A colorimetric assay for releasable plasminogen activator.

1982 ◽  
Vol 28 (5) ◽  
pp. 1125-1128 ◽  
Author(s):  
E E Campbell ◽  
M A Shifman ◽  
J G Lewis ◽  
J J Pasqua ◽  
S V Pizzo
Keyword(s):  
Plasminogen Activator ◽  
Colorimetric Assay ◽  
Systolic Pressure ◽  
Venous Occlusion ◽  
Clin Pathol ◽  
Before And After ◽  
Report Data ◽  
Human Plasminogen ◽  
Form Blood Vessel ◽  
Radioactive Substrate

Abstract We describe an equilibrium assay for measuring release of plasminogen activator form blood-vessel walls and report data from 125 individuals free of overt thromboembolic disease. Excess human plasminogen is added to the euglobulin fraction of plasma obtained before and after venous occlusion at mean systolic pressure. To measure plasmin generation in these samples, we used the chromogenic plasmin substrate D-Val-Leu-Lys-p-nitroanilide, which liberates p-nitroaniline upon cleavage. Releasable plasminogen activator in 24 subjects was determined by this colorimetric assay and by the radiocasein assay previously reported by this laboratory (Am. J. Clin. Pathol. 76,403-409, 1981), and the results were compared. The correlation coefficient was 0.97. The colorimetric assay offers several advantages over the radiocasein assay: shorter incubation (6 vs 16 h) and no preparation or quantification of a radioactive substrate and its cleavage products.

Binding Of Plasminogen And Vascular Plasminogen Activator To Fibrin And The Fibrin Alpha-Chain

1981 ◽  
Author(s):  
D A Lloyd ◽  
S A Cederholm-William ◽  
A A Sharp
Keyword(s):  
Plasminogen Activator ◽  
Binding Sites ◽  
Venous Occlusion ◽  
Plasminogen Activation ◽  
Catalytic Surface ◽  
Alpha Chain ◽  
Fibrin Monomer ◽  
Human Plasminogen ◽  
Early Degradation ◽  
Fibrin Structure

Investigation of the mechanism of plasminogen activation in the presence of fibrin has shown that fibrin acts as a regulatory and catalytic surface for both the reaction between activator and fibrin and between activator and plasminogen. Plasminogen (mol wt 84,000) and vascular activator form stable complexes with fibrin. We have carried out studies to determine the location of these binding sites in the fibrin structure.METHODS: Human plasminogen (mol wt 84,000) was iodinated with 125-I and vascular plasminogen activator was partially purified from venous occlusion plasma by chromatography on lysine-Sepharose. Sepharose coupled fibrinogen, fibrin monomer, fragments E and D and isolated fibrin alpha chain were prepared and the interactions between plasminogen and vascular activator studied.RESULTS: Plasminogen (mol wt 84,000), plasmin and vascular activator each showed affinity for Sepharosebound fibrin monomer and fibrin alpha chain. In addition the affinity of the vascular activator for fibrinogen and fragment E was increased by treatment with thrombin.CONCLUSION: The alpha chain of fibrin brings together plasminogen activator and plasminogen in such a way as to produce plasminogen activation resulting in the early degradation steps of fibrin.

scholarly journals Plasminogen activator inhibitor 1: development of a radioimmunoassay and observations on its plasma concentration during venous occlusion and after platelet aggregation

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1645-1653 ◽  
Author(s):  
EK Kruithof ◽  
G Nicolosa ◽  
F Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Plasminogen Activator ◽  
Platelet Rich Plasma ◽  
Plasminogen Activator Inhibitor ◽  
Venous Occlusion ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Bovine Endothelial Cell ◽  
Before And After ◽  
Pai 1

