MEASUREMENT OF PLASMINOGEN ACTIVATOR INHIBITOR 1 (PAI-1) IN PLASMA WITH AN ELISA BASED ON TWO MURINE MONOCLONAL ANTIBODIES

1987 ◽  
Author(s):  
P J Declerck ◽  
M C Alessi ◽  
M Verstreken ◽  
E K O Kruithof ◽  
I Juhan-Vague ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Pregnant Women ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
First Trimester ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasminogen Activator Inhibitor 1 ◽  
Murine Monoclonal Antibodies ◽  
Activator Inhibitor ◽  
Pai 1

An enzyme-linked immunosorbent assay (ELISA) for quantitation of plasminogen activator inhibitor 1 (PAI-1) was developed, based on two murine monoclonal antibodies (MA-7D4B7 and MA-7F5), raised against purified PAI-1 from HT-1080 fibrosarcoma cells, which react with non-overlapping epitopes. MA-7D4B7 was coated on microtiter plates and bound PAI-1 antigen was quantitated with MA-7F5 conjugated with horseradish peroxidase.In normal plasma, collected on citrate at pH 7.4, the PAI-1 level is 27 ± 16 ng/ml (mean ± SD, n=ll), with a corresponding value of 19 ± 11 ng/ml (n=12) in plasma collected on acid citrate pH 4.5, which inhibits the release of PAI-1 from platelets. The lower limit of the assay in plasma is 2 ng/ml; the intra-assay, inter-assay and inter-dilution coefficients of variation are 5.2%, 8.0% and 7.1% respectively.This ELISA was used to measure PAI-1 levels in plasma (collected on citrate, pH 7.4) of non-pregnant women and of women at different stages of pregnancy. A progressive increase is observed : before: 20±9 ng/ml, n=7; first trimester: 25 ± 12 ng/ml, n=5; second trimester: 40 ± 25 ng/ml, n=ll; third trimester: 98 ± 46 ng/ml, n=13. A correlation coefficient of 0.70 is found between the duration of pregnancy and the PAI-1 level.Preliminary data indicate that the PAI-1 antigen level is increased in several disease states, including myocardial infarction and deep vein thrombosis.Thus, this newly developed ELISA allows a direct measurement of the fast-acting inhibitor of plasminogen activator in plasma. Application of this assay to plasma of non-pregnant and pregnant women substantiates previous results obtained with the use of functional assays. In order to quantitate PAI-1 antigen circulating in plasma, blood should be collected under conditions that prevent platelet stimulation.

scholarly journals Measurement of plasminogen activator inhibitor 1 in biologic fluids with a murine monoclonal antibody-based enzyme-linked immunosorbent assay

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 220-225 ◽  
Author(s):  
PJ Declerck ◽  
MC Alessi ◽  
M Verstreken ◽  
EK Kruithof ◽  
I Juhan-Vague ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Immunosorbent Assay ◽  
Healthy Subjects ◽  
Plasminogen Activator Inhibitor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasminogen Activator Inhibitor 1 ◽  
Platelet Poor Plasma ◽  
Activator Inhibitor ◽  
Pai 1

