Immunoassays for the Quantitation of Porcine PAI-1 Antigen and Activity in Biological Fluid Samples

2000 ◽  
Vol 84 (12) ◽  
pp. 1082-1086 ◽  
Author(s):  
Henry M. Leng ◽  
Els Brouwers ◽  
Isabelle Knockaert ◽  
Paul Declerck
Keyword(s):  
Biological Fluid ◽  
Platelet Rich Plasma ◽  
Active Form ◽  
Plasminogen Activator Inhibitor ◽  
Experimental Models ◽  
Activity Levels ◽  
Plasminogen Activator Inhibitor 1 ◽  
Antigen Elisa ◽  
Pathophysiological Role ◽  
Pai 1

SummaryTwo monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) for the quantitation of porcine plasminogen activator inhibitor-1 (PAI-1) antigen and activity in plasma were constructed and validated. The intra-assay, interassay, and interdilution coefficients of variation were 4.3, 13, and 8%, respectively, for the antigen ELISA and 5, 16, and 11% for the activity assay. Assay recoveries, in the antigen ELISA, of either latent or active recombinant porcine PAI-1 (10 and 50 ng/ml) added to plasma were 86 ± 9% and 92 ± 22%, respectively, for the latent form and 89 ± 9% and 87 ± 7% for the active form (mean ± SD, n = 3 to 4). In the immunofunctional assay, recoveries for the same concentrations of active PAI-1 were 108 ± 16% and 92 ± 21%, respectively. In male porcine plasma the level of PAI-1 antigen was 31 ± 11 ng/ ml and the activity, 34 ± 16 ng/ml (mean ± SD, n = 10). In female plasma PAI-1 antigen levels were 20 ± 5.2 ng/ml and the PAI-1 activity 42 ± 17 ng/ml (n = 13). A linear correlation was found between PAI-1 antigen and activity levels in male (r = 0.60) and female (r = 0.70) plasma. Immunodepletion resulted in a decrease of >95% of the original PAI-1 antigen or activity levels. Incubation of plasma samples at 37° C for 16 h resulted in a significant decrease (70 to 85%) of PAI-1 activity. Under these conditions (37° C, 16 h) PAI-1 antigen levels remained unchanged in males whereas the response of the female samples in the PAI-1 antigen assay increased two-fold.In lysed platelet-rich plasma males had 990 ± 470 ng/ml antigen and 160 ± 80 ng/ml activity and females, 920 ± 500 ng/ml antigen and 150 ± 98 ng/ml activity corresponding to 2.1 ± 0.77 fg PAI-1 antigen per platelet. Only 16% of PAI-1 released from platelets was found to be active. Linear correlations between PAI-1 antigen and activity were found for both males (r = 0.61) and females (r = 0.67).The assays are both sensitive and specific and may, therefore, aid the elucidation of the pathophysiological role of PAI-1 in swine experimental models of atherosclerosis and other thrombotic disorders.

Plasminogen Activator Inhibitor 1 Is Elevated in the Children of Men with Premature Myocardial Infarction

1996 ◽  
Vol 76 (03) ◽  
pp. 417-421 ◽  
Author(s):  
Loukianos S Rallidis ◽  
Ageliki A Megalou ◽  
Nikos H Papageorgakis ◽  
Athanasios G Trikas ◽  
Grammatiki I Chatzidimitriou ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Plasminogen Activator ◽  
Apolipoprotein B ◽  
Plasminogen Activator Inhibitor ◽  
Activity Levels ◽  
Plasminogen Activator Inhibitor 1 ◽  
History Of ◽  
Premature Myocardial Infarction ◽  
Young Subjects ◽  
Pai 1

SummaryTo assess whether plasminogen activator inhibitor 1 (PAI-1) activity is elevated in the progeny of young coronary men, 193 young subjects were recruited and divided into two groups. Group A consisted of 104 children whose fathers had suffered a myocardial infarction before the age of 55 (“cases”). Eighty-nine young subjects matched for age, sex, body mass index (BMI) and smoking habits without familial history of coronary artery disease (CAD) served as controls (group B). Children with a family history of diabetes mellitus or hypertension were excluded from both groups. We measured PAI-1 activity, tissue-type plasminogen activator (t-PA) antigen, a2-antiplasmin, fibrinogen, lipids and apolipoproteins in both groups. PAI-1 activity levels were also determined in the men who suffered a premature myocardial infarction 4 months after their discharge. PAI-1 activity levels were higher in cases compared to controls (3.13 ± 1.9 vs 2.17 ± 1.9 U/ml, p = 0.0014). t-PA antigen and a2-antiplasmin did not differ significantly between the two groups, while fibrinogen, total cholesterol, low-den-sity lipoprotein cholesterol, apolipoprotein B and lipoprotein(a) were significantly higher in group A. PAI-1 was positively correlated with triglycerides (r = 0.22, p = 0.024), apolipoprotein B (r = 0.21, p = 0.039) and fibrinogen (r = 0.22, p = 0.029 ) in cases and with BMI in both cases (r = 0.37, p = 0.0003) and controls (r = 0.23, p = 0.044). In stepwise multiple regression analysis, only apolipoprotein B (p = 0.008) and BMI (p = 0.0014) were significant determinants of PAI-1 activity in cases. There was also a positive correlation between PAI-1 activity levels of the affected fathers and their children (r = 0.30, p = 0.01). The present data support the hypothesis that elevated PAI-1 levels in the offspring of men with premature myocardial infarction impair their fibrinolytic capacity contributing to their familial predisposition to CAD.

