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The determination of recombinant human tissue-type plasminogen activator activity by turbidimetry using a microcentrifugal analyzer
ANALYTICAL
BIOCHEMISTRY
168,428-435
(1988)
The Determination of Recombinant Human Tissue-Type Plasminogen Activator Activity by Turbidimetry Using a Microcentrifugal Analyzer RONALD H. CARLSON,
ROBERT
L. GARNICK,
ANDREW
J. S. JONES, AND ANN M. MEUNIER
Genentech, Inc.. 460 Point San Bruno Boulevard, South San Francisco, California 94080 Received July 2, 1987 A method is described for determining the activity of recombinant human tissue-type plasminogen activator @t-PA) by turbidimetry using a microcentrifugal analyzer (MCA). A mixture of thrombin and r&PA is centrifuged into a mixture of fibrinogen and plasminogen to initiate clot formation and subsequent clot dissolution. The resultant profile of absorbance versus time is analyzed to determine the assay endpoint. Different r&PA assay concentration ranges were studied in conjunction with profile endpoints for assay optimization. Spiked placebo recovery studies were used to evaluate assay accuracy and precision, which were determined to be 99.5 and 5% (relative standard deviation or RSD), respectively. Assay sensitivity was 0.5 @ml. Typical analysis time, including calculations, for a standard curve plus 14 samples was less than 30 min. The application of turbidimetry with the MCA for determining rt-PA activity provides rapid sample analysis and high throughput while maintaining accuracy and precision. 8 1988 Academic KEY
Press, Inc.
WORDS: clot lysis assay: t-PA activity assay; tissue plasminogen activator; clot lysis activity; clot lysis; turb&metric assay.
Coronary thrombosis, which is responsible for approximately 90% of myocardial infarctions (I), ranks as the leading cause of death and disability in the United States (2). Tissue plasminogen activator (t-PA)’ is an enzyme that converts plasminogen (a zymogen) to active plasmin that, in turn, degrades insoluble fibrin to soluble by-products (3-5). Recombinant DNA technology has made possible a method for the production of t-PA on a large scale (3), which has led to great interest in the use of the recombinant form of t-PA (r&PA) as a pharmaceutical for thrombolytic therapy in humans. t-PA has a high affinity for fibrin as does r&PA, which becomes most active when it forms a complex with fibrin (6-8). Plasminogen binds readily to the t-PA-fibrin complex, but not as strongly to ’ Abbreviations used: r&PA, recombinant human tissue-type plasminogen activator; L-TF’CK, tosyl+phenylalanine chloromethyl ketone; MCA, microcentrifugal analyzer; t-PA, tissue plasminogen activator; EP, endpoint. 0003-2697188 $3.00 Copyri&t 8 1988 by Academic Press, Inc. AU rights of reproduction in any fom reserved.
428
circulating t-PA (8). Thus, the generation of plasmin by t-PA is localized to the fibrin clot (8). A potency or activity assay for pharmaceutical drug use should meet the following criteria: (a) possess a degree of accuracy and precision to determine the activity within 90-l 10% of its actual value; (b) be stability indicating; (c) require minimal specialized technical skills; and (d) be highly reliable in day-to-day use. In addition, it is desirable that the method be automated to minimize technique-dependent assay performance and to maximize sample throughput. The assay described in this report was developed to meet the above criteria in determining the activity (potency) of rt-PA. The assay is based on a modified approach of the one used in a 1985 collaborative study to establish an international t-PA standard (9). The major modification in the present assay is the manner in which the lysis time (endpoint) of the synthetic fibrin clot is measured (A. M. Meunier et al., manuscript in prepa-
TURBIDIMETRIC
ASSAY
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ration). The collaborative study utilized the release of entrapped air bubbles (“bubble release” method) from the fibrin clot or the dropping of glass beads through the fibrin clot to measure lysis time (9-l 3). The current study bases the lysis time endpoint determination on a turbidimetric measurement using a microcentrifugal analyzer (MCA), a commercially available clinical instrument. The advantage of this approach in developing an rt-PA activity assay is that the MCA provides automation; it can pipet and mix samples and reagents, load cuvettes, record and transfer absorbance data, analyze data, and perform calculations. Both the bubble release and MCA methods are i-t-PA clot matrix methods; i.e., &PA is at the site of the clot initially. MATERIALS
AND
METHODS
Materials. All water used in this study met USP XXI specifications for purified water. The buffer used throughout was 0.06 M sodium phosphate (reagent grade), pH 7.4, containing 0.01% (v/v) Tween 80 (practical grade) and 0.01% (w/v) sodium azide. Human thrombin (Calbiochem, Catalog No. 605 195) was reconstituted to 1000 units/vial with water and then diluted to 33 units/ml with phosphate buffer. Fibrinogen (Calbiothem, Catalog No. 34 1576) was dissolved in phosphate buffer to a concentration of 2.0 mg/ml clottable protein, chilled on wet ice to precipitate fibronectin ( 14), and then gravity filtered (Whatman No. 1 filter paper). Plasminogen, prepared according to the method of Deutsch and Mertz (15) or purchased from Kabi Vitrum (Catalog No. 5304), was diluted to 1.0 mg/ml with water. Trypsin degradation samples were prepared by the addition of 1 mg L-TPCK (tosyl-L-phenylalanine chloromethyl ketone) trypsin (Cooper) to a 1 mg/ml r&PA solution. Oxidative degradation was performed by bubbling water-saturated air through reconstituted t-t-PA solutions at ambient temperature. The plasmin used (Sigma, 10 units/vial) was reconstituted with 0.01% sodium azide and
PLASMINOGEN
ACTIVATOR
429