Abstract To study the effect of plasminogen activator inhibitors (PAI) on fibrinolysis it is essential to be able to specifically measure these proteins in plasma. To this end PAI-1 was purified from cortisol- stimulated HT 1080 fibrosarcoma cells and antisera raised in rabbits. The immunologic relationship of the purified inhibitor to PAI-1 in plasma and platelet extracts was established by immunoblotting and regular and reverse fibrin zymography. Furthermore, the purified product could be immunoprecipitated with antibodies to human or bovine endothelial cell-derived PAI-1. A radioimmunoassay was developed that measures both free and tissue-type plasminogen activator (t-PA)-bound PAI-1 in plasma and has an effective range of 8 to 250 ng/mL. PAI-1 antigen levels showed a twofold increase after 20 minutes of venous occlusion, partially due to hemoconcentration. Approximately one quarter of PAI-1 before and after venous occlusion is derived from platelets. After correction for hemoconcentration and the contribution of platelets to plasma PAI-1 levels, a still significant increase in PAI-1 levels was noted during venous occlusion, which suggests that the local vascular bed releases PAI-1. Concomitant with PAI-1, t-PA antigen levels increased eightfold and fibrinolytic activity 18-fold after 20 minutes of venous occlusion. PAI-1 and t-PA levels tend to augment with age: in a group of older healthy volunteers (mean age, 53 years) PAI-1 levels were twice and t-PA levels 1.7 times higher than those in a group with a mean age of 29 years. Determination of PAI-1 antigen levels before and after platelet aggregation demonstrated that 85% of PAI-1 in platelet-rich plasma is associated with platelets. The average amount of PAI-1 per platelet was 0.3 fg/platelet, ie, 4,000 molecules per platelet.

scholarly journals Plasminogen activator inhibitor 1: development of a radioimmunoassay and observations on its plasma concentration during venous occlusion and after platelet aggregation

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1645-1653
Author(s):  
EK Kruithof ◽  
G Nicolosa ◽  
F Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Plasminogen Activator ◽  
Platelet Rich Plasma ◽  
Plasminogen Activator Inhibitor ◽  
Venous Occlusion ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Bovine Endothelial Cell ◽  
Before And After ◽  
Pai 1

To study the effect of plasminogen activator inhibitors (PAI) on fibrinolysis it is essential to be able to specifically measure these proteins in plasma. To this end PAI-1 was purified from cortisol- stimulated HT 1080 fibrosarcoma cells and antisera raised in rabbits. The immunologic relationship of the purified inhibitor to PAI-1 in plasma and platelet extracts was established by immunoblotting and regular and reverse fibrin zymography. Furthermore, the purified product could be immunoprecipitated with antibodies to human or bovine endothelial cell-derived PAI-1. A radioimmunoassay was developed that measures both free and tissue-type plasminogen activator (t-PA)-bound PAI-1 in plasma and has an effective range of 8 to 250 ng/mL. PAI-1 antigen levels showed a twofold increase after 20 minutes of venous occlusion, partially due to hemoconcentration. Approximately one quarter of PAI-1 before and after venous occlusion is derived from platelets. After correction for hemoconcentration and the contribution of platelets to plasma PAI-1 levels, a still significant increase in PAI-1 levels was noted during venous occlusion, which suggests that the local vascular bed releases PAI-1. Concomitant with PAI-1, t-PA antigen levels increased eightfold and fibrinolytic activity 18-fold after 20 minutes of venous occlusion. PAI-1 and t-PA levels tend to augment with age: in a group of older healthy volunteers (mean age, 53 years) PAI-1 levels were twice and t-PA levels 1.7 times higher than those in a group with a mean age of 29 years. Determination of PAI-1 antigen levels before and after platelet aggregation demonstrated that 85% of PAI-1 in platelet-rich plasma is associated with platelets. The average amount of PAI-1 per platelet was 0.3 fg/platelet, ie, 4,000 molecules per platelet.

On the Role of the Stable Plasminogen Activator of the Venous Wall in Occlusion Induced Fibrinolytic State

1969 ◽  
Vol 22 (03) ◽  
pp. 544-551 ◽  
Author(s):  
R Constantini ◽  
F Spöttl ◽  
F Holzknecht ◽  
H Braunsteiner
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Venous Blood ◽  
Venous Occlusion ◽  
Histochemical Method ◽  
Before And After ◽  
Venous Wall

SummaryThe fibrinolytic activity respectively the PA activity of normal veins before and after venous occlusion is measured by the histochemical method of Todd. The results show marked decreases of PA of the adventitia and an increase of PA of the endothelial type, after venous occlusion. This suggests a transfer of PA through the venous wall into the intravascular lumen, and may be the cause of increased fibrinolytic activity in venous blood after occlusion. Contrary assumptions are discussed.