An enzyme-linked immunosorbent assay for plasminogen activator inhibitor-1 (PAI-1) in biologic fluids was developed on the basis of two murine monoclonal antibodies raised against PAI-1 purified from HT- 1080 fibrosarcoma cells. The lower limit of sensitivity of the assay in plasma is 2 ng/mL. The assay is 12 times less sensitive toward the PAI- 1/human tissue-type plasminogen activator (t-PA) complex as compared with free PAI-1. The intraassay, interassay, and interdilution coefficients of variation are 5.2%, 8.0%, and 7.1%, respectively. The level of PAI-1 in platelet-poor plasma of healthy subjects is 18 +/- 10 ng/mL (mean +/- SD, n = 45). In platelet-rich plasma after freezing and thawing, 92% of PAI-1 antigen is released from platelets, whereas only 8% is found in the corresponding platelet-poor plasma. In platelet-poor plasma from healthy subjects, a linear correlation (r = 0.80) was found between PAI activity and PAI-1 antigen. In plasma approximately two thirds of the PAI-1 antigen was functionally active, whereas only 5% of the PAI-1 antigen released from platelets was active. During pregnancy a progressive increase of PAI-1 antigen levels up to three- to sixfold the control value was observed. In plasma of patients with recurrent deep vein thrombosis, PAI-1 levels were 44 +/- 20 ng/mL (mean +/- SD, n = 7), during a clinically silent phase. Four of these patients had a level above 38 ng/mL (mean +/- 2 SD of normal). The present assay, based on stable and reproducible reagents, allows the specific determination of PAI-1 antigen in biologic fluids. It may facilitate interlaboratory comparisons and be useful for further investigations of the role of PAI-1 in clinical conditions associated with impaired fibrinolysis and/or a tendency to thrombosis and investigations of the role of PAI-1 in platelets.

scholarly journals Cell-Free mRNA Concentrations of Plasminogen Activator Inhibitor-1 and Tissue-Type Plasminogen Activator Are Increased in the Plasma of Pregnant Women with Preeclampsia

2007 ◽  
Vol 53 (3) ◽  
pp. 399-404 ◽  
Author(s):  
Yuditiya Purwosunu ◽  
Akihiko Sekizawa ◽  
Keiko Koide ◽  
Antonio Farina ◽  
Noroyono Wibowo ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Maternal Plasma ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Background: Detection of placental mRNA in maternal plasma has been reported in high-risk pregnancies. We attempted to investigate the concentrations of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (tPA) mRNA in maternal plasma in preeclampsia. Methods: Peripheral blood samples were obtained from healthy pregnant women before and after delivery and also from women with or without preeclampsia. Plasma was isolated from these samples, and RNA was extracted. Plasma PAI-1 and tPA mRNA concentrations were then measured by use of reverse transcription PCR assays. The concentrations were converted into multiples of the median (MoM) of the controls adjusted for gestational age. Data were stratified and analyzed according to the clinical severity of preeclampsia and quantitative distribution of blood pressure and proteinuria. Results: The median (minimum–maximum) PAI-1 mRNA MoM values for women with preeclampsia and controls were 2.48 (0.82–8.53) and 1.00 (0.41–2.33), respectively, whereas the median (minimum–maximum) tPA mRNA MoM values were 3.33 (1.01–10.58) and 1.00 (0.95–1.20), respectively. The concentrations of both PAI-1 and tPA mRNA were significantly increased in cases of preeclampsia, compared with controls (P <0.0001). The MoM values of both mRNA species were directly correlated with the severity of preeclampsia and were greatest among a subgroup of hemolysis, increased liver enzymes, and low platelets pregnancies. Conclusion: Maternal plasma PAI-1 and tPA mRNAs are significantly increased in patients with preeclampsia and are positively correlated with the severity of preeclampsia.

scholarly journals Measurement of plasminogen activator inhibitor 1 in biologic fluids with a murine monoclonal antibody-based enzyme-linked immunosorbent assay

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 220-225 ◽  
Author(s):  
PJ Declerck ◽  
MC Alessi ◽  
M Verstreken ◽  
EK Kruithof ◽  
I Juhan-Vague ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Immunosorbent Assay ◽  
Healthy Subjects ◽  
Plasminogen Activator Inhibitor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasminogen Activator Inhibitor 1 ◽  
Platelet Poor Plasma ◽  
Activator Inhibitor ◽  
Pai 1

