[Show abstract][Hide abstract] ABSTRACT: Vascular fibrinolysis, a major natural defense mechanism against thrombosis, is a highly regulated process. The aim of this study was to evaluate the effect of endotoxin, tumor necrosis factor-alpha (TNFalpha) and interleukin-1alpha (IL-1alpha), on the fibrinolytic potential of cultured human umbilical vein endothelial cells (HUVEC).
Samples of stimulated conditioned media were collected over a period of 24 hours to determine: plasminogen activator (PA) and plasminogen activator inhibitor (PAI) activity, PAI-1 mRNA, tissue-type plasminogen activator (t-PA) antigen and urokinase-type plasminogen activator (u-PA) antigen.
Similar changes were observed after endotoxin and cytokine stimulation: there was a significant increase of PAI activity (p<0.01), starting at 6 hours, which remained 24 hours after stimulation. PAI-1 mRNA also showed an important rise with these agents, although cytokines induced an earlier and more intense inhibitor response (up to 6-fold increase). PA activity increased significantly at 6 hours (p<0.01) to drop at 24 hours and was mainly related to the presence of u-PA.
We conclude that endotoxin,+TNFalpha and IL-1alpha induce profound alterations in the fibrinolytic potential of HUVEC, characterized by an initial rise of activators (u-PA) followed by a strong increase of PAI-1. These changes may be of pathophysiologic significance for thrombosis and inflammatory reactions.
[Show abstract][Hide abstract] ABSTRACT: Cultured human endothelial cells synthesize and secrete plasminogen activators tissue-type and urokinase-type plasminogen activators (t-PA and u-PA) and their inhibitor plasminogen activator inhibitor-1 (PAI-1). We have studied the effects of increased concentrations of unfractionated heparin (UFH), low molecular weight heparin (LMWH) and recombinant hirudin (Revasc™) on the fibrinolytic potential of human umbilical vein endothelial cells (HUVEC). Samples from the conditioned media were collected before and 1,6,24 and 48 h after addition to assess the levels of PAI activity and antigen, t-PA activity and antigen and u-PA antigen. Fibrin autography was also performed in samples collected 24 h after stimulation. UFH induced a significant increase of u-PA with respect to controls at 24 and 48 h (P<0.01), whereas a dose-dependent decrease in PAI activity and antigen was observed (P<0.01), starting one hour after the addition of UFH. There were no changes in t-PA antigen, and t-PA activity was not detected. Fibrin autography revealed no differences in t-PA/PAI-1 complexes between UFH-stimulated and control cultures. LMWH induced a significant increase (P<0.01) of u-PA antigen at 24 and 48 h. PAI activity was found to be slightly increased (P<0.05) whereas PAI-1 and t-PA antigen did not change and increase formation of t-PA/PAI-1 complexes was demonstrated. Finally, hirudin induced a moderate increase of t-PA activity at 24 and 48 h (P<0.01), without changes in other parameters analyzed.Our results show that UFH could potentiate the fibrinolytic activity of HUVEC by increasing u-PA and by decreasing PAI-1 levels. LMWH lacks the ability to reduce the PAI-1 concentration in the conditioned media but could contribute to fibrinolysis activation by increasing u-PA. Hirudin might have some delay profibrinolytic effect by increasing t-PA activity.
