, respectively. Labeling at position 303 results in a 3.3- and 1.9-fold increase of the SI value for the large and the small label, respectively. At position 305, the SI values are 3.1-fold increased for both labels. The effect (on SI and on serpin activity) of the manipulations at these positions is in good agreement with the effect exerted by MA-8H9D4. In conclusion, our study provides proof of concept for the proposed approach in evaluating whether targeting a functional epitope with a small synthetic compound may be a feasible strategy in rational drug design.

Immunoassay of Murine t-PA, u-PA and PAI-1 Using Monoclonal Antibodies Raised in Gene-inactivated Mice

1995 ◽  
Vol 74 (05) ◽  
pp. 1305-1309 ◽  
Author(s):  
Paul J Declerck ◽  
Maria Verstreken ◽  
Désiré J Collen
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Fold Increase ◽  
Tissue Type ◽  
Wild Type ◽  
Tissue Extracts ◽  
Type Plasminogen Activator ◽  
Murine Tissue ◽  
Pai 1

SummaryThree enzyme-linked immunosorbent assays for the quantitation of murine tissue-type plasminogen activator (t-PA), urokinase-type plasminogen aetivator (u-PA) and plasminogen activator inhibitor I (PAI-1), were developed using monoclonal antibodies raised against the autologous proteins in gene-inactivated mice. Dose-response was linear for t-PA and PAI-1 between 5 and 0.1 ng/ml and for u-PA between 50 and 1 ng/ml, with intra-assay, inter-assay and inter-dilution coefficients of variation of 6 to 14%. Assay recoveries of proteins (5 to 100 ng/ml) added to plasma were 73 to 95% for t-PA and PAI-1. Linear correlations (r = 0.65, r = 0.91 and r = 0.92, for t-PA, u-PA and PAI-1 respectively) were found between antigen and activity in plasma, urine and tissue extracts. Levels of t-PA and PAI-1 antigen in murine plasma were 2.5 ± 1.0 ng/ml (mean ± SD, n = 9) and 1.9 ± 0.6 ng/ml (mean ± SD, n = 8), respectively, in wild-type mice and undetectable in gene-inactivated mice. Bradykinin injection in mice provoked a 12-fold increase (p <0.0002) of t-PA and endotoxin injection an 80-fold increase (p <0.005) of PAI-1 levels. u-PA antigen levels in urine from wild-type mice ranged between 0.2 and 8.2 μ;g/ml (1.8 ± 1.9 μg/ml, mean ± SD, n = 17) and were undetectable in gene-inactivated mice.Thus, these assays may be useful for studies on the role of these proteins in tissue remodeling, atherosclerosis, embryogenesis, etc., in established mouse models. Gene-inactivated mice may constitute a general approach for the generation of monoclonal antibodies against the deficient translation products and for the development of specific immunoassays for murine proteins.

scholarly journals Colistin dampens fibrinolysis and endothelial activation during endotoxaemia

2017 ◽  
Vol 117 (09) ◽  
pp. 1714-1721 ◽  
Author(s):  
Christian Schoergenhofer ◽  
Peter Matzneller ◽  
Marion Mußbacher ◽  
Johannes A. Schmid ◽  
Petra Jilma-Stohlawetz ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Fold Increase ◽  
Endothelial Activation ◽  
Tissue Type ◽  
Coagulation System ◽  
Plasminogen Activator Inhibitor 1 ◽  
Double Blind ◽  
Beneficial Effects ◽  
Pai 1

SummaryColistin electrostatically interacts with lipopolysaccharides (LPS). Preclinical studies demonstrated beneficial effects of colistin on LPS-induced coagulation and fibrinolysis. The objective of this trial was to investigate the effects of colistin during experimental endotoxaemia. In this randomised, double-blind, placebo-controlled, crossover trial 16 healthy volunteers received a 2 ng/kg LPS bolus after infusion of 2.5 million IU colistin or placebo. Plasma levels of F1+2 prothrombin fragments, thrombin-antithrombin complexes (TAT), von Willebrand factor antigen levels (vWF), E-selectin, plasmin-antiplasmin complexes (PAP), tissue-type plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor-1 (PAI-1) were measured. Infusion of colistin significantly reduced peak concentrations of PAP complexes by 70 %, t-PA antigen levels by 63 % and t-PA activity by 48 %, while PAI-1 levels decreased numerically by 63 %. Two hours after the LPS bolus F1+2 levels and TAT complexes were slightly reduced in the colistin period, but peak concentrations were similar in both periods. Colistin blunted the LPS induced four-fold increase in soluble E-Selectin levels by ∼50 % and the two-fold increase in vWF antigen levels by ∼70 %. The LPS-scavenging actions of colistin significantly reduce endothelial activation and fibrinolytic response in the human endotoxaemia model, while the activation of the coagulation system remains largely unaffected.Note: This work was conducted at the Medical University of Vienna.EudraCT-Nr.: 2014–00285720Supplementary Material to this article is available online at http://www.thrombosis-online.com