The Increase of Leg Fibrinolytic Potential After Reduction of Hydrostatic Stimulus

1983 ◽  
Vol 50 (03) ◽  
pp. 731-734 ◽  
Author(s):  
Dušan Keber
Keyword(s):  
Plasminogen Activator ◽  
Venous Occlusion ◽  
Clot Lysis ◽  
The Third ◽  
Lysis Time ◽  
Before And After ◽  
Euglobulin Clot Lysis Time ◽  
The Difference ◽  
Fibrinolytic Potential ◽  
Clot Lysis Time

SummaryEleven patients were studied sequentially from the beginning of recumbency due to trauma up to the complete mobilization. The first blood sampling was performed 12 hr to 4 days after injury, the second after 12 to 33 days of recumbency and the third after one or more months of mobilization. The blood was drawn each time before and after venous occlusion of the arm and the leg for 20 min. Fibrinolytic potential was calculated as the difference between post- and preocclusion values of plasminogen activator activity, measured with the euglobulin clot lysis time (ECLT) and on fibrin plates. The results showed that fibrinolytic potential of legs after the period of recumbency was approaching that of the arms, being ten times higher as measured with ECLT and five times higher as measured on fibrin plates in comparison with the period after mobilization. It was concluded that hydrostatic pressure was the main, if not the only factor responsible for the difference in the content of plasminogen activator in veins of arms and legs and their fibrinolytic potential.

IDENTIFICATION OF A FIBRINOLYTIC DEFECT IN TWO FAMILIES WITH A TENDENCY TO THROMBOSIS

1987 ◽  
Author(s):  
H Ostermann ◽  
S Koenig ◽  
H Pollmann ◽  
U Schmitz-Huebner
Keyword(s):  
Venous Thrombosis ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Venous Occlusion ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Normal Range ◽  
Before And After ◽  
Euglobulin Clot Lysis Time

We identified two families in whom one member each suffered from deep venous thrombosis and subsequent pulmonary emboli at the age of 15 and 17. We were able to investigate several members of both families with regard to their fibrinolytic system. Blood sampling was done before and after ten minutes of venous occlusion. Parameters measured were euglobulin clot lysis time (ECLT), tissue-type plasminogen activator (t-PA) activity, t-PA concentration and plasminogen activator inhibitor (PAI) activity, besides several other constituents of the coagulation and fibrinolytic system commonly associated with thrombophilia.In the first family six persons could be evaluated. A prolonged ECLT was found in four probands. Three of them had no measurable t-PA activity after stasis, t-PA concentration after stasis was below the normal range in two and borderline in one of them.In the second family 13 members could be invest-gated. Three adults were found to have a prolonged ECLT. Two of these had very low t-PA activity after stasis, with normal increase in t-PA antigen. Their PAI was increased above the normal range. Six children showed no measurable PAI and increased levels of t-PA activity before stasis. After stasis four children had a prolonged ECLT. Two of them had low t-PA antigen levels, while all of them showed normal t-PA activity increases.These findings suggest, that there may be hereditary defects related to activators and inhibitors of the fibrinolytic system in the investigated families. These could possibly be responsible for the occurence of venous thrombosis early in life. However, results in the second family show that not all of the prolonged ECLT values can be explained by changes of t-PA and PAI.

Fibrinolytic Activity in Chinese Patients with Diabetes or Hyperlipidemia in Comparison with Healthy Controls

1991 ◽  
Vol 65 (01) ◽  
pp. 003-006 ◽  
Author(s):  
Chao Hung Ho ◽  
Tjin Shing Jap
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Plasminogen Activator Inhibitor ◽  
Venous Occlusion ◽  
Chinese Patients ◽  
Tissue Type ◽  
Diabetic Patients ◽  
Healthy Controls ◽  
Before And After ◽  
Patients With Diabetes

SummaryPlasma tissue-type plasminogen activator (tPA), plasminogen activator inhibitor (PAI) and euglobulin lysis time (ELT) were determined before and after the venous occlusion test (VOT) in 3 groups of patients with mean age about 60 years: 29 diabetic patients (D group), 8 hyperlipidemic patients (H group) and 19 healthy controls (C group). In the D and H groups, the mean of morning tPA was significantly higher than that of the C grogp, but the means of PAI were not significantly different among the 3 groups. ELI was significantly shortened and tPA was markedly increased after the VOT in all 3 groups whereas PAI had not significantly changed. In conclusion, high tPA activity and good fibrinolytic response without significant change of PAI .activity were found in the diabetic and hyperlipidemic patients, and no definite impairment of the fibrinolytic activity could be found in the Chinese patients with diabetes and hyperlipidemia. This might be one of the reasons why the Chinese has low incidence of thromboembolic diseases.