Abstract An enzyme-linked immunosorbent assay for plasminogen activator inhibitor-1 (PAI-1) in biologic fluids was developed on the basis of two murine monoclonal antibodies raised against PAI-1 purified from HT- 1080 fibrosarcoma cells. The lower limit of sensitivity of the assay in plasma is 2 ng/mL. The assay is 12 times less sensitive toward the PAI- 1/human tissue-type plasminogen activator (t-PA) complex as compared with free PAI-1. The intraassay, interassay, and interdilution coefficients of variation are 5.2%, 8.0%, and 7.1%, respectively. The level of PAI-1 in platelet-poor plasma of healthy subjects is 18 +/- 10 ng/mL (mean +/- SD, n = 45). In platelet-rich plasma after freezing and thawing, 92% of PAI-1 antigen is released from platelets, whereas only 8% is found in the corresponding platelet-poor plasma. In platelet-poor plasma from healthy subjects, a linear correlation (r = 0.80) was found between PAI activity and PAI-1 antigen. In plasma approximately two thirds of the PAI-1 antigen was functionally active, whereas only 5% of the PAI-1 antigen released from platelets was active. During pregnancy a progressive increase of PAI-1 antigen levels up to three- to sixfold the control value was observed. In plasma of patients with recurrent deep vein thrombosis, PAI-1 levels were 44 +/- 20 ng/mL (mean +/- SD, n = 7), during a clinically silent phase. Four of these patients had a level above 38 ng/mL (mean +/- 2 SD of normal). The present assay, based on stable and reproducible reagents, allows the specific determination of PAI-1 antigen in biologic fluids. It may facilitate interlaboratory comparisons and be useful for further investigations of the role of PAI-1 in clinical conditions associated with impaired fibrinolysis and/or a tendency to thrombosis and investigations of the role of PAI-1 in platelets.

Secretion of α2-macroglobulin, α2-antiplasmin, and plasminogen activator inhibitor-1 by glioblastoma multiforme in primary organ culture

1990 ◽  
Vol 73 (2) ◽  
pp. 234-241 ◽  
Author(s):  
Mary E. Keohane ◽  
Scott W. Hall ◽  
Scott R. VandenBerg ◽  
Steven L. Gonias
Keyword(s):  
Glioblastoma Multiforme ◽  
Organ Culture ◽  
Plasminogen Activator ◽  
Culture System ◽  
Proteinase Inhibitors ◽  
Plasminogen Activator Inhibitor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

✓ Explants from a glioblastoma multiforme were maintained for 4 weeks in a three-dimensional Gelfoam matrix culture in order to study the synthesis of α2-antiplasmin (α2AP), α2-macroglobulin (α2M), plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (t-PA). Since the organ culture system promotes cellular differentiation in gliomas with increasing time in vitro, secretion of the proteinase inhibitors and t-PA was examined at weekly intervals. Increased immunohistochemical staining for glial fibrillary acidic protein, a marker of astroglial differentiation, was observed in the explants with advancing time in culture. The proteinase inhibitors α2AP and α2M were secreted into the medium in all 4 weeks, while PAI-1 was detected at significant concentrations by an enzyme-linked immunosorbent assay (ELISA) in Weeks 3 and 4 only. The quantity of each inhibitor secreted into fresh medium during a 24-hour interval increased with the age of the tumor explants in the Gelfoam culture system. At no time was a sensitive ELISA able to detect t-PA in the culture medium. This study demonstrates that glioblastoma multiforme cells in primary organ culture can secrete three major fibrinolysis proteinase inhibitors. The appearance of PAI-1 only after extensive culturing of the explants suggests a possible correlation with neoplastic astroglial maturation.

scholarly journals Regulation of fibrinolysis by platelet-released plasminogen activator inhibitor 1: light scattering and ultrastructural examination of lysis of a model platelet-fibrin thrombus

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1290-1299 ◽  
Author(s):  
JV Braaten ◽  
S Handt ◽  
WG Jerome ◽  
J Kirkpatrick ◽  
JC Lewis ◽  
...  
Keyword(s):  
Light Scattering ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Enzyme Linked Immunosorbent Assay ◽  
Scanning Electron Micrographs ◽  
Plasminogen Activator Inhibitor 1 ◽  
Kinetic Measurements ◽  
Activator Inhibitor ◽  
Pai 1