added to a 1 mg/ml r&PA solution. One vial of plasmin was used per 50 mg &PA. The carboxypeptidase B used (Worthington, in suspension) was added to a reconstituted rt-PA solution at 45 units per 50 mg r&PA. The pronase used (Calbiochem) was prepared as a 1 mg/ml solution in 0.0 1% sodium azide and added to a reconstituted rt-PA solution at 1 mg pronase per 50 mg t-t-PA. The rt-PA was reconstituted to 1 mg/ml with water and then diluted to assay levels in phosphate buffer. All reagents were stored on wet ice until used. The activity of rt-PA throughout this report is expressed as nanograms per milliliter of the internal reference material and not as international units (IU). The activity of the internal reference material in IU is still under investigation; its approximate activity is 500,000 IU/mg. Equipment. The MCA was an Instrumentation Laboratory (IL) Model 0773 Multistat III Plus Microcentrifugal Analyzer system. Rotors, which contain the cuvettes for mounting in the MCA, were purchased from IL. The loader was set to dispense 20 ~1 of i-t-PA as the sample, 20 ~1 of thrombin as the secondary reagent, and 200 ~1 of 50: 1 (v/v) fibrinogen:plasminogen mixture as the primary reagent. The analyzer was blanked against water and the analyzer chamber temperature was set at 37°C. The absorbance/ time program was used with a 5-min incubation time, 340-nm filter, and 90 interval readings. The analyzer delay and interval times were set according to the assay range as specified under Results and Discussion. An IBM-PC/XT, via the RS-232C ports, was interfaced to the analyzer to allow for automation of the analysis of the analytical data generated by the MCA as well as for archival purposes. Data analysis. Absorbance/time data were transferred from the analyzer to the IBMPC/XT using a commercially available program named “Perfect Link” (manufactured by Perfect Software). A program was written at Genentech in the language “C” for data analysis and calculations. This program analyzed the data according to the endpoint cri-
430
CARLSON
teria (defined under Results and Discussion) for the endpoint regression of the standard of log time versus log concentration. RESULTS AND DISCUSSION
A profile of clot absorbance (A) versus time is shown in Fig. 1. The formation of the clot is due to the formation of insoluble fibrin, which causes light to be scattered (turbidity) and is measured as an absorbance at 340 nm. The kinetics of clot formation are very rapid, resulting in a firm clot within 40 s. The absorbance plateau (between 0.28 and 0.30 A) indicates formation of a stable clot. Lysis becomes evident at about 140 s, with complete dissolution of the fibrin clot shortly after 300 s. It is interesting to note the shoulder that occurs between 260 and 280 s. The significance of this phenomenon has not been thoroughly established, although it may be related to the final collapse of the clot from the centrifugal force generated by the MCA. This would be consistent with the observation, made during bubble release experiments performed in test tubes, that the bubbles rise just prior to total transparency. The time-absorbance profile obtained with the MCA turbidimetry method provides a means of quantitating r&PA activity and is a source of diagnostic information about the assay. The profile provides a rapid
B
*-=EP4 ..*
-=EP2
AND 3
-*... *. *.
00
visual check of the raw data and verification that the endpoint has been correctly determined by the computer.
Choice of Endpoint
Absorbance ProJile
.8
ET AL.
1. ‘.FSEP, “....a .a.,.. 380
SXONDS FIG. 1. Clot lysis absorbance versus time profile (950 @ml rt-PA).
The time-absorbance profile was analyzed to determine which endpoint (EP) had the best correlation to &PA activity. The four endpoints (Fig. 1) investigated were EPl, the time at which the absorbance approached the baseline value; EP2, the time of the maximum decrease in absorbance (time of first derivative maximum); EP3, the slope of the maximum decrease in absorbance (slope of first derivative maximum); and EP4, the time of the one-half maximum absorbance (the average between the highest and lowest absorbance values). A slope threshold criterion (AA) was established for EPl to determine when the absorbance had reached a baseline value. To distinguish between the plateau at the beginning of clot formation from the plateau after lysis, only absorbances co.03 (the absorbance cutoff) were evaluated for their slope. The baseline was considered to be reached when AA < 0.003. Other values for the absorbance cutoff (0.02-0.04 A) and slope thresholds (AA of 0.002-0.004) were investigated. The combination that yielded the best linearity (correlation coefficient) for five standard concentrations over the ranges studied was considered optimum. The criteria for the choice of endpoint were based on linearity of the standard curve (five points), accuracy of recovery (a known concentration equivalent to the middle standard of the five-point curve), and precision of recovery (RSD). Results of the endpoint study are shown in Table 1. Evaluation of endpoints based on a first derivative approach requires a distinct inflection point. As the r-t-PA concentration decreases, a shallower slope is observed in the time-absorbance profile. This shallower slope complicated the reliable identification of the inflection point that correlated with r-t-PA activity. Thus, the usefulness of EP2 and EP3 was
TURBIDIMETRIC
ASSAY FOR TISSUE PLASMINOGEN
ACTIVATOR
431
TABLE 1 ENDPOINT COMPARISON No. of curves
Percentage recovery
Percentage RSD
No. of replicates
-0.9986 -0.9989 -0.9993
4 4 4
102.4 99.8 97.6
3.6 2.9 3.7
56 56 55
950-3000 127-400 32-100
-0.9954
4
101.7 Inflection point problem Inflection point problem
2.8
56
3 3 3
950-3000 127-400 32-100
-0.9972
4
97.2 Inflection point problem Inflection point problem
5.0
56
4 4 4
950-3000 127-400 32-100
-0.9995 -0.9982 -0.999 1
2 2 2
4.3 9.3 12.8
28 28 28
Cone range”
Average
EP 1 1 1
950-3000 127-400 32-100
2 2 2
r
103.3 104.8 99.6
LIConcentration range of rt-PA in the assay in nanograms per milliliter.
restricted to high r-t-PA concentration ranges. Less precise recoveries were obtained for EP4. EP 1 demonstrated utility in all concentration ranges; its linearity, accuracy, and precision were better than or equal to those of the other endpoints. Therefore, EPl was selected for further studies. The absorbance readings are collected only at discrete time intervals, but the endpoint can occur at any time. The endpoint is therefore measured with some finite resolution, defined by the time between adjacent data points. This introduces an uncertainty in the measured concentration that is related by the standard curve to the uncertainty in the endpoint, with the error decreasing as the r&PA concentration decreases for a given interval time. The analyzer collects a fixed number of data points per analysis (in the case of the Multistat, this number is 90). There is thus a compromise between the desire to use the highest resolution possible and the convenience of a wide standard curve range. Preliminary experiments indicated that an RSD of approximately 5% was obtained in the assay. The standard curve ranges and interval times (described below) were selected to
keep the uncertainty in concentration, introduced by the resolution error, to less than 3%, while yielding a useful working range of fivefold in the standard curve.
