Plasminogen Activator and Plasminogen Activator ...

Plasminogen Activator and Plasminogen Activator Inhibitor Activities in a Reference Population CHITRA KRISHNAMURTI, PH.D., DOUGLAS B. TANG, PH.D., CHARLES F. BARR, B.S., AND BARBARA M. ALVING, M.D.

ASSESSMENT of the fibrinolytic activity of human plasma includes measurement of tissue plasminogen activator (t-PA) as well as quantitation of the specific t-PA inhibitor-plasminogen activator inhibitor-1 (PAI-1).9-19-23 Both t-PA and PAI are synthesized and released by endothelial cells18; levels of t-PA are influenced by the quantity of PAI, which, in some clinical situations, can be increased by as much as 40-fold.7 Although multiple reports have described decreased levels of t-PA and elevated levels of PAI in patients with thrombosis,19,23 infection,7 lupus erythematosus,8 and history of myocardial infarction," there has been no study that clearly identifies those factors that influence levels of t-PA and PAI in a reference population, a group essential for the establishment of guidelines for normal ranges. In this report we have used sensitive and specific assays to determine the effect of age and sex, as well as aspirin ingestion, on levels of t-PA and PAI in a reference population of 35 men and 35 women between the ages of 20 and 65 years. The study indicates that factors such as age and sex may influence the values of t-PA and PAI in such a population.

Departments of Hematology and Biometrics, Walter Reed Army Institute of Research, Washington, D.C.

Methods Reagents Purified two-chain human t-PA with a specific activity of 500,000 IU/mg protein was obtained from American Diagnostica, Inc., Greenwich, Connecticut. The plasmin substrate S-2251 was purchased from Kabi Diagnostica, Stockholm, Sweden. Human fibrinogen and Cohn fraction III paste were obtained from Cutter Laboratories, Berkeley, California. Plasminogen was purified from the paste by affinity chromatography5 on lysine-Sepharose® (Pharmacia Fine Chemicals, Uppsala, Sweden); the specific activity was 20 CTA U/mg. Healthy Volunteers

Received August 24, 1987; received revised manuscript and accepted for publication November 10, 1987. Address reprint requests to Dr. Krishnamurti: Department of Hematology, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100.

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The protocol for measurement of PAI and t-PA in normal volunteers was approved by the Human Use Committee at the Walter Reed Army Medical Center. Thirty-five male and 35 female volunteers (20-65 years) who had no prior history of venous thrombosis, angina, myocardial infarction, diabetes, or hyperlipidemia were chosen for these studies. Women were excluded if they were pregnant or taking birth control pills. All blood samples were obtained from fasting persons between 8 and 10 A.M. The preocclusion samples were obtained from an antecubital vein that had been occluded by a tourniquet for less than 1 minute. The postocclusion samples were drawn from the opposite arm after 5 minutes of venous occlusion by a blood pressure cuff inflated to a pressure midway between systolic and diastolic pressure. Preliminary study in our laboratory as well as work by others20 indicated that this duration of venous occlusion was well tolerated and would cause a reproducible increase in t-PA. In 40 of these volunteers (20 men and 20 women), a double-blind randomized study was conducted to determine the effect of a single dose of aspirin on PAI and t-PA values. After undergoing blood drawing for measurement of t-PA and PAI on the morning of day 1,

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The influence of age, gender, and aspirin ingestion on plasma levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activities was studied in a reference population of 35 men and 35 women between the ages of 20 and 65 years. The t-PA values (mean ± SD) in the women before and after 5 minutes of venous occlusion were 3.8 ± 1 . 4 and 7.8 ± 4.4 /ig/L, respectively; in men these values were 3.3 ± 1 . 2 and 8.8 ± 8.9 jxg/L, Men had higher mean PAI levels than did women (5.0 vs. 2.5 kU/L). T-PA showed an inverse relationship to PAI in both sexes. There was a negative correlation of t-PA levels with age, whereas PAI levels were positively correlated. The ingestion of a single dose of aspirin (650 mg) did not alter PAI or t-PA activities. This study indicates that factors such as age and sex may need to be considered when reference populations are developed for clinical studies of fibrinolysis. (Key words: Tissue plasminogen activator; Plasminogen activator inhibitor-1) Am J Clin Pathol 1988; 89:747-752

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KRISHNAMURTI ET AL.

these volunteers ingested a tablet containing either 650 mg aspirin or a placebo containing lactose powder between 3-4 P.M. on the same day. They returned 18 hours later on the morning of day 2 for repeat measurements oft-PAandPAI. Blood was mixed with 0.1 mol/L citrate anticoagulant in a ratio of 9:1 and immediately centrifuged at 22 °C in an Eppendorf® microfuge 3200 (Brinkman Instruments, Westbury, NY) for 1 minute at 12,500 X rpm. Plasma for PAI determination was aliquoted and frozen immediately at -70 °C. Plasma for t-PA determinations was acidified with an equal volume of 1 mol/L sodium acetate for 15 minutes at 22 °C, aliquoted, and frozen at -70 °C until analyzed. Measurement of t-PA Activity