PARALLEL VARIATIONS OF PLASMA INSULIN, TRIGLYCERIDE AND PLASMINOGEN ACTIVATOR INHIBITOR 1 (PAI 1) LEVELS IN OBESE NON DIABETIC SUBJECTS

1987 ◽  
Author(s):  
I Juhan-Vague ◽  
P Vague ◽  
M C Alessi ◽  
C Atlan ◽  
J Valadier ◽  
...  
Keyword(s):  
Plasma Insulin ◽  
Direct Action ◽  
Plasminogen Activator Inhibitor ◽  
Activity Levels ◽  
Metformin Treatment ◽  
Obese Women ◽  
Plasminogen Activator Inhibitor 1 ◽  
Obese Subjects ◽  
High Level ◽  
Pai 1

We had shown in non diabetic healthy subjects with Body Mass Index (BMI) varying largely, a significant correlation (r) between PAI activity levels and BMI : (r=0.66) and insulinemia (r=0.52).PAI levels were then studied in non diabetic Obese women (0)(n=50) and in age matched healthy women with normal BMI (N) (m ± SEM) = 0 versus N : BMI : 33.4± 0.8/ 20.2± 0.8 ; plasma insulin (Ins -uu/ml) : 22.7±1.5/12±1; Triglyceride(TG-mmol/1):1.240.09/0.8±0.1.The low value of euglobulin fibrinolytic activity (EFA) in Obese was due to high levels of PAI 1 : 0 versus N : EFA (mm) : 5.2 ±0.3/ 9 ±0.3 ; PAI activity (PAIact.- Verheijen's method-u/ml) :14 ± 2.1/5.04 ±0.6 -(p 0.01). In 10 0 and 10 N PAIact and PAI 1 antigen (PAI 1 Ag- Kruithof's method-ng/ml) were determined in parallel : 0 versus N : PAIact.: 29.4±3.8/ 5.8±0.9 ; PAI 1 Ag : 100±14.2/19.7 ±1.7, (p0.01). In Obesegroup correlations between, PAIact. and BMI (r=0.51), and insulin (r=0.69) and Triglyceride (r=0.48) were significant (p 0.01).When in Obese subjects, insulinemia was decreased by 24 hours Fast (n=10) or by 15 days Metformin treatment (1.7g/day) (n=9), PAI activity (measured on fibrin plates)decreased significantly in a parallel way : compared to initial values -after 24 hours Fast =Ins : 75 %, PAIact. 73% -after Metformin treatment=Ins :75%, PAIact. :57%, TG :73%.From these results, a direct action of insulin on the synthesis cells of PAI 1 could be evoked. An effect of Triglyceride levels cannot be excluded, the variation of Triglyceride, insulin, PAI levels being parallel.As hyperinsulinemia, hypertriglyceridemia are risk factors for atherothrombosis, as there is a link between insulin, triglyceride, PA Inhibitor 1 levels, the pathogenic role of hyperinsulinemia and hypertriglyceridemia in the development of atherothrombosis could be in part mediated by plasma hypofibrinolysis due to high level of PAI 1.