Spiked Placebo Recovery Studies Most of the work described in this paper was completed before the activity of an internal t-t-PA reference was correlated with the WHO t-PA standard. A batch of r-t-PA was specially filled and lyophilized at 1 mg/ vial and a fresh vial was used each day to prepare the standard curves. For this internal standard, an internal unitage was arbitrarily assigned where 1 unit = 1 mg. Spiked placebo samples were prepared at 1.5-0.5 mg/ml t-t-PA to evaluate the accuracy and precision of a +50% working assay range. Two assay ranges were investigated. Five standard concentrations were prepared for each range: 200- 1000 (advised delay time = 280 s, interval time = 4 s, analysis time = 28 min) and 40-200 rig/ml (advised delay time = 500 s, interval time = 8 s, analysis time = 39 min). The spiked placebos were diluted 1667:l (300-900 rig/ml) and 8333: 1 (60-180 rig/ml) for the appropriate ranges.
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ET AL.
TABLE 2 SPIKED PLACEBO RECOVERY DATA Standard curve range h/ml) 200-loo0 200-1000 200-loo0 200-loo0 200-loo0
Spike W/ml) 0.5 0.8 1.0 1.2 1.5
Percentage recovery
Perceutage RSD
No. of replicates
91.7 98.8 100.4 100.4 100.2
4.4 5.1 4.4 4.2 7.8
100 60 157 60 loo
Mean recovery = 99.5 40-200 40-200 40-200
0.5 1.0 1.5
Mean RSD = 5.2
95.4 99.1 100.3
5.4 3.4 7.0
Mean recovery = 98.3
The results are shown in Table 2. Average recoveries of 99.5 (RSD = 5.2%) and 98.3% (RSD = 5.3%) were obtained for the high and low assay ranges, respectively. The average correlation coefficients were -0.999 1 (40 standard curves) and -0.9988 (24 standard curves) for the high and low concentration ranges, respectively. The rapid analysis times and high accuracy of this method made the 200-1000 rig/ml range the preferred choice. This is the range used throughout the report unless another range is specified. The spiked placebo recovery data were collected by three analysts over the course of many rotors and days. Small variances were observed between days, analysts, and rotors. A one-way analysis of variance was performed to determine if these variances were statistically significantly different. The P values, listed in Table 3, indicate that in some cases the variance could be linked to the day, the rotor, or the analyst. Optimal precision was obtained for replicates run on the same rotor (and thus also run on the same day and by the same analyst); the average RSD was 3.4 instead of 5.2% ‘(the unweighted mean for all samples of all three analysts). Conversely, optimal accuracy for a limited number of replicates can be obtained by spreading the replicates out over different
93 92 93 Mean RSD = 5.3
rotors, days, and analysts, where possible. It was desired that the MCA method not only minimize, but eliminate, technique dependence. Although a total elimination of technique dependence was not achieved, it was found that the RSD of 5.2% compared favorably to an RSD of 9% obtained for a bubble release study performed by three analysts over several days. A precision of 6% can be obtained for bubble release if a single analyst performs the experiments (A. M. Meunier et al., manuscript in preparation).
TABLE 3 P VALUESFROMANALYSISOFVARIANCE Range/spike”
Rotor
Analyst
Day
High/O.5 High/O.8 High/ 1.O High/l.2 High/l .5
0.02 NS 0.02 NS 0.01
Low/o.5 Low/l.0 Low/l.5
NS NS
a Spiked placebo concentrations are nanograms per milliliter r&PA. * NS, Not significant (P > 0.05).
TURBKXMETRIC
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In addition to the bubble release or dropping glass bead techniques (9- 13 and A. M. Meunier et al., manuscript in preparation) to determine t-PA activity, other common approaches include the fibrin plate technique (16-20), the use of chromogenic (or fluorogenie) substrates ( 16,2 l-24), and isotopic labeling (16). The method adapted for the MCA offers advantages over all of these established methods. The control over mixing times, monitoring times, and temperature afforded by the MCA is superior to what can be obtained by bubble release, fibrin plates, or other kinetic-based assays. The MCA method does not require continuous visual monitoring as does the bubble release method nor is it as susceptible to subjective error (11,12). The MCA is free from major fibrin-plate drawbacks such as technical problems in preparation and use of the plates, quantitation of the method, and length of time in setup and incubation (16-19). The MCA method offers a better stability-indicating model than chromogenic or immunologic-based assays because chromogenic assays are less specific in their proteolytic activity and immunologic-based assays may not be stability indicating. Isotopic labeling possesses the fundamental problem of radioactive handling and disposal. Methods involving solid phase surfaces have the same drawback of plate preparation as does the fibrin plate technique (7). Reports of semiautomated clot lysis apparatus have appeared in the literature ( 11,12). These devices were not commercially available as was the Multistat III Plus MCA. The Multistat III Plus MCA has the capability of analyzing 19 samples at a time (or a fivepoint standard curve and 14 samples). An automated fibrinolytic assay performed in microtitration plates has been reported recently (25). However, the plate reader used was limited to readings at I-min intervals, whereas the MCA is capable of readings every 3 s. This resolution factor as well as the control over the addition of reagents inherent in the MCA design could explain why
PLASMINOGEN
ACTIVATOR
433
better precision was observed in testing r&PA with the MCA method (5.2%) than with the automated plate method (13.8%). It is also worth noting the similarity between the endpoint described (time at 50% lysis) and EP4. Thus, development of the rt-PA activity assay using an MCA apparatus to measure turbidimetry allows for automation while maintaining the required high degree of precision and accuracy.