acetate, pH 3.9, was added. Samples were diluted 1/25 in TRIS-Triton, pH 8.8, and residual t-PA activity was determined in triplicate as described above. One unit (U) of inhibitor is defined as the amount that inhibits 1 IU t-PA in 10 minutes. Assay Reproducibility To obtain estimates of intraassay (within-day) and interassay (between-day) variation of means based on triplicates, a blood sample was drawn from each of four subjects, aliquoted, processed, and stored. On each of five days, three aliquots from each subject were assayed. The intraassay and interassay coefficients of variation for PA activity measurements were 5.4% and 10.4%, respectively. PAI measurements showed intraassay and interassay variations of 17% and 21.4%, respectively. The degree of variation did not depend on the level of PA or PAI measured. Statistical Methods Distribution of t-PA (before and after venous occlusion) and PAI in men (n = 35) and women (n = 35) was summarized graphically with the use of boxplots.12 Mean levels were compared with the use of Student's unpaired Mest (after transformation to reduce skewness) and the Wilcoxon two-sample rank sum test.1 Differences in distribution were also examined by use of the Kolmogrov-Smirnov test. This procedure is aimed at detecting broader degrees of differences rather than only shifts in locations (e.g., means or medians).13 Because overall conclusions were similar, results from the more familiar Mest are reported. Changes in mean t-PA and PAI after subjects' ingestion of aspirin or placebo were examined by use of both the Wilcoxon signed rank test and Student's paired Mest. Relationships between variables (e.g., t-PA and PAI) were examined graphically for general structure and consistent patterns. Correlation coefficients (product moment unless otherwise stated) are provided for reference to direction. All reported P values are two-sided unless otherwise stated. Significance levels (P values) from formal analyses are provided as descriptive information. Overall conclusions are not (should not be) based solely on the statistical significance (or lack of it) in any particular case. Results

Measurement of PAI Activity Inhibitor activity was determined according to the method of Chmielewska and colleagues.6 t-PA (10 klU/L,final)was added to plasma or dilutions of plasma in 0.02 mol/L sodium phosphate, pH 7.3, containing 0.1 mol/L sodium chloride. After incubation for 10 minutes at 22 °C, an equal volume of 1 mol/L sodium

PA Activity Figure 1 summarizes the distribution of t-PA before and after venous occlusion in men (n = 35) and women (n = 35). Although the differences in distribution between men and women were not statistically significant,

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The assay was performed according to the method of Wiman and associates.22 The acidified plasma was diluted to 1% (v/v) with 0.05 mol/L TRIS-HC1, pH 8.8, containing 0.1 mol/L sodium chloride and 0.1% Triton X-100®. The diluted sample (100 fiL) was pipetted into a 96-well flat-bottom microtiter plate. An equal volume of substrate containing 0.3 g/L plasminogen and 1 mmol/ L S-2251 in the same TRIS-Triton buffer was added, followed by 5 nL soluble fibrin (final concentration 0.07 g/L). Plates were incubated at 37 °C in a humidified atmosphere, and the change in absorbance was recorded 3-8 hours later at 405 nm in a Titertek® spectrophotometer (Flow Laboratories Inc, CA). Standard curves were obtained by diluting the t-PA in acidified and diluted pooled plasma from male or female subjects. Each sample was tested in triplicate, and the mean value was compared with the activity of the t-PA standard. Wiman and associates22 and Levin and associates17 had found respective decreases of 50% and 30% in endogenous t-PA activity in normal resting plasma that had been stored frozen without prior acidification. We also measured a 21 % decrease in t-PA activity in samples that had been frozen without acidification. Further, plasma samples left in ice for 2 hours, then acidified and assayed, showed a 25% loss of activity. However, when compared with fresh samples, those that were frozen at - 7 0 °C after acidification for 15 minutes at 22 °C showed no decrease in t-PA activity. Therefore, all samples measured in this report were treated in this fashion.

A.J.C.P. • June 1988

vol.89.No.6

PLASMINOGEN ACTIVATOR AND INHIBITOR

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FIG. 2. Distribution of resting PAI activities in 35 men and 35 women. The dashed line indicates the median. The solid horizontal bars indicate the upper and lower quartiles. The tenth and ninetieth percentiles are indicated by the vertical lines.

women had values that were generally shifted higher than for men for both preocclusion and postocclusion levels. Resting levels in men ranged (10-90%) from 1.5 to 6.2 Mg/L (median = 2.9; mean = 3.3; SD = 1.2). In comparison, postocclusion levels varied from 1.8 to 41.0 ngJL (median = 6.2; mean = 8.8; SD = 8.9). Preocclusion levels in women ranged from 0.9 to 6.2 /*g/L (median = 3.5; mean = 3.8; SD = 1.4) with postocclusion levels from 0.8 to 18.5 ^g/L (median = 7.7; mean = 7.8; SD = 4.4).

14.9 kU/L (median = 4.2; mean = 5.0; SD = 3.4), whereas in women they ranged from undetectable to 8.5 kU/L (median = 1.8; mean = 2.5; SD = 2.0). The difference in mean levels as well as overall shift to lower values in women is statistically significant (t = 3.8; P = 0.0003; 95% confidence interval: 1.2-3.9 kU/L). There was no difference between preocclusion and postocclusion PAI levels when measured in ten men and ten women; therefore, all PAI values are reported on plasma obtained before venous occlusion.

PAI Activity

Relationship Between t-PA and PAI Activities

The distribution of PAI levels is summarized in Figure 2. For men the levels ranged from undetectable to

The relationship between t-PA and PAI levels is summarized in Figures 2>A (before occlusion) and B (after

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