Fibrinolysis in Normal Subjects - Comparison Between Plasminogen Activator Inhibitor and Other Components of the Fibrinolytic System

1988 ◽  
Vol 59 (02) ◽  
pp. 299-303 ◽  
Author(s):  
Grazia Nicoloso ◽  
Jacques Hauert ◽  
Egbert K O Kruithof ◽  
Guy Van Melle ◽  
Fedor Bachmann
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Plasminogen Activator Inhibitor ◽  
Venous Occlusion ◽  
Venous Stasis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Plate Assay ◽  
Fibrin Plate ◽  
Activator Inhibitor ◽  
Pai 1

SummaryWe analyzed fibrinolytic parameters in 20 healthy men and 20 healthy women, aged from 25 to 59, before and after 10 and 20 min venous occlusion. The 10 min post-occlusion fibrinolytic activity measured directly in diluted unfractionated plasma by a highly sensitive 125I-fibrin plate assay correlated well with the activity of euglobulins determined by the classical fibrin plate assay (r = 0.729), but pre-stasis activities determined with these two methods did not correlate (r = 0.084). The enhancement of fibrinolytic activity after venous occlusion was mainly due to an increase of t-PA in the occluded vessels (4-fold increase t-PA antigen after 10 min and 8-fold after 20 min venous occlusion). Plasminogen activator inhibitor (PAI) activity and plasminogen activator inhibitor 1 (PAI-1)1 antigen levels at rest showed considerable dispersion ranging from 1.9 to 12.4 U/ml, respectively 6.9 to 77 ng/ml. A significant increase of PAI-1 antigen levels was observed after 10 and 20 min venous occlusion. At rest no correlation was found between PAI activity or PAI-1 antigen levels and the fibrinolytic activity measured by 125I-FPA. However, a high level of PAI-1 at rest was associated with a high prestasis antigen level of t-PA and a low fibrinolytic response after 10 min of venous stasis. Since the fibrinolytic response inversely correlated with PAI activity at rest, we conclude that its degree depends mainly on the presence of free PAI.

A Plasma Clot Lysis Assay Based on the Release of Fibrin Degradation Products: Application to the Diagnosis of Hypofibrinolytic States

1990 ◽  
Vol 63 (01) ◽  
pp. 076-081 ◽  
Author(s):  
Pascale Gaussem ◽  
Sophie Gandrille ◽  
Pascale Molho-Sabatier ◽  
Loïc Capron ◽  
Jean-Noël Fiessinger ◽  
...  
Keyword(s):  
Degradation Products ◽  
Venous Occlusion ◽  
Lower Limbs ◽  
Clot Lysis ◽  
Plasma Clot ◽  
Vein Thrombosis ◽  
Fibrin Degradation Products ◽  
Before And After ◽  
Euglobulin Clot Lysis Time ◽  
Pai 1

SummaryUsing a monoclonal antibody-based assay, we measured the fibrin degradation product release in the supernatant of plasma clots obtained before and after venous occlusion (VO) in 30 patients with definite or suspected vascular thrombosis (19 definite and 2 suspected deep vein thrombosis, 6 recurrent superficial thrombophlebitis, 3 arterial occlusions of lower limbs). tPA and PAI-1 concentrations were determined using ELISA assays; the post-occlusion values were corrected for haemoconcentration. The increase in tPA during VO was correlated with haemoconcentration (r = 0.74), but 3 patients had ineffective VO (<2% increase in proteins). The fibrinolytic response to VO was evaluated using the shortening of the time necessary for the release of 200 μg of fibrin degradation products per mg of fibrinogen (Δ T 200). Two among the 27 patients with effective VO were bad responders with a Δ T 200 <3 h (whereas all the others had Δ T 200 >10 h). These patients had respectively a deficient tPA release (Δ tPA = 1 ng/ml) and an elevated PAI-1 level at rest (33 ng/ml). Several other patients were bad responders in terms of tPA release or of shortening of the euglobulin clot lysis time but they had a normal Δ T 200. This plasma clot test reflects the ability of free tPA to bind to fibrin (the amount of which depends on the level of tPA and PAI-1), and may be useful in the diagnosis of a hypofibrinolytic state.

Sign in / Sign up

Export Citation Format

Share Document