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At least 325 records · Page 18Linked to original sources
PubMed1990

D-dimer and TAT measurement in patients with deep venous thrombosis: utility in diagnosis and judgement of anticoagulant treatment effectiveness.

The plasma levels of thrombin-antithrombin III-complexes (TAT) and the fibrin split product D-Dimer were measured in 39 patients with phlebographically proven acute DVT: 34 patients had proximal DVT, 5 had calf DVT. The sensitivity of D-Dimer and TAT measurements in the diagnosis of proximal DVT was found to be dependent on the duration of symptoms: 0 to 7 days (n = 27): elevated D-Dimer levels (greater than 120 ng/ml) = 1, D-Dimer Latex test positive (greater than 500 ng/ml) = 1, elevated TAT levels (greater than 6 ng/ml) = 0.88. Eight to 14 days (n = 7): elevated D-Dimer levels = 1, D-Dimer Latex test positive = 0.33, elevated TAT levels = 0.66; specificity: elevated D-Dimer: 0.48, D-Dimer Latex test: 1, elevated TAT: 0.76. Calf DVT patients (n = 5) had elevated D-Dimer levels, negative Latex tests and 3 of them had normal TAT values. Hemostatic and fibrinolytic parameters were also determined in 13 patients during heparin treatment of proximal DVT. Elevated D-Dimer and TAT levels rapidly decreased after initiation of anticoagulant therapy. In 2 of 13 patients a marked increase in D-Dimer and TAT levels was observed in periods of ineffective heparinization, documented by normal or only slightly prolonged thrombin clotting times. We conclude from our results that 1) D-Dimer EIA measurement, in contrast to TAT measurement, shows a very high sensitivity in the diagnosis of DVT, 2) due to low specificity this test can only be used to exclude thrombosis in patients with suspected DVT, and 3) the determination of the plasma levels of D-Dimer and TAT may be useful for judging the effect of anticoagulant treatment on thrombotic processes.

Adult↗
PubMed1989

[Quantification of a specific inhibitor (PAI-1) of tissue-type plasminogen activator (t-PA) in the plasma].

In human plasma, the activation of plasminogen by tissue plasminogen activator (t-PA) is a fibrin localized process which allows the specific dissolution of thrombi. Most of the t-PA circulates as a complex with its inhibitor, PAI-1, which thereby regulates its activity. In the present work the authors have studied the kinetics of inhibition of t-PA by PAI-1 and have developed an assay for its specific detection. The assay is performed in microtitration plates containing a solid-phase fibrin network, as follows: the source of inhibitor is mixed with solutions containing increasing amounts of t-PA, then the residual t-PA is separated by means of a solid-phase fibrin support and detected with a coupled reaction using a plasmin selective chromogenic substrate. The change in absorbance is measured in a microtiter plate reader and converted to t-PA activity by reference to a standard curve. The residual t-PA activity is inversely proportional to the concentration of PAI-1. The quantitation of PAI-1 is based on the variation of the dissociation constant of the fibrin/t-PA interaction obtained in the presence of the inhibitor. Since other serine-protease inhibitors do not interfere with the assay, the method is specific for PAI-1 and can be safely used in other biological fluids.

Adult↗
PubMed1989

Influence of nicotine and cotinine on the expression of plasminogen activator activity in bovine aortic endothelial cells.

The effects of nicotine and its major metabolite, cotinine, were evaluated on the secretion of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) in cultured bovine aortic endothelial cells. Both compounds increased PA secretion, determined by 125I fibrin plate assay, in a time- and dose-dependent manner. Maximum effects after 24 hr incubation were observed for nicotine at 10(-8) M and for cotinine at 10(-7) M, which corresponded to about 2.6-fold increases over control for both compounds. The pharmacological PA stimulation required both RNA and protein syntheses, as evidenced by inhibition by actinomycin D and cycloheximide. Both control and treated cells produced multiple forms of PA, as evaluated by SDS-PAGE zymography, and a single form of PAI, as evidenced by reverse fibrin autography. Although activities of all species of PA were enhanced by nicotine and cotinine, these compounds had no significant effects on the release of PAI. These results thus suggest that nicotine and cotinine may have fibrinolytic activity in vivo.