Correlation between Clot Lysis Activity and Protein Concentration as Determined by Spectrophotometric Scan The correlation between the activity determined by the clot lysis assay and the protein concentration (mg/ml) determined by the spectrophotometric scan assay was studied. Assuming the r&PA samples tested have the same activity as the reference material, there should be a correlation between the activity and the protein concentration. The r&PA activity determined by clot lysis was compared to the protein concentration determined by spectrophotometric scan (performed at approximately 0.5 mgfml protein, 240-500 nm). The six samples studied (Table 4) had an average activity of 99% (2% RSD) relative to the concentration determined by spectrophotometric scan.
Stability Indication The clot lysis activity assay was evaluated for its stability-indicating properties by measuring r&PA degraded by several modes of physical, chemical, and enzymatic degradation (Table 5). These conditions were chosen to achieve significant amounts of degradation during the study. The results of these experiments demonstrate that the assay is stability indicating (Table 5). The least severe condition studied (pH = 5.0 at 30°C) caused only a 20% loss of r&PA clot lysis activity after 4 weeks; a pH of 7.5 at the same temperature caused a 50% loss of activity over the same time period. The thermal and carboxypeptidase B studies showed a 50% loss of activity in 7 to 12 days. The
434
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ET AL.
TABLE 4 COMPARISONOFCLOTLYSISTOUVSPECTROPHOTOMETRY Clot lysis (U/ml)n
uv spectrophotometry (mg/ml)
Percentage specific activity
1.035 0.999 0.929 0.921 1.009 0.927
1.048 0.999 0.937 0.968 1.008 0.92 1
98.8 100.0 99.2 95.1 100.1 100.7 X = 99.0 (RSD = 2.0%)
’ Relative activity of an internal Genentech standard where 1 U = 1 mg of reference material.
other proteases, as well as the oxidative mode of degradation, caused a more rapid loss of activity. Sensitivity To work at sample levels of approximately 1 rig/ml, the reagent volumes and concentrations needed to be adjusted so that the sample would lyse in a reasonable time frame and the standard curve would remain linear. The adjustments were sample volume = 90 ~1, secondary reagent = 10 ~1 (66 units/ml thrombin), and primary reagent = 140 ~1 (2.86 mg/ml clottable protein of fibrinogen
and 1.4 mg/ml plasminogen). Advised analyzer delay and interval times were 900 and 55 s, respectively. Flushing the loader lines with assay buffer to remove any adsorbed r-t-PA incurred during testing with higher r-t-PA concentrations (e.g., 1000 rig/ml) was the only special precautionary measure. Standard curves in the range of 0.5-2.5 rig/ml had an average linearity of 0.9987 (n = 10). Recoveries for 0.5 and 1.O rig/ml were 103.9 (RSD = 3.9%, 12 = 30) and 97.9% (RSD = 2.4%, n = 40), respectively. Under the given conditions, the factor that limits sensitivity is the lysis time. Greater sensitivity is possible if longer lysis times are tolera-
TABLE 5 PERCENTAGEACTIVI~REMAININGAFTERACCELERATEDDEGRADATIONOF~~-PA Time Sample”
Temperature
1 &Y
1 week
2 weeks
3 weeks
4 weeks
Thermal Acidic Slightly basic Oxidative Carboxypeptidase B Plasmin Trypsin Pronase
37°C 3o”c 30°C Ambient 30°C 30°C 30°C 30°C
92.7 97.6 93.9 89.6 94.0 82.6 27.5 <306
59.0 94.0 89.3 <306 56.0 39.5 <306 <3d
44.3 92.0 75.5 <3Ob 36.7 <3Ob <30b <30b
36.6 88.8 69.8 <3d <3Ob <30b <30b <30b
29.6 78.5 54.4 <30b <3Ob 1306 <3Ob 130”
Note. Control activity was 1.03 U/ml. LIr&PA degraded by various chemical, physical, and enzymatic modes. ’ Sample had less than 30% activity remaining.
TIJRBIDIMETRIC
ASSAY FOR TISSUE PLASMINGGEN
ble and if care is taken to choose interval times yielding the best resolution. In conclusion, modification of the bubble release clot lysis assay to a turbidimetric method enabled the assay to be performed using an MCA. The time-absorbance profile generated in the turbidimetric assay provided a means of quantitation of clot lysis and a source of diagnostic information (i.e., being able to relate the fibrinolytic phenomena to the profile shape). The automation with the MCA simplified, accelerated, and better controlled the manipulations (e.g., pipetting and mixing) normally performed manually, thus minimizing dependence on technician technique. Computer control of the MCA made possible automatic data acquisition, data analysis, and calculations of results. Computer control thus eliminated the need for constant visual monitoring and subjectivity of the endpoint occurrence (lysis time) associated with the bubble release method ( 11,12). In addition, the turbidimetric MCA method was observed to have an accuracy of 99.5% with a precision of 5% (RSD). Although the focus of this paper was development of an automated assay to measure rt-PA activity, it should be noted that, like the bubble release assay, this turbidimetric methodology is applicable to the study of other fibrinolytic components. ACKNOWLEDGMENTS The authors thank Allen Lam for performing many of the experiments, Fred Miller for writing the data analysis computer program, and Mary Jean Pramik and Patricia A. Papa for their assistance in preparing and editing the manuscript.
REFERENCES 1. DeWood, M. A., Spores, J., Notske, R., Mouser, L. T., Burroughs, R., Golden, M. S., and Lang, H. T. (1980) N. En@. J. Med. 303,897-902. 2. Solomon, E. P., and Davis, P. W. (1983) Human Anatomy and Physiology, p. 497, CBS College Publishing, New York. 3. Pennica, D., Holmes, W. E., Kohr, W. J., Harkins, R. N.. Vehar, G. A., Ward, C. A., Bennett, W. F., Yelverton, E., Seeburg, P. H., Heyneker, H. L.,
4. 5.