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 in poorly controlled vs well controlled Type-2 Diabetes Mellitus patients: A case-control study in a district hospital in Ghana

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250090
Author(s):  
Charles Nkansah ◽  
Otchere Addai-Mensah ◽  
Kofi Mensah ◽  
Michael Owusu ◽  
Richard K. D. Ephraim ◽  
...  
Keyword(s):  
Glycemic Control ◽  
District Hospital ◽  
Plasminogen Activator Inhibitor ◽  
Activity Level ◽  
Activity Levels ◽  
Plasminogen Activator Inhibitor 1 ◽  
Good Glycemic Control ◽  
Activator Inhibitor ◽  
Pai 1

Background Hypofibrinolysis resulting from the up-regulation of plasminogen activator inhibitor-1 (PAI-1) usually occurs in patients with type 2 diabetes mellitus (T2DM), rendering them hypercoagulable. This study assessed the plasma antigen and activity levels of the PAI-1 enzyme in T2DM patients in a district hospital in Ghana. Methods This was a hospital-based case-control study conducted from December 2018 to May 2019 at Nkenkaasu District Hospital. Sixty subjects with T2DM (30 T2DM subjects with good glycemic control and 30 with poor glycemic control), and 30 apparently healthy blood donors were recruited into the study. Blood specimens were collected for complete blood count, lipid profile, PAI-1 Ag and PAI-1 activity levels. A pre-tested questionnaire was used to obtain demographic and clinical information. The data was analyzed using SPSS version 22.0. Results Elevated PAI-1 Ag and activity levels were observed in the T2DM subjects compared to the healthy controls, with the levels and activity significantly higher (PAI-1 Ag; p< 0.001, PAI-1 activity level; p = 0.004) in the T2DM subjects with poor glycemic control in comparison to those with good glycemic control. A significant positive correlation was observed between HbA1c and PAI-1 enzymes. PAI-1 Ag levels significantly increased along with increased total cholesterol (Β = 0.262, p = 0.033), triglyceride (Β = -0.273, p = 0.034) and HbA1c (Β = 0.419, p = 0.001). Similarly, PAI-1 activity level was associated with total cholesterol (Β = 0.325, p = 0.009), triglyceride (Β = -0.262, p = 0.042), HbA1c (Β = 0.389, p = 0.003) and VLDL-c (Β = -0.227, p = 0.029). Conclusion PAI-1 antigen/activity is enhanced in poorly controlled Ghanaian T2DM subjects. The hypercoagulable state of the affected individuals put them at higher risk of developing cardiovascular diseases. Good glycemic control to regulate plasma PAI-1 levels is essential during T2DM lifelong management. Markers of fibrinolysis should be assessed in these individuals and appropriate anticoagulants given to prevent thrombosis and adverse cardiovascular diseases.

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.

Monoclonal Antibody-based Immunoassays for the Specific Quantitation of Rat PAI-1 Antigen and Activity in Biological Samples

1998 ◽  
Vol 79 (04) ◽  
pp. 808-812 ◽  
Author(s):  
Thu-Hoa Ngo ◽  
S. Verheyen ◽  
I. Knockaert ◽  
P. J. Declerck
Keyword(s):  
Biological Fluids ◽  
Gel Filtration ◽  
Plasminogen Activator Inhibitor ◽  
Fold Increase ◽  
Rat Plasma ◽  
Activity Levels ◽  
Activity Assay ◽  
Plasminogen Activator Inhibitor 1 ◽  
Two Peaks ◽  
Pai 1

SummaryTwo enzyme-linked immunosorbent assays (ELISAs) for the quantitation of rat plasminogen activator inhibitor-1 (PAI-1) antigen and activity, respectively, in biological fluids were developed using monoclonal antibodies raised against recombinant rat PAI-1. These assays had a lower limit of sensitivity in plasma of 0.3 and 0.15 ng/ml, respectively. The intra-assay, inter-assay and inter-dilution coefficients of variation were 9, 14 and 9%, respectively, for the antigen assay and 8, 17 and 13%, respectively for the activity assay. Assay recoveries of recombinant rat PAI-1 (5 to 20 ng/ml) added to plasma were 73 to 88% and 89 to 93% for the antigen and the activity assay, respectively. The level of PAI-1 antigen in rat plasma was 1.8 ± 0.9 ng/ml (mean ± SD, n = 18), with a corresponding value of 1.0 ± 0.5 ng/ml for PAI-1 activity. In lysed platelet-rich rat plasma PAI-1 antigen was 6.0 ± 1.0 ng/ml (n = 8) and PAI-1 activity was 2.3 ± 0.4 ng/ml (n = 8). Endotoxin injection (0.5 mg/kg) induced a time-dependent increase of both PAI-1 antigen and PAI-1 activity levels in rat plasma, eventually resulting in a 100- to 200-fold increase (p <0.0001 vs. baseline). A linear correlation was found between PAI-1 antigen and activity levels in normal plasma (r = 0.63, n = 18, p <0.01) and in plasma from endotoxin-treated rats (r = 0.90, n = 35, p <0.001). Application of these assays for the analysis of gel filtration experiments of plasma from endotoxin-treated rats demonstrated that PAI-1 antigen eluted as two peaks (with corresponding Mr of ~430 kDa and 61 kDa) whereas PAI-1 activity eluted as a single peak corresponding with the high molecular weight antigen form.Thus, these unique assays allowing the specific determination of rat PAI-1 antigen and rat PAI-1 activity may constitute important tools for further investigations on the pathophysiological role of PAI-1 in a variety of experimental rat models.