[Show abstract][Hide abstract] ABSTRACT: The biosynthesis of plasminogen activator inhibitor (PA1-I) by endotheliai cells is increased by a variety of physiologic and pathologic stimuli, such as endotoxin and cytokines. We have analyzed the PAI-I gene expression by human umbilical vein endotheliai cells (HUVEC) using Northern blotting and reverse transcription-polymerase chain reaction (RT-PCR) with biotinylated primers. The PAI-1 antigen concentration was assessed in the supernates by ELISA assay. Cultures were stimulated with endotoxin(LPS. 100 ng/ml), tumor necrosis factor a (TNF-a, 500 U/ml) and interleukin l-a (IL-I, 500 U/ml). mRNA was prepared from HUVEC before and 2, 6 and 24 hours after stimulation. The restriction enzymes Sacl and Bell were used to identify the amplified cDNA fragment. A human PAI-1 cDNA probe prepared by PCR was used in the Northern analysis. The amount of mRNA starting material was 400 ng for Northern and 0.03-0.1 ug for RT-PCR. Treatment of HUVEC with endotoxin, TNF and IL-1 markedly enhanced the PAI-1 mRNA levels. In the case of endotoxin increases were detectable by both techniques at 6 hours with a good correlation (r=0.96). Maximal effect was observed al 24 hours (3-fold increase as compared to baseline). TNF and IL-1 also induced an increase in PAI-1 mRNA by both methods (r=0.80), starting earlier, at 2 hours, with a peak within 4 and 6 hours (3-13 fold increase) to decline by 24 hours. A significant increase of PAI-1 antigen was observed with the three inflammatory mediators 24 hours after stimulation, more evident specilly in the case of IL-1 stimulated cultures. These data indicate that endotoxin and inflammatory mediators induce an increase in the PAI-1 gene expression by HUVEC, being IL-1 the cytokine which induced a more significant enhancement of mRNA and protein synthesis. Both RT-PCR and Northern allowed the mRNA detection with a similar specificity but the RT-PCR seems more appropriate as screening procedure.
[Show abstract][Hide abstract] ABSTRACT: To analyse the possible correlation between the plasma levels of the extrinsic pathway inhibitor (TFPI) in patients with infection along with two cytokines mediating in the action of bacterial endotoxin on the vascular endothelium, namely, tumour factor (TNF) and interleukin-1 (IL-1).
Twenty-five patients with infection, none of them showing septic shock or disseminated intravascular coagulation, were studied. Plasma TFPI concentration was assessed by a chromogenic substrate technique; TNF was determined with immunoradiometric methods and IL-1 was estimated with ELISA. The results were compared with those of a group of 25 healthy subjects matched for age and sex.
Positive blood cultures were found in 15 of the 25 patients (60%), 12 due to gram-negative and 3 to gram-positive germs. A significant increase of TFPI (p < 0.004), TNF and IL-1 (p < 0.001) was found in infection patients with respect to the control subjects. No significant correlation between TFPI and TNF or IL-1 was found.
Increased TFPI, unrelated to cytokines, is present in patients with sepsis.
[Show abstract][Hide abstract] ABSTRACT: Development of monoclonal antibodies capable of inhibiting the specific binding of t-PA to fibrin.
After immunization of Balb/c mice with recombinant t-PA (rt-PA) we selected the monoclonal antibody MA3B5 by its ability to inhibit the binding of t-PA to fibrin, and the MA2C1 devoid of this property. The influence of such antibodies was evaluated on fibrin plates, amidolytic assays and clot lysis assays. Furthermore, their interference with the activator bound to fibrin was assayed with a spectrophotometric solid phase assay (SOFIA).
The results showed that MA3B5 totally inhibited the t-PA induced fibrinolytic activity on fibrin plates, reduced amidolytic activity by 86.5% and inhibited the clot lysis induced by t-PA in a dynamic system. In contrast, the MA2C1 showed no inhibition. By assessing the binding of t-PA to fibrin with the SOFIA assay we could demonstrate that only the MA3B5 reduced significantly (up to 90% with 100 micrograms/mL of antibody) the amount of t-PA bound to fibrin surface.
We have purified and characterized a monoclonal antibody which specifically blocks the fibrin binding site of t-PA.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the possible role played by activation of the coagulation system in the early occlusion of grafts in patients subjected to aortocoronary bypass.
This study includes 30 patients subjected to coronary revascularization surgery. The plasma rates of thrombin-anti-thrombin complexes (TAT) and 1+2 prothrombin fragments (F1+2) were measured before and after surgery. The studies were performed by enzyme immunoassay techniques. Selective angiography was performed in every patient on the 10th day after surgery. Descriptive statistics, Student's t test and Mann-Whitney U test were used for comparison of means ans statistical analysis.
Occlusive lesion was shown by angiography in 15 of the 30 patients. The TAT and F1+2 levels showed a statistically significant increase (p less than 0.001) along the postoperative period, without any correlation regarding the presence or absence of occlusion. The preoperative concentration of TAT was significantly higher in patients with thrombotic occlusion (p less than 0.05).
1) Marked activation of the coagulation system is present in patients subjected to coronary revascularization surgery. 2) The pre-operative determination of TAT may be of value for predicting early graft occlusion.