6. 7. 8.
9. 10. 11. 12. 13.
14.
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Goeddel, D. V., and Collen, D. (1983) Nature (London) 301,2 14-22 1. Collen, D. (1980) Thromb. Haemostasis 43,77-89. Wiman, B., and Collen, D. (1978) Nature (London) 272,549-550. Thorsen, S., Glas-Greenwalt, P., and Astrup, T. (1972) Thromb. Diath. Haemorrh. 28, 65-74. Angles-Cane, E. ( 1986) Anal. B&hem. 153.201-2 10. Van De Werf, F., Ludbrook, P. A., Bergmann, S. R., Tiefenbrunn, A. J., Fox, K. A. A., Hilaire de Geest, H., Verstraete, M., Collen, D., and Sobel, B. E. (1984) N. Engl. J. Med. 310, 609-613. Gaffney, P. J., and Curtis, A. D. (1985) Thromb. Haemostasis 53, 134- 136. Philo, R. D., and Gaffney, P. J. (1981) Thromb. Haemostasis 45, 107- 109. Wilkens, H. J., and Back, N. (1976) Amer. J. Clin. Pathol. 66, 124- 13 1. Collen, D., Tytgat, G., and Verstraete, M. (1968) J. Clin. Pathol. 21, 705-107. Johnson, A. J. (1964) in Blood Coagulation, Hemorrhage and Thrombosis (Tocantins, L. M., and Kazal, L. A., Eds.), pp. 458-459, Grune & Stratton, New York. Mosher, D. F., and Johnson, R. B. (1983) Ann. N. Y. Acad.
Sci. 408,583-594.
15. Deutsch, D. G., and Mertz, E. T. (1970) Science 170, 1095-1096. 16. Marsh, N. ( 198 1) Fibrinolysis, pp. 206-24 1, Wiley, New York. 17. Marsh, N. A., and GaIfney, P. J. (1977) Thromb. Haemostasis 38, 545-55 1. 18. Astrup, T., and Jespersen, J. (1983) in Progress in Fibrinolysis (Davidson, J. F., Bachmann, F., Bouvier, C. A., and Kruithof, E. K. O., Eds.), Vol. 6, pp. 197-201, Churchill Livingstone, New York. 19. Jespersen, J., and Astrup, T. (1983) Haemostasis 13,301-315. 20. Ploug, J., and Kjeldgaard, N. 0. (1957) Biochim. Biophys.
Acta 24,278-282.
21. Egberg, N. (1984) Clin. Chem. Newsl. 4, 88-90. 22. Lijnen, H. R., and Cohen, D. (1982) Acta Chir. Stand. Suppl. 509, 129-132. 23. Wijngaards, G., and Nieuwenhuizen, W. (1980) in Synthetic Substrates in Clinical Blood Coagulation Assays (Lijnen, H. R., Collen, D., and Verstraete, M., Eds.), pp. 123- 13 1, Martinus Nijhoff Publishers, Boston. 24. Smith, D. S., Harmon, J., and Owen, W. G. (1985) Thromb. Rex 37, 533-541. 25. Beebe, D. P., and Aronson, D. L. (1987) Thromb. Rex 47, 123-128.
BIOCHEMISTRY
168,428-435
(1988)
The Determination of Recombinant Human Tissue-Type Plasminogen Activator Activity by Turbidimetry Using a Microcentrifugal Analyzer RONALD H. CARLSON,
ROBERT
L. GARNICK,
ANDREW
J. S. JONES, AND ANN M. MEUNIER
Genentech, Inc.. 460 Point San Bruno Boulevard, South San Francisco, California 94080 Received July 2, 1987 A method is described for determining the activity of recombinant human tissue-type plasminogen activator @t-PA) by turbidimetry using a microcentrifugal analyzer (MCA). A mixture of thrombin and r&PA is centrifuged into a mixture of fibrinogen and plasminogen to initiate clot formation and subsequent clot dissolution. The resultant profile of absorbance versus time is analyzed to determine the assay endpoint. Different r&PA assay concentration ranges were studied in conjunction with profile endpoints for assay optimization. Spiked placebo recovery studies were used to evaluate assay accuracy and precision, which were determined to be 99.5 and 5% (relative standard deviation or RSD), respectively. Assay sensitivity was 0.5 @ml. Typical analysis time, including calculations, for a standard curve plus 14 samples was less than 30 min. The application of turbidimetry with the MCA for determining rt-PA activity provides rapid sample analysis and high throughput while maintaining accuracy and precision. 8 1988 Academic KEY
Press, Inc.
WORDS: clot lysis assay: t-PA activity assay; tissue plasminogen activator; clot lysis activity; clot lysis; turb&metric assay.
Coronary thrombosis, which is responsible for approximately 90% of myocardial infarctions (I), ranks as the leading cause of death and disability in the United States (2). Tissue plasminogen activator (t-PA)’ is an enzyme that converts plasminogen (a zymogen) to active plasmin that, in turn, degrades insoluble fibrin to soluble by-products (3-5). Recombinant DNA technology has made possible a method for the production of t-PA on a large scale (3), which has led to great interest in the use of the recombinant form of t-PA (r&PA) as a pharmaceutical for thrombolytic therapy in humans. t-PA has a high affinity for fibrin as does r&PA, which becomes most active when it forms a complex with fibrin (6-8). Plasminogen binds readily to the t-PA-fibrin complex, but not as strongly to ’ Abbreviations used: r&PA, recombinant human tissue-type plasminogen activator; L-TF’CK, tosyl+phenylalanine chloromethyl ketone; MCA, microcentrifugal analyzer; t-PA, tissue plasminogen activator; EP, endpoint. 0003-2697188 $3.00 Copyri&t 8 1988 by Academic Press, Inc. AU rights of reproduction in any fom reserved.