Animals↗
PubMed1987

Enzyme secretion by human endothelial cells infected with Rickettsia conorii.

When cultured endothelial cells from the human umbilical vein were infected with rickettsia conorii, a greater increase was noticed in the inhibitor of plasminogen activator than in tissue type plasminogen activator. This could be linked to clinical observation of coagulation abnormalities in Boutonneuse fever (BF). Otherwise, transaminase levels increased significantly in the supernatant of R. conorii infected endothelial cells which could explain the increased levels of these enzyme in BF.

Alanine Transaminase↗
PubMed1987

Plasminogen activator in normal subjects after exercise and venous occlusion: t-PA circulates as complexes with C1-inhibitor and PAI-1.

Exercise to exhaustion was associated with the appearance in plasma of plasminogen activator (PA) in several mol wt forms, as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with zymography. A number of active bands, all immunologically identified as tissue-type PA (t-PA), were observed. The major form had an apparent mol wt of approximately 60,000 and is due to free t-PA. The other strong bands had apparent mol wts of approximately 110,000 and 180,000. The 110,000 band, also present in pre-exercise samples, represents t-PA complexed with its major inhibitor (PAI-1), and the 180,000 band is due to t-PA complexed with C1 inhibitor. The released forms of t-PA were cleared rapidly after cessation of exercise at exhaustion. Urokinase-type PA (u-PA) activity was also identified in pre- and postexercise samples at an apparent mol wt of approximately 50,000. This is consistent with its being free u-PA; no complexed forms of u-PA were observed. Qualitatively similar changes in plasma PA were observed after venous occlusion. Small quantities of plasmin were generated after strenuous exercise, as observed by detection of plasmin-alpha 2-antiplasmin complex by two-dimensional immunoelectrophoresis in three of five subjects. This complex was cleared rapidly after cessation of exercise. Plasmin-alpha 2-antiplasmin complex was not detected in any of the subjects after venous occlusion.

Fibrinolysin↗
PubMed1986

Monoclonal antibodies towards the fast inhibitor of tissue plasminogen activator from human plasma and serum.

Hybridoma cells were produced by fusing mouse myeloma cells (SP 2/0-Ag 14) with spleen cells from a Balb/c mouse, previously immunized with the partially purified complex between tissue plasminogen activator (t-PA) and its fast inhibitor from human plasma (serum). Screening with a radioimmunoassay revealed a number of hybridomas secreting antibodies directed towards the complex. Of these, about 1/3 reacted both with the complex and t-PA, whereas about 2/3 reacted only with the complex. Three of the latter hybridomas, producing antibodies directed towards the inhibitor-moiety in the complex have been cloned and the antibodies were studied in detail. PA-inhibitor activity in plasma or serum and t-PA/PA-inhibitor complex could be specifically adsorbed on all three insolubilized monoclonal antibodies (MC1, MC2 and MC3). None of the antibodies seems to be directed against structures of vital importance for the functional activity of the PA-inhibitor. In accordance with this finding the antibody with the highest avidity (MC1) reacts equally well with the PA-inhibitor alone or in complex with t-PA. A radioimmunoassay was developed with this antibody and significant displacement was obtained with samples with PA-inhibitor concentrations above 2 AU/mL. In 13 plasma samples with different levels of PA-inhibitory activity a significant correlation was obtained when comparing this activity with the PA-inhibitor antigen as measured with the radioimmunoassay (r = 0.88).

Antibodies, Monoclonal↗
PubMed1987

Secretion of tissue-type plasminogen activator and plasminogen activator inhibitor by cultured human endothelial cells: modulation by thrombin, endotoxin, and histamine.

