Determination of tissue plasminogen activator and its "fast" inhibitor in plasma.

1986 ◽  
Vol 32 (3) ◽  
pp. 482-485 ◽  
Author(s):  
J Chmielewska ◽  
B Wiman
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Plasma Samples ◽  
Inhibitor Concentration ◽  
Blood Samples ◽  
Analytical Recovery ◽  
Specific Determination ◽  
Tissue Plasminogen ◽  
Coupled Assay

Abstract We describe efficient, accurate methods for specific determination of tissue plasminogen activator (t-PA, EC 3.4.21.31) and its "fast" inhibitor in plasma. In this coupled assay, a sample containing t-PA is incubated with plasminogen, a plasmin (EC 3.4.21.7) substrate of low Km and high Kcat, and fibrin as a stimulator. The inhibitor of t-PA is determined by incubating the sample with a known amount of t-PA in excess, then determining the residual t-PA. Both t-PA and t-PA inhibitor can be determined in many samples simultaneously within a few hours. These assays are modifications of procedures described by us (Clin Chim Acta 1983;127:279-88 and Thromb Res 1983;31:427-36). Their accuracy as assessed by analytical recovery of pure t-PA added to blood samples (91 +/- 4%) or of partly purified inhibitor added to plasma samples (102 +/- 10%) is satisfactory, as is their precision. For the t-PA assay the CV was 1.6% (within run) or 4.1% (between run). The corresponding values for the inhibitor assay were 4.5% (within run) or 8.4% (between run) if the inhibitor concentration exceeded 3 arb. units/mL.

Immunoreactivity of Tissue Plasminogen Activator and of Its Inhibitor Complexes

1989 ◽  
Vol 61 (03) ◽  
pp. 409-414 ◽  
Author(s):  
M Rånby ◽  
G Nguyen ◽  
P Y Scarabin ◽  
M Samama
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Degradation Products ◽  
Gel Filtration ◽  
Free Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Significant Difference ◽  
Tissue Plasminogen ◽  
Pai 1

SummaryAn enzyme linked immunosorbent assay (ELISA) based on goat polyclonal antibodies against human tissue plasminogen activator (tPA) was evaluated. The relative immunoreactivity of tPA in free form and tPA in complex with inhibitors was estimated by ELISA and found to be 100, 74, 94, 92 and 8l% for free tPA and tPA in complex with PAI-1, PAI-2, α2-antiplasmin and C1-inhibitor, respectively. Addition of tPA to PAI-1 rich plasma resulted in rapid and total loss of tPA activity without detectable loss of ELISA response, indicating an immunoreactivity of tPA in tPA/PAI-1 complex of about l00%. Three different treatments of citrated plasma samples (acidification/reneutralization, addition of 5 mM EDTA or of 0.5 M lysine) prior to determination by ELISA all resulted in increased tPA levels. The fact that the increase was equally large in all three cases along with good analytical recovery of tPA added to plasffi, supported the notion that all tPA antigen present in plasma samples is measured by the ELISA. Analysis by ELISA of fractions obtained by gel filtration of plasma from a patient undergoing tPA treatment identified tPA/inhibitor complexes and free tPA but no low molecular weight degradation products of tPA. Determinations of tPA antigen were made at seven French clinical laboratories on coded and randomized plasma samples with known tPA antigen content. For undiluted samples there was no significant difference between the tPA levels found and those known to be present. The between-assay coefficient of variation was 7 to 10%. In conclusion, the ELISA appeared suited for determination of total tPA antigen in human plasma samples.

An Enzyme Linked Immunosorbent Assay for Determination of Tissue Plasminogen Activator Applied to Patients with Thromboembolic Disease

1983 ◽  
Vol 50 (03) ◽  
pp. 740-744 ◽  
Author(s):  
Nils Bergsdorf ◽  
Torbjörn Nilsson ◽  
Per Wallén
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Specific Activity ◽  
Thromboembolic Disease ◽  
Tissue Type ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type Plasminogen Activator ◽  
Tissue Plasminogen ◽  
Normal Human

SummaryUtilizing the immunoglobulin fraction from a goat antiserum against human uterine tissue plasminogen activator, an enzyme- linked immunoassay for tissue-type plasminogen activator in human plasma has been developed. With the new method, the concentration of t-PA in normal human acidified plasma is found to be 4.0 ± 1.8 (SD) ng/ml. It increases to 12 ng/ml after a tomiquet test, and to 14 ng/ml after strenous physical exercise. In a group of patients with idiopathic thromboembolic disease, the resting t-PA concentration was 5 ng/ml and the post-occlusion value 16 ng/ml. Furthermore, the patients also exhibited a normal post-occlusion rise in the concentration of plasmin-α2-antiplasmin complex. However, in 37% of the post-occlusion patient plasmas, virtually no increase in t-PA could be detected by a specific activity assay. The results indicate that the reason for a defective post-occlusion fibrinolytic activity in a majority of cases may be the presence of increased concentrations of a fast-acting specific t-PA inhibitor.