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circulating t-PA (8). Thus, the generation of plasmin by t-PA is localized to the fibrin clot (8). A potency or activity assay for pharmaceutical drug use should meet the following criteria: (a) possess a degree of accuracy and precision to determine the activity within 90-l 10% of its actual value; (b) be stability indicating; (c) require minimal specialized technical skills; and (d) be highly reliable in day-to-day use. In addition, it is desirable that the method be automated to minimize technique-dependent assay performance and to maximize sample throughput. The assay described in this report was developed to meet the above criteria in determining the activity (potency) of rt-PA. The assay is based on a modified approach of the one used in a 1985 collaborative study to establish an international t-PA standard (9). The major modification in the present assay is the manner in which the lysis time (endpoint) of the synthetic fibrin clot is measured (A. M. Meunier et al., manuscript in prepa-
TURBIDIMETRIC
ASSAY
FOR
TISSUE
ration). The collaborative study utilized the release of entrapped air bubbles (“bubble release” method) from the fibrin clot or the dropping of glass beads through the fibrin clot to measure lysis time (9-l 3). The current study bases the lysis time endpoint determination on a turbidimetric measurement using a microcentrifugal analyzer (MCA), a commercially available clinical instrument. The advantage of this approach in developing an rt-PA activity assay is that the MCA provides automation; it can pipet and mix samples and reagents, load cuvettes, record and transfer absorbance data, analyze data, and perform calculations. Both the bubble release and MCA methods are i-t-PA clot matrix methods; i.e., &PA is at the site of the clot initially. MATERIALS
AND
METHODS
Materials. All water used in this study met USP XXI specifications for purified water. The buffer used throughout was 0.06 M sodium phosphate (reagent grade), pH 7.4, containing 0.01% (v/v) Tween 80 (practical grade) and 0.01% (w/v) sodium azide. Human thrombin (Calbiochem, Catalog No. 605 195) was reconstituted to 1000 units/vial with water and then diluted to 33 units/ml with phosphate buffer. Fibrinogen (Calbiothem, Catalog No. 34 1576) was dissolved in phosphate buffer to a concentration of 2.0 mg/ml clottable protein, chilled on wet ice to precipitate fibronectin ( 14), and then gravity filtered (Whatman No. 1 filter paper). Plasminogen, prepared according to the method of Deutsch and Mertz (15) or purchased from Kabi Vitrum (Catalog No. 5304), was diluted to 1.0 mg/ml with water. Trypsin degradation samples were prepared by the addition of 1 mg L-TPCK (tosyl-L-phenylalanine chloromethyl ketone) trypsin (Cooper) to a 1 mg/ml r&PA solution. Oxidative degradation was performed by bubbling water-saturated air through reconstituted t-t-PA solutions at ambient temperature. The plasmin used (Sigma, 10 units/vial) was reconstituted with 0.01% sodium azide and
PLASMINOGEN
ACTIVATOR
429
added to a 1 mg/ml r&PA solution. One vial of plasmin was used per 50 mg &PA. The carboxypeptidase B used (Worthington, in suspension) was added to a reconstituted rt-PA solution at 45 units per 50 mg r&PA. The pronase used (Calbiochem) was prepared as a 1 mg/ml solution in 0.0 1% sodium azide and added to a reconstituted rt-PA solution at 1 mg pronase per 50 mg t-t-PA. The rt-PA was reconstituted to 1 mg/ml with water and then diluted to assay levels in phosphate buffer. All reagents were stored on wet ice until used. The activity of rt-PA throughout this report is expressed as nanograms per milliliter of the internal reference material and not as international units (IU). The activity of the internal reference material in IU is still under investigation; its approximate activity is 500,000 IU/mg. Equipment. The MCA was an Instrumentation Laboratory (IL) Model 0773 Multistat III Plus Microcentrifugal Analyzer system. Rotors, which contain the cuvettes for mounting in the MCA, were purchased from IL. The loader was set to dispense 20 ~1 of i-t-PA as the sample, 20 ~1 of thrombin as the secondary reagent, and 200 ~1 of 50: 1 (v/v) fibrinogen:plasminogen mixture as the primary reagent. The analyzer was blanked against water and the analyzer chamber temperature was set at 37°C. The absorbance/ time program was used with a 5-min incubation time, 340-nm filter, and 90 interval readings. The analyzer delay and interval times were set according to the assay range as specified under Results and Discussion. An IBM-PC/XT, via the RS-232C ports, was interfaced to the analyzer to allow for automation of the analysis of the analytical data generated by the MCA as well as for archival purposes. Data analysis. Absorbance/time data were transferred from the analyzer to the IBMPC/XT using a commercially available program named “Perfect Link” (manufactured by Perfect Software). A program was written at Genentech in the language “C” for data analysis and calculations. This program analyzed the data according to the endpoint cri-
430
CARLSON
teria (defined under Results and Discussion) for the endpoint regression of the standard of log time versus log concentration. RESULTS AND DISCUSSION
A profile of clot absorbance (A) versus time is shown in Fig. 1. The formation of the clot is due to the formation of insoluble fibrin, which causes light to be scattered (turbidity) and is measured as an absorbance at 340 nm. The kinetics of clot formation are very rapid, resulting in a firm clot within 40 s. The absorbance plateau (between 0.28 and 0.30 A) indicates formation of a stable clot. Lysis becomes evident at about 140 s, with complete dissolution of the fibrin clot shortly after 300 s. It is interesting to note the shoulder that occurs between 260 and 280 s. The significance of this phenomenon has not been thoroughly established, although it may be related to the final collapse of the clot from the centrifugal force generated by the MCA. This would be consistent with the observation, made during bubble release experiments performed in test tubes, that the bubbles rise just prior to total transparency. The time-absorbance profile obtained with the MCA turbidimetry method provides a means of quantitating r&PA activity and is a source of diagnostic information about the assay. The profile provides a rapid
B
*-=EP4 ..*
-=EP2
AND 3
-*... *. *.
00
visual check of the raw data and verification that the endpoint has been correctly determined by the computer.