Primary cultures of pooled endothelial cells obtained from four to six human umbilical cord veins were grown to confluency in M 199 cell culture medium containing 20% human serum. The secretion of tissue-type plasminogen activator antigen (t-PA Ag) and fast-acting plasminogen activator inhibitor activity (PA-I activity) was measured in the conditioned medium after incubation of confluent cultures with serum-free medium for 24 hours (1.5 ml/25 cm2 cell surface). The baseline production of t-PA Ag was 2.7 +/- 1.4 ng/ml (mean +/- SD) and of PA-I activity 36 +/- 18 IU/ml. Stimulation with thrombin resulted in a dose-dependent and time-dependent increase of the secretion of both components. At 1 NIH U thrombin per milliliter, a fourfold increase of t-PA Ag and a twofold increase of PA-I activity were observed. This effect was dependent on a free active site in thrombin and specific protein synthesis (inhibited by cycloheximide and dactinomycin) but unrelated to prostacyclin synthesis (no effect of aspirin or indomethacin). No free t-PA activity could be demonstrated on fibrin plates in conditioned medium from either stimulated or nonstimulated endothelial cell cultures. Endotoxin stimulation resulted in a specific secretion of PA-I activity but not of t-PA Ag, whereas histamine stimulation at concentrations of 0.1 to 1 mumol/L caused a specific secretion of t-PA Ag but not of PA-I. These findings indicate that the secretion of t-PA Ag and PA-I activity are not necessarily coupled.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1986

[Thromboembolic disease and anomalies of fibrinolysis].

Quantitative abnormalities of fibrinolytic system factors (tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PA-inhibitor) are often found in the patients exhibiting an idiopathic thromboembolic pathology. Exploration of fibrinolytic system is performed by taking blood samples prior and after stimulation (after venous occlusion or DDAVP injection). Patients can be "good responders" (that is presenting with an increase of fibrinolytic activity after stimulation) or "bad responders". Among these latter (that is 30 p. 100 of cases), there are two groups: patients exhibiting an increase of PA inhibitor level, this concealing fibrinolytic role of t-PA released by stimulation (20 p. 100 of cases), patients failing to present any t-PA release through endothelial cells stimulation (10 p. 100 of cases). Furthermore, an hypofibrinolysis was demonstrated a long time ago, in certain thrombogenic conditions (post-operative period, obesity, elderly patients). Hypofibrinolysis was recently demonstrated, according to such conditions, as liable to an increase in the PA inhibitor levels. As pathogenic role of hypofibrinolysis is then demonstrated, therapeutic studies reducing the PA inhibitor level or increasing the t-PA rate produced and released by endothelial cells are to be developed.

Fibrinolysis↗
PubMed1987

Release pattern of the vascular plasminogen activator and its inhibitor in human postvenous occlusion plasma as assessed by a spectrophotometric solid-phase fibrin-tPA activity assay.

Vascular or tissue-type plasminogen activator (plasma t-PA) is the circulating physiological fibrinolytic enzyme of endothelial cell origin which function is regulated by fibrin and a specific inhibitor (PAI). To study the pattern of release of t-PA and the behavior of t-PA-PAI complexes in plasma we determined t-PA activity in 44 healthy subjects before and after 10 min of forearm venous occlusion using a new spectrophotometric solid-phase fibrin-tPA activity assay. The assay is based on 1) the high affinity binding of t-PA to fibrin, and 2) the detection of fibrin-bound t-PA by measuring the release of pNA from a chromogenic substrate in the presence of plasminogen. Values at rest were rather undetectable in plasma (0.05 +/- 0.03 IU/ml, in 23 out of 44 samples) but were positively detected in all the euglobulins: 0.88 +/- 0.68 IU/ml. After venous occlusion the majority of plasmas (36 out of 44) showed a slight increase in t-PA activity (0.65 +/- 0.63 IU/ml) as compared to the important level observed in all the euglobulins (9.78 +/- 9.58 IU/ml). So, the ratio plasma/euglobulin t-PA activity was very low (0.06) and remained identical in both pre- and postocclusion samples. However, when diluted plasmas were tested the inhibitory effect disappeared and t-PA activity increased indicating that although t-PA circulates in a neutralized state it can be available for fibrinolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Immunoaffinity purification of HTC rat hepatoma cell plasminogen activator-inhibitor-1.

Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits tissue-type plasminogen activator activity by inducing a specific plasminogen activator-inhibitor (PAI-1). Using immobilized polyclonal antibodies raised against HT-1080 human fibrosarcoma PAI-1, we have purified HTC PAI-1 from serum-free medium conditioned by dexamethasone-treated HTC hepatoma cells and shown it to be antigenically related to human PAI-1. Greater than 100-fold purification with greater than 75% yield was achieved in a single step. The purified PAI-1 migrates on SDS-polyacrylamide gels as a single major band of 49 kDa with a minor band of 46 kDa. Digestion of PAI-1 with endoglycosidase F causes a shift toward faster migrating species which retain inhibitory activity. The purified PAI-1 was stable at pH 2.5, lost 50% of its activity after 15 min at 45 degrees C, and showed marked activation after treatment with SDS or guanidine-HCl. Purified PAI-1 rapidly inhibited and formed complexes with both tissue-type and urokinase-type plasminogen activators. Polyclonal rabbit antirat PAI-1 antibodies were raised which immunoprecipitate both free and complexed PAI-1.

Amino Acids↗
PubMed1988

Tissue plasminogen activator and its inhibitor following major surgery in relation to ventilatory pattern.

Based on animal data it has been hypothesised that lungs may regulate systemic fibrinolysis. To test this hypothesis 12 patients undergoing infrarenal aortic reconstruction due to arteriosclerotic disease were evaluated for influence of the respiratory pattern on release of tissue plasminogen activator (t-PA) and its inhibitor (PAI) in systemic and pulmonary arterial blood. Blood samples were drawn simultaneously from the pulmonary artery and a radial artery at defined times before and during surgery. Samples were also collected during application of positive end expiratory pressure (PEEP) of 5, 10 and 15 cmH2O. The blood samples were analysed for euglobulin clot lysis time (ECLT), t-PA activity, t-PA antigen and PAI. ECLT was not altered during surgery and t-PA activity was below the detectable limit. Before surgery t-PA antigen was 14.5 +/- 1.3 ng/ml and PAI 15.1 +/- 2.8 U/ml (mean +/- SEM). PAI was significantly elevated at end of surgery. There were no differences between samples from systemic and pulmonary arteries. Following application of 5, 10 and 15 cmH2O PEEP the fibrinolytic parameters were unchanged. Thus, in the present study on arteriosclerotic patients undergoing abdominal aortic reconstructions no influence from the respiratory pattern on systemic fibrinolysis could be demonstrated.

Aged↗
PubMed1988

Residual plasminogen activator inhibitor activity after venous stasis as a criterion for hypofibrinolysis: a study in 83 patients with confirmed deep vein thrombosis.

In eighty-three patients with confirmed deep vein thrombosis, the fibrinolytic system was studied before and after a 10-minute venous occlusion. Blood was collected at least 3 months after the last acute episode, and PAI-1 antigen and activity, as well as tissue-type plasminogen activator (t-PA) antigen, urokinase-type plasminogen activator (u-PA) antigen, and fibrinolytic activity were measured in these samples. During venous stasis, plasminogen activator inhibitor (PAI) activity decreased in almost all patients (81 of 83), from a median value of 8.2 to 2.9 U/mL (P less than .001, Wilcoxon signed-rank test). Because PAI-1 antigen augmented from a median value of 16 to 19.2 ng/mL (P less than .001), the decline in PAI activity was attributed to an increase in t-PA antigen from a median value of 10 to 21.7 ng/mL (P less than .001). Neutralization of PAI activity thus reflects the patient's capacity to overcome basal inhibitory potential through t-PA release. Based on residual PAI activity after 10-minute stasis, patients were classified as good or bad responders (PAI activity below detection limit, ie, less than or equal to 1.0 and greater than 1.0 U/ml, respectively). Good responders had a significantly higher fibrinolytic response after stasis than bad responders (median euglobulin clot lysis time 60 v 180 minutes; dilute whole blood clot lysis time 60 v 120 minutes; fibrinolytic activity on fibrin plates 7.7 v 0 U/mL). Furthermore, good responders, as compared with bad responders, had higher t-PA release (median 16.5 v 11.5 ng/mL), lower basal PAI activity (median 4.8 v 11.2 U/mL), and lower basal PAI-1 (median 11 v 21 ng/mL) and u-PA antigen (median 7.9 v 9.0 ng/mL, P less than .02). Hypofibrinolysis, as defined by the inability of released t-PA to overcome PAI-1 basal inhibitory potential, was observed in 45 of 83 patients (54%) and resulted either from an insufficient release of t-PA or from an increased basal PAI activity.

Adult↗