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Abstract
Current studies show a more than 50-fold variation in the estimated level of tissue-type
plasminogen activator (t-PA) activity in normal resting blood samples that result
from major differences in the methods used to sample, preserve, and assay t-PA activity
in blood. In this study we developed optimized methods for stabilizing and measuring
t-PA activity in plasma by using a coupled plasminogen-chromogenic substrate (amidolytic)
assay. To maximize the recovery of t-PA activity, blood should be acidified within
60 seconds after being drawn by adding 2 parts whole blood to 1 part 0.5 mol/L sodium
acetate, pH 4.2. This method prevents hemolysis and eliminates 70% of the α2-plasmin inhibitor. Optimum conditions for measuring t-PA activity are pH 8.0 to 8.3
(37°C), ionic strength 0.02 to 0.04, 0.5 μmol/L plasminogen, 80 μg/ml CNBr-cleaved
fibrinogen, and a chromogenic substrate concentration of 0.65 mmol/L d-valyl-leucyl-lysyl-p-nitroanilide, 0.25 mmol/L d-valyl-phenylalanyl-lysyl-p-nitroanalide, or 0.2 mmol/L d-norleucyl-hexahydrotyrosyl-lysyl-p-nitroanilide. The final assay is linear with respect to added one-chain t-PA, two-chain
t-PA, and acidified plasma. There was no difference in t-PA activity measured with
ethylenediaminetetraacetic acid anticoagulant versus that measured with citrate anticoagulant
after correction for dilution effects (average resting t-PA activity in plasma from
20 healthy individuals = 1.59 IU/ml). We conclude that assay conditions can have major
effects on the measurement of t-PA activity in plasma and that suboptimal conditions
may result in a significant underestimation of t-PA activity.
Abbreviations:
Abs405 (absorbance at 405 nm), CNBr (cyanogen bromide), EDTA (ethylenediaminetetraacetic acid), HOAc (acetic acid), M (relative molecular mass), SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), S-2251 (D-valyl-leucyl-lysyl-p- nitroanilide), S-2390 (d-valyl-phenylalanyl-lysyl-p-nitroanilide), t-PA (tissue-type plasminogen activator)To read this article in full you will need to make a payment
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References
- Plasminpgen activator release during venous stasis and exercise as determined by a new specific assay.Clin Chem Acta. 1983; 127: 279-288
- A sensitive assay for tissue plasminogen activator activity in plasma, using adsorption on lysine-sepharose.Thromb Res. 1984; 35: 547-558
- Sensitive assays for tissue plasminogen activator (t-PA) and its specific inhibitor (t-PA/INH).in: Davidson JF Donati MB Coccheri S Progress in fibrinolysis VII. Churchhill Livingstone, London1985: 174-178
- Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction.N Engl J Med. 1985; 313: 1557-1563
- Two different mechanisms in patients with venous thrombosis and defective fibrinolysis: low concentrations of plasminogen activator or increased concentration of plasminogen activator inhibitor.Br Med J. 1985; 290: 1453-1456
- The role of the fibrinolytic system in deep vein thrombosis.J Lab Clin Med. 1985; 105: 265-270
- Release of tissue plasminogen activator and its fast-acting inhibitor in defective fibrinolysis.Arch Intern Med. 1986; 146: 188-191
- Measurement of tissue plasminogen activator in plasma. A comparison of 3 methods and description of a new improved technique.Thromb Res. 1987; 46: 213-223
- Atrophie blanche, a disorder associated with defective release of tissue plasminogen activator.Arch Pathol Lab Med. 1986; 110: 517-519
- Plasminogen activator and plasminogen activator inhibitor activities in a reference population.Am J Clin Pathol. 1988; 89: 747-752
- Desmopressin stimulates parallel norepinephrine and tissue plasminogen activator release in normal subjects and patients with diabetes mellitus.Thromb Haemost. 1988; 59: 269-272
- Evidence for a rapid inhibitor to tissue plasminogen activator in plasma.Thromb Res. 1983; 31: 427-436
- Fibrin plate method with reagents purified by affinity chromatography and its use for determination of fibrinolytic and other proteolytic activity in saliva, bile and plasma.Haemostasis. 1975; 4: 110-124
- A sensitive assay for tissue plasminogen activator.Thromb Res. 1982; 27: 743-749
- The effect of citrate on euglobulin methods of estimating fibrinolytic activity.J Clin Pathol. 1958; 11: 403-405
- Chromogenic peptide substrates— their use for the assay of factors in the fibrinolytic and plasma kallikrein-kinin systems. Methods for the measurement of plasminogen in plasma.Scand J Clin Lab Invest. 1982; 42: 49-54
- Physiology of plasminogen: with special reference to activation and degradation.Haemostasis. 1988; 18: 25-35
- A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma.Thromb Haemost. 1982; 48: 266-269
- A collaborative study of a proposed international standard for tissue plasminogen activator (t-PA).Thromb Haemost. 1985; 53: 134-136
- Chromogenic peptide substrates—their use for the assay of factors in the fibrinolytic and plasma kallikrein-kinin systems. Methods for the measurement of antiplasmin in plasma.Scand J Clin Lab Invest. 1982; 42: 41-48
- Control of human plasminogen activation.Haemostasis. 1988; 18: 15-23
- Probleme bei der messung von parametern des fibrinolytischen systems: circadianne rhythmik von gewebe-plasminogen-aktivator und plasminogen-aktivatorinhibitor.Klin Wochenschr. 1988; 66: 62-67
- A solid-phase radioassay for the quantitative determination of antiplasmin activity in human plasma.Br J Haematol. 1975; 30: 117-122
- Hydrogen ion buffers for biological research.Biochem. 1966; 5: 467-477
- Studies on the kinetics of plasminogen activation by tissue plasminogen activator.Biochim Biophys Acta. 1982; 704: 461-469
- Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator.J Biol Chem. 1982; 257: 2920-2925
- Daytime fluctuations in blood of tissue-type plasminogen activator (t-PA) and its fast-acting inhibitor (PAI-1).Thromb Haemost. 1988; 59: 329-332
- Desmopressin stimulates parallel norepinephrine and tissue plasminogen activator release in normal subjects and patients with diabetes mellitus.Thromb Haemostas. 1988; 59: 269-272
- Determination of tissue plasminogen activator and its “fast” inhibitor in plasma.Clin Chem. 1986; 32: 482-485
Article info
Publication history
Accepted:
November 18,
1988
Received in revised form:
October 20,
1988
Received:
May 3,
1988
Footnotes
☆Supported in part by the American Heart Association, Washington Affiliate, and the Medical Research Service of the Veterans Administration.
Identification
Copyright
© 1989 Published by Elsevier Inc.