Choice of Endpoint
Absorbance ProJile
.8
ET AL.
1. ‘.FSEP, “....a .a.,.. 380
SXONDS FIG. 1. Clot lysis absorbance versus time profile (950 @ml rt-PA).
The time-absorbance profile was analyzed to determine which endpoint (EP) had the best correlation to &PA activity. The four endpoints (Fig. 1) investigated were EPl, the time at which the absorbance approached the baseline value; EP2, the time of the maximum decrease in absorbance (time of first derivative maximum); EP3, the slope of the maximum decrease in absorbance (slope of first derivative maximum); and EP4, the time of the one-half maximum absorbance (the average between the highest and lowest absorbance values). A slope threshold criterion (AA) was established for EPl to determine when the absorbance had reached a baseline value. To distinguish between the plateau at the beginning of clot formation from the plateau after lysis, only absorbances co.03 (the absorbance cutoff) were evaluated for their slope. The baseline was considered to be reached when AA < 0.003. Other values for the absorbance cutoff (0.02-0.04 A) and slope thresholds (AA of 0.002-0.004) were investigated. The combination that yielded the best linearity (correlation coefficient) for five standard concentrations over the ranges studied was considered optimum. The criteria for the choice of endpoint were based on linearity of the standard curve (five points), accuracy of recovery (a known concentration equivalent to the middle standard of the five-point curve), and precision of recovery (RSD). Results of the endpoint study are shown in Table 1. Evaluation of endpoints based on a first derivative approach requires a distinct inflection point. As the r-t-PA concentration decreases, a shallower slope is observed in the time-absorbance profile. This shallower slope complicated the reliable identification of the inflection point that correlated with r-t-PA activity. Thus, the usefulness of EP2 and EP3 was
TURBIDIMETRIC
ASSAY FOR TISSUE PLASMINOGEN
ACTIVATOR
431
TABLE 1 ENDPOINT COMPARISON No. of curves
Percentage recovery
Percentage RSD
No. of replicates
-0.9986 -0.9989 -0.9993
4 4 4
102.4 99.8 97.6
3.6 2.9 3.7
56 56 55
950-3000 127-400 32-100
-0.9954
4
101.7 Inflection point problem Inflection point problem
2.8
56
3 3 3
950-3000 127-400 32-100
-0.9972
4
97.2 Inflection point problem Inflection point problem
5.0
56
4 4 4
950-3000 127-400 32-100
-0.9995 -0.9982 -0.999 1
2 2 2
4.3 9.3 12.8
28 28 28
Cone range”
Average
EP 1 1 1
950-3000 127-400 32-100
2 2 2
r
103.3 104.8 99.6
LIConcentration range of rt-PA in the assay in nanograms per milliliter.
restricted to high r-t-PA concentration ranges. Less precise recoveries were obtained for EP4. EP 1 demonstrated utility in all concentration ranges; its linearity, accuracy, and precision were better than or equal to those of the other endpoints. Therefore, EPl was selected for further studies. The absorbance readings are collected only at discrete time intervals, but the endpoint can occur at any time. The endpoint is therefore measured with some finite resolution, defined by the time between adjacent data points. This introduces an uncertainty in the measured concentration that is related by the standard curve to the uncertainty in the endpoint, with the error decreasing as the r&PA concentration decreases for a given interval time. The analyzer collects a fixed number of data points per analysis (in the case of the Multistat, this number is 90). There is thus a compromise between the desire to use the highest resolution possible and the convenience of a wide standard curve range. Preliminary experiments indicated that an RSD of approximately 5% was obtained in the assay. The standard curve ranges and interval times (described below) were selected to
keep the uncertainty in concentration, introduced by the resolution error, to less than 3%, while yielding a useful working range of fivefold in the standard curve.
Spiked Placebo Recovery Studies Most of the work described in this paper was completed before the activity of an internal t-t-PA reference was correlated with the WHO t-PA standard. A batch of r-t-PA was specially filled and lyophilized at 1 mg/ vial and a fresh vial was used each day to prepare the standard curves. For this internal standard, an internal unitage was arbitrarily assigned where 1 unit = 1 mg. Spiked placebo samples were prepared at 1.5-0.5 mg/ml t-t-PA to evaluate the accuracy and precision of a +50% working assay range. Two assay ranges were investigated. Five standard concentrations were prepared for each range: 200- 1000 (advised delay time = 280 s, interval time = 4 s, analysis time = 28 min) and 40-200 rig/ml (advised delay time = 500 s, interval time = 8 s, analysis time = 39 min). The spiked placebos were diluted 1667:l (300-900 rig/ml) and 8333: 1 (60-180 rig/ml) for the appropriate ranges.
432
CARLSON
ET AL.
TABLE 2 SPIKED PLACEBO RECOVERY DATA Standard curve range h/ml) 200-loo0 200-1000 200-loo0 200-loo0 200-loo0
Spike W/ml) 0.5 0.8 1.0 1.2 1.5
Percentage recovery
Perceutage RSD
No. of replicates
91.7 98.8 100.4 100.4 100.2
4.4 5.1 4.4 4.2 7.8
100 60 157 60 loo
Mean recovery = 99.5 40-200 40-200 40-200
0.5 1.0 1.5
Mean RSD = 5.2
95.4 99.1 100.3
5.4 3.4 7.0
Mean recovery = 98.3
The results are shown in Table 2. Average recoveries of 99.5 (RSD = 5.2%) and 98.3% (RSD = 5.3%) were obtained for the high and low assay ranges, respectively. The average correlation coefficients were -0.999 1 (40 standard curves) and -0.9988 (24 standard curves) for the high and low concentration ranges, respectively. The rapid analysis times and high accuracy of this method made the 200-1000 rig/ml range the preferred choice. This is the range used throughout the report unless another range is specified. The spiked placebo recovery data were collected by three analysts over the course of many rotors and days. Small variances were observed between days, analysts, and rotors. A one-way analysis of variance was performed to determine if these variances were statistically significantly different. The P values, listed in Table 3, indicate that in some cases the variance could be linked to the day, the rotor, or the analyst. Optimal precision was obtained for replicates run on the same rotor (and thus also run on the same day and by the same analyst); the average RSD was 3.4 instead of 5.2% ‘(the unweighted mean for all samples of all three analysts). Conversely, optimal accuracy for a limited number of replicates can be obtained by spreading the replicates out over different
93 92 93 Mean RSD = 5.3
rotors, days, and analysts, where possible. It was desired that the MCA method not only minimize, but eliminate, technique dependence. Although a total elimination of technique dependence was not achieved, it was found that the RSD of 5.2% compared favorably to an RSD of 9% obtained for a bubble release study performed by three analysts over several days. A precision of 6% can be obtained for bubble release if a single analyst performs the experiments (A. M. Meunier et al., manuscript in preparation).