scholarly journals Enhancement of the fibrinolytic activity of sheep endothelial cells by retroviral vector-mediated gene transfer

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 533-541 ◽  
Author(s):  
DA Dichek ◽  
O Nussbaum ◽  
SJ Degen ◽  
WF Anderson
Keyword(s):  
Endothelial Cells ◽  
Gene Transfer ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Fold Increase ◽  
Retroviral Vector ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Pai 1

Abstract In an attempt to enhance the fibrinolytic activity of endothelial cells (EC), a retroviral vector containing the human tissue-type plasminogen activator (t-PA) cDNA was constructed. Sheep EC were stably transduced with the vector, resulting in a 30-fold increase in t-PA activity over that detected in EC transduced with a control vector. Southern and Northern analyses confirmed the presence of both the vector sequence and the appropriate mRNA transcripts. Secretion of high levels of recombinant human t-PA continued in vitro for the duration of the experiments, up to 11 weeks after transduction, although the rate of t- PA secretion decreased in some of the EC lines. Zymographic analysis of conditioned medium from t-PA-transduced EC showed the presence of two new molecular species with plasminogen activator activity that could be specifically immunoprecipitated with a monoclonal antihuman t-PA antibody. The relative molecular masses of these species (60 to 80 and 110 Kd) suggest that they represent recombinant human t-PA both free and bound to sheep plasminogen activator inhibitor 1 (PAI-1). Consistent with this interpretation, the 110-Kd species could be specifically immunoprecipitated with antiserum to PAI-1. These studies demonstrate that retroviral vector-mediated gene transfer may be used to increase total EC fibrinolytic activity.

scholarly journals Enhancement of the fibrinolytic activity of sheep endothelial cells by retroviral vector-mediated gene transfer

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 533-541
Author(s):  
DA Dichek ◽  
O Nussbaum ◽  
SJ Degen ◽  
WF Anderson
Keyword(s):  
Endothelial Cells ◽  
Gene Transfer ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Fold Increase ◽  
Retroviral Vector ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Pai 1

In an attempt to enhance the fibrinolytic activity of endothelial cells (EC), a retroviral vector containing the human tissue-type plasminogen activator (t-PA) cDNA was constructed. Sheep EC were stably transduced with the vector, resulting in a 30-fold increase in t-PA activity over that detected in EC transduced with a control vector. Southern and Northern analyses confirmed the presence of both the vector sequence and the appropriate mRNA transcripts. Secretion of high levels of recombinant human t-PA continued in vitro for the duration of the experiments, up to 11 weeks after transduction, although the rate of t- PA secretion decreased in some of the EC lines. Zymographic analysis of conditioned medium from t-PA-transduced EC showed the presence of two new molecular species with plasminogen activator activity that could be specifically immunoprecipitated with a monoclonal antihuman t-PA antibody. The relative molecular masses of these species (60 to 80 and 110 Kd) suggest that they represent recombinant human t-PA both free and bound to sheep plasminogen activator inhibitor 1 (PAI-1). Consistent with this interpretation, the 110-Kd species could be specifically immunoprecipitated with antiserum to PAI-1. These studies demonstrate that retroviral vector-mediated gene transfer may be used to increase total EC fibrinolytic activity.

Differential Role of Components of the Fibrinolytic System in the Formation and Removal of Thrombus Induced by Endothelial Injury

1999 ◽  
Vol 81 (04) ◽  
pp. 601-604 ◽  
Author(s):  
Hiroyuki Matsuno ◽  
Osamu Kozawa ◽  
Masayuki Niwa ◽  
Shigeru Ueshima ◽  
Osamu Matsuo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Thrombus Formation ◽  
Endothelial Injury ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

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