scholarly journals Role of Shear Stress and tPA Concentration in the Fibrinolytic Potential of Thrombi

2021 ◽  
Vol 22 (4) ◽  
pp. 2115
Author(s):  
Claire S. Whyte ◽  
Hadj Ahmed. Mostefai ◽  
Kim M. Baeten ◽  
Andrew J. Lucking ◽  
David E. Newby ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Ex Vivo ◽  
Active Form ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
High Shear ◽  
Plasminogen Activator Inhibitor 1 ◽  
Fibrinolytic Potential ◽  
Pai 1 ◽  
Low Shear

The resolution of arterial thrombi is critically dependent on the endogenous fibrinolytic system. Using well-established and complementary whole blood models, we investigated the endogenous fibrinolytic potential of the tissue-type plasminogen activator (tPA) and the intra-thrombus distribution of fibrinolytic proteins, formed ex vivo under shear. tPA was present at physiologically relevant concentrations and fibrinolysis was monitored using an FITC-labelled fibrinogen tracer. Thrombi were formed from anticoagulated blood using a Chandler Loop and from non-anticoagulated blood perfused over specially-prepared porcine aorta strips under low (212 s−1) and high shear (1690 s−1) conditions in a Badimon Chamber. Plasminogen, tPA and plasminogen activator inhibitor-1 (PAI-1) concentrations were measured by ELISA. The tPA–PAI-1 complex was abundant in Chandler model thrombi serum. In contrast, free tPA was evident in the head of thrombi and correlated with fibrinolytic activity. Badimon thrombi formed under high shear conditions were more resistant to fibrinolysis than those formed at low shear. Plasminogen and tPA concentrations were elevated in thrombi formed at low shear, while PAI-1 concentrations were augmented at high shear rates. In conclusion, tPA primarily localises to the thrombus head in a free and active form. Thrombi formed at high shear incorporate less tPA and plasminogen and increased PAI-1, thereby enhancing resistance to degradation.

Immunological Quantitation of Rabbit Plasminogen Activator Inhibitor-1 in Biological Samples

1999 ◽  
Vol 82 (11) ◽  
pp. 1510-1515 ◽  
Author(s):  
Thu-Hoa Ngo ◽  
Paul Declerck
Keyword(s):  
Plasminogen Activator ◽  
Biological Samples ◽  
Plasminogen Activator Inhibitor ◽  
Rabbit Serum ◽  
Activity Levels ◽  
Rabbit Plasma ◽  
Plasminogen Activator Inhibitor 1 ◽  
Rabbit Platelets ◽  
Activator Inhibitor ◽  
Pai 1

SummaryTwo immunoassays for the specific quantitation of rabbit plasminogen activator inhibitor-1 (PAI-1) antigen and activity in biological samples were developed and applied for the evaluation of PAI-1 in rabbits. Levels of PAI-1 antigen in rabbit plasma were 9.8 ± 4.6 ng/ml (mean ± SD, n = 6), with a corresponding value of 20.5 ± 13.5 ng/ml for PAI-1 activity. In rabbit serum PAI-1 antigen was 11.8 ± 4.9 ng/ml (n = 6) and PAI-1 activity was 2.9 ± 2.0 ng/ml (n = 6). Endotoxin injection (20 μg/kg, iv) induced a time-dependent increase of both PAI-1 antigen and PAI-1 activity levels in rabbit plasma, eventually resulting in a 40- to 90-fold increase (p <0.0001 vs baseline). A linear correlation was found between PAI-1 antigen and activity levels in normal plasma (r = 0.90, n = 6, p <0.05) and in plasma from endotoxin-treated rabbits (r = 0.98, n = 20, p <0.001). Analysis of PAI-1 antigen and activity in lysates of washed rabbit platelets revealed the absence of PAI-1 (i.e. <0.03 ng/108 platelets).In conclusion, development of specific immunological assays allowed the quantitation of PAI-1 in rabbit samples. In striking contrast to other species (human, rat, mouse, pig) rabbit platelets lack detectable amounts of PAI-1 (i.e. >100-1000 fold lower vs other species studied). This observation may have important implications for the use of experimental rabbit models especially in studies on the role of platelets in various pathological conditions including thrombosis and atherosclerosis.

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