Abstract We have investigated the role of plasminogen activator inhibitor 1 (PAI- 1) in the regulation of fibrinolysis using a model thrombus composed of thrombin-stimulated platelets, fibrin(ogen), plasminogen, and recombinant tissue-type plasminogen activator. Laser light scattering kinetic measurements showed that clot lysis was significantly delayed both by thrombin-stimulated platelets and their cell-free releasate. This delay in lysis was almost fully reversed by the addition of a PAI- 1-specific monoclonal antibody that blocks the ability of PAI-1 to inhibit plasminogen activators. Lysis half-times exhibited a linear dependence on the concentration of PAI-1 antigen present, as determined by enzyme-linked immunosorbent assay (ELISA). Sodium dodecylsulfate- polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting confirmed the presence of PAI-1 antigen in the platelet releasates. Scanning electron micrographs of the model thrombus components sampled late in lysis showed considerable unproteolyzed fibrin still attached to platelets. Immunogold cytochemistry detected large amounts of PAI-1 antigen in the partially lysed platelet-fibrin thrombi. This PAI-1 appeared to be bound to the fibrin network rather than to the platelet surface itself. We conclude that the residual clots observed late in lysis represent platelet-associated fibrin to which platelet-released PAI-1 has bound, rendering it less susceptible to degradation.

scholarly journals Regulation of fibrinolysis by platelet-released plasminogen activator inhibitor 1: light scattering and ultrastructural examination of lysis of a model platelet-fibrin thrombus

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1290-1299
Author(s):  
JV Braaten ◽  
S Handt ◽  
WG Jerome ◽  
J Kirkpatrick ◽  
JC Lewis ◽  
...  
Keyword(s):  
Light Scattering ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Enzyme Linked Immunosorbent Assay ◽  
Scanning Electron Micrographs ◽  
Plasminogen Activator Inhibitor 1 ◽  
Kinetic Measurements ◽  
Activator Inhibitor ◽  
Pai 1

We have investigated the role of plasminogen activator inhibitor 1 (PAI- 1) in the regulation of fibrinolysis using a model thrombus composed of thrombin-stimulated platelets, fibrin(ogen), plasminogen, and recombinant tissue-type plasminogen activator. Laser light scattering kinetic measurements showed that clot lysis was significantly delayed both by thrombin-stimulated platelets and their cell-free releasate. This delay in lysis was almost fully reversed by the addition of a PAI- 1-specific monoclonal antibody that blocks the ability of PAI-1 to inhibit plasminogen activators. Lysis half-times exhibited a linear dependence on the concentration of PAI-1 antigen present, as determined by enzyme-linked immunosorbent assay (ELISA). Sodium dodecylsulfate- polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting confirmed the presence of PAI-1 antigen in the platelet releasates. Scanning electron micrographs of the model thrombus components sampled late in lysis showed considerable unproteolyzed fibrin still attached to platelets. Immunogold cytochemistry detected large amounts of PAI-1 antigen in the partially lysed platelet-fibrin thrombi. This PAI-1 appeared to be bound to the fibrin network rather than to the platelet surface itself. We conclude that the residual clots observed late in lysis represent platelet-associated fibrin to which platelet-released PAI-1 has bound, rendering it less susceptible to degradation.

scholarly journals Human Plasminogen Activator Inhibitor-1 (PAI-1) Deficiency: Characterization of a Large Kindred With a Null Mutation in the PAI-1 Gene

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 204-208 ◽  
Author(s):  
William P. Fay ◽  
Andrew C. Parker ◽  
Lorraine R. Condrey ◽  
Amy D. Shapiro
Keyword(s):  
Plasminogen Activator ◽  
Clinical Manifestations ◽  
Plasminogen Activator Inhibitor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasminogen Activator Inhibitor 1 ◽  
Oligonucleotide Hybridization ◽  
Abnormal Bleeding ◽  
Activator Inhibitor ◽  
Pai 1