A Comparison of Thromboelastography with Heparinase or Protamine Sulfate Added In Vitro during Heparinized Cardiopulmonary Bypass

1997 ◽  
Vol 78 (02) ◽  
pp. 820-826 ◽  
Author(s):  
Bruce D Spiess ◽  
Michael H Wall ◽  
Bruce S Gillies ◽  
Jane C K Fitch ◽  
Louise O Soltow ◽  
...  
Keyword(s):  
Cardiopulmonary Bypass ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Postoperative Hemorrhage ◽  
Sensitive Indicator ◽  
Blood Samples ◽  
Significant Difference ◽  
Tissue Plasminogen ◽  
Heparin Neutralization

SummaryThromboelastography (TEG) has been used after cardiopulmonary bypass (CPB) to diagnose excessive postoperative hemorrhage. Conventional TEG during CPB is not possible due to the sensitivity of the TEG to even small amounts of heparin, which produces a nondiagnostic tracing. The purpose of this study was to compare heparin neutralization using heparinase or protamine in TEG blood samples obtained during CPB. TEG testing was performed on 48 patients before, during and after CPB. Tissue plasminogen activator activity and antigen were measured on a subset of 32 patients. We found: 1) heparinase neutralized at least 10 IU/ml heparin while 1.6 ug/ml protamine neutralized up to 7 IU/ml heparin, 2) in samples with complete heparin neutralization by both methods, there was no significant difference in the R values, 3) while there was good correlation for other TEG parameters between heparinase and protamine treated samples, heparinase treatment produced shorter K values and higher angle, MA and A60, 4) while fibrinolysis was detected using both methods, heparinase treatment suppressed fibrinolysis in the TEG in both samples from patients and after in vitro addition of tissue plasminogen activator, 5) TEG was not a sensitive indicator of t-PA activity, detecting only 21% of samples with increased t-PA activity during bypass, and 5) heparinase was at least 100 times more expensive than protamine. We conclude that while both heparinase and protamine can be used to neutralize heparin in TEG samples obtained during CPB, protamine neutralization is more sensitive to fibrinolysis and less expensive, but the protamine dose must be carefully selected to match the heparin level used at individual institutions.

Abstract WP287: Deterioration in Recombinant Tissue Plasminogen Activator After Repeated Freezing and Thawing Cycles for Thromboelastography

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Sean Calo ◽  
Anja-Kathrin Jaehne ◽  
Kelly A Keenan ◽  
Jun Xu ◽  
Baruch Tawil ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Human Blood ◽  
Whole Blood ◽  
Recombinant Tissue Plasminogen Activator ◽  
Healthy Human ◽  
Clot Strength ◽  
Blood Samples ◽  
Freeze Dried ◽  
Tissue Plasminogen

Background and Purpose: Thromboelastography (TEG) is often used to measure coagulation dynamics in the setting of acute ischemic stroke and thrombolytic therapy. The stability of thrombolytics has not been investigated in TEG. We conducted an experimental series to test the effects of recombinant tissue plasminogen activator (rtPA) on fibrinolysis in normal blood samples using TEG. Methods: Freeze dried rtPA powder was reconstituted in normal saline containing 0.2% bovine serum albumin (100 mg/24 mL), divided into 1 mL aliquots, and diluted to enable using a relatively large volume for complete mixing with blood samples. Aliquots and dilutions were frozen at -20°C. The same rtPA dilution was thawed to ambient temperature before each use and refrozen until the next use over 4 testing days. Blood was drawn into 3.2% sodium citrated collection tubes. rtPA (100 μL) was added to 1 mL whole blood to achieve a 636 ng/mL rtPA TEG sample concentration. Control-whole blood and rtPA-whole blood TEG was performed for 3 h on 4 healthy human blood samples. Maximum clot amplitude (mm) and absolute clot strength (dynes/cm 2 ) were measured. Data (mean±SD) were analyzed by t-tests and significance inferred at p <0.05. Results: Clot amplitude increased with thawing and refreezing (28±3, p=0.004; 35±2 p=0.01; 50±3, p=0.02; and 55±3, p=0.30; for testing cycles 1, 2, 3 and 4, respectively) compared to untreated samples (63±4). Clot strength also increased over the 4 cycles (2±0.3, p=0.007; 3±0.2, p=0.02; 5±05, p=0.01 and 6±0.7, p=0.30) compared to untreated (9±1.4). Lysis initiation time was gradually longer over the 4 tests (red arrows, Figure 1) suggesting delayed fibrinolysis. Conclusions: One repeatedly thawed and refrozen rtPA stock showed a delay in fibrinolysis in healthy human blood, suggesting a loss of potency. Thus, rtPA should be aliquoted for 1-time use for experiments using TEG. Further investigation into rtPA potency deterioration with storage after reconstitution is warranted.

A plasmin generation method for the determination of tissue plasminogen activator (t-PA) activity in blood

1989 ◽  
Vol 67 (3) ◽  
pp. 197-203 ◽  
Author(s):  
SCL Koh ◽  
R Yuen ◽  
OAC Viegas ◽  
SE Chua ◽  
BL Ng ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Tissue Plasminogen
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