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue- and urokinase-type plasminogen activators, is considered a critical regulator of the fibrinolytic system. We previously reported a child with abnormal bleeding and complete PAI-1 deficiency caused by a frame-shift mutation in exon 4 of the PAI-1 gene. The purpose of this study was to provide genetic and clinical data on the extended pedigree of the original proband to better define the phenotype associated with PAI-1 deficiency. Allele-specific oligonucleotide hybridization was used to genotype individuals, and serum PAI-1 antigen was measured by enzyme-linked immunosorbent assay. By this approach we have identified 19 individuals who are heterozygous for the PAI-1 null allele and 7 homozygous individuals with complete PAI-1 deficiency. Clinical manifestations of PAI-1 deficiency were restricted to abnormal bleeding, which was observed only after trauma or surgery in homozygous affected individuals. A spectrum of bleeding patterns was observed, including intracranial and joint bleeding after mild trauma, delayed surgical bleeding, severe menstrual bleeding, and frequent bruising. Fibrinolysis inhibitors, including ε-aminocaproic acid and tranexamic acid, were effective in treating and preventing bleeding episodes. Other than abnormal bleeding, no significant developmental or other abnormalities were observed in homozygous PAI-1–deficient individuals. Heterozygous PAI-1 deficiency was not associated with abnormal bleeding, even after trauma or surgery. These observations define the clinical spectrum of PAI-1 deficiency and provide additional evidence to support the hypothesis that the primary function of plasminogen activator inhibitor-1 in vivo is to regulate vascular fibrinolysis.

scholarly journals Identification of and partial characterization of platelet vitronectin: evidence for complex formation with platelet-derived plasminogen activator inhibitor-1

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 1989-1996 ◽  
Author(s):  
KT Preissner ◽  
S Holzhuter ◽  
C Justus ◽  
G Muller-Berghaus
Keyword(s):  
Complex Formation ◽  
Plasminogen Activator ◽  
Immunosorbent Assay ◽  
Plasminogen Activator Inhibitor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasminogen Activator Inhibitor 1 ◽  
Platelet Factor ◽  
Adhesive Proteins ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Vitronectin (VN; = complement S-protein), a plasma glycoprotein that is also associated with extracellular sites, was identified in washed human platelets contaminated with less than 0.05% of plasma VN. A specific enzyme-linked immunosorbent assay (ELISA) for VN has been developed and was used to detect and to quantitate VN in detergent extracts of washed platelets with 8.1 +/- 4.6 micrograms/10(9) platelets (n = 10), representing about 0.8% of the plasma VN pool. Platelet and plasma VN were similar by immunochemical criteria using Western-blot analysis, although platelet VN was mainly found as partially proteolyzed polypeptide. Total release of platelet VN occurred at optimal doses of Ca-ionophore 23187 or thrombin, whereas no VN was released by platelet treatment with digitonin or Staphylococcus alpha-toxin. During stimulation of washed platelets with various concentrations of thrombin, the nearly concomitant release of VN and plasminogen activator inhibitor-1 (PAI-1) together with platelet factor 4 indicated the association of VN with inner-platelet storage granules. Furthermore, platelet VN and PAI-1 in Ca-ionophore releasates comigrated during ultracentrifugation in high mol wt fractions of sucrose density gradients, indicating a possible association of both components. Complex formation of platelet VN and PAI-1 was verified by a sensitive enzyme-linked immunosorbent assay (ELISA) and accounts at least in part for a high molecular form of platelet VN. The identification of platelet VN and its binding to platelet PAI-1 raises the possibility that VN, in contrast to other adhesive proteins, may participate in localized regulatory functions of blood coagulation and fibrinolysis in platelet-matrix interactions and the protection of the matrix against proteolysis.

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