TABLE 3 P VALUESFROMANALYSISOFVARIANCE Range/spike”
Rotor
Analyst
Day
High/O.5 High/O.8 High/ 1.O High/l.2 High/l .5
0.02 NS 0.02 NS 0.01
Low/o.5 Low/l.0 Low/l.5
NS NS
a Spiked placebo concentrations are nanograms per milliliter r&PA. * NS, Not significant (P > 0.05).
TURBKXMETRIC
ASSAY
FOR
TISSUE
In addition to the bubble release or dropping glass bead techniques (9- 13 and A. M. Meunier et al., manuscript in preparation) to determine t-PA activity, other common approaches include the fibrin plate technique (16-20), the use of chromogenic (or fluorogenie) substrates ( 16,2 l-24), and isotopic labeling (16). The method adapted for the MCA offers advantages over all of these established methods. The control over mixing times, monitoring times, and temperature afforded by the MCA is superior to what can be obtained by bubble release, fibrin plates, or other kinetic-based assays. The MCA method does not require continuous visual monitoring as does the bubble release method nor is it as susceptible to subjective error (11,12). The MCA is free from major fibrin-plate drawbacks such as technical problems in preparation and use of the plates, quantitation of the method, and length of time in setup and incubation (16-19). The MCA method offers a better stability-indicating model than chromogenic or immunologic-based assays because chromogenic assays are less specific in their proteolytic activity and immunologic-based assays may not be stability indicating. Isotopic labeling possesses the fundamental problem of radioactive handling and disposal. Methods involving solid phase surfaces have the same drawback of plate preparation as does the fibrin plate technique (7). Reports of semiautomated clot lysis apparatus have appeared in the literature ( 11,12). These devices were not commercially available as was the Multistat III Plus MCA. The Multistat III Plus MCA has the capability of analyzing 19 samples at a time (or a fivepoint standard curve and 14 samples). An automated fibrinolytic assay performed in microtitration plates has been reported recently (25). However, the plate reader used was limited to readings at I-min intervals, whereas the MCA is capable of readings every 3 s. This resolution factor as well as the control over the addition of reagents inherent in the MCA design could explain why
PLASMINOGEN
ACTIVATOR
433
better precision was observed in testing r&PA with the MCA method (5.2%) than with the automated plate method (13.8%). It is also worth noting the similarity between the endpoint described (time at 50% lysis) and EP4. Thus, development of the rt-PA activity assay using an MCA apparatus to measure turbidimetry allows for automation while maintaining the required high degree of precision and accuracy.
Correlation between Clot Lysis Activity and Protein Concentration as Determined by Spectrophotometric Scan The correlation between the activity determined by the clot lysis assay and the protein concentration (mg/ml) determined by the spectrophotometric scan assay was studied. Assuming the r&PA samples tested have the same activity as the reference material, there should be a correlation between the activity and the protein concentration. The r&PA activity determined by clot lysis was compared to the protein concentration determined by spectrophotometric scan (performed at approximately 0.5 mgfml protein, 240-500 nm). The six samples studied (Table 4) had an average activity of 99% (2% RSD) relative to the concentration determined by spectrophotometric scan.
Stability Indication The clot lysis activity assay was evaluated for its stability-indicating properties by measuring r&PA degraded by several modes of physical, chemical, and enzymatic degradation (Table 5). These conditions were chosen to achieve significant amounts of degradation during the study. The results of these experiments demonstrate that the assay is stability indicating (Table 5). The least severe condition studied (pH = 5.0 at 30°C) caused only a 20% loss of r&PA clot lysis activity after 4 weeks; a pH of 7.5 at the same temperature caused a 50% loss of activity over the same time period. The thermal and carboxypeptidase B studies showed a 50% loss of activity in 7 to 12 days. The
434
CARLSON
ET AL.
TABLE 4 COMPARISONOFCLOTLYSISTOUVSPECTROPHOTOMETRY Clot lysis (U/ml)n
uv spectrophotometry (mg/ml)
Percentage specific activity
1.035 0.999 0.929 0.921 1.009 0.927
1.048 0.999 0.937 0.968 1.008 0.92 1
98.8 100.0 99.2 95.1 100.1 100.7 X = 99.0 (RSD = 2.0%)
’ Relative activity of an internal Genentech standard where 1 U = 1 mg of reference material.
other proteases, as well as the oxidative mode of degradation, caused a more rapid loss of activity. Sensitivity To work at sample levels of approximately 1 rig/ml, the reagent volumes and concentrations needed to be adjusted so that the sample would lyse in a reasonable time frame and the standard curve would remain linear. The adjustments were sample volume = 90 ~1, secondary reagent = 10 ~1 (66 units/ml thrombin), and primary reagent = 140 ~1 (2.86 mg/ml clottable protein of fibrinogen
and 1.4 mg/ml plasminogen). Advised analyzer delay and interval times were 900 and 55 s, respectively. Flushing the loader lines with assay buffer to remove any adsorbed r-t-PA incurred during testing with higher r-t-PA concentrations (e.g., 1000 rig/ml) was the only special precautionary measure. Standard curves in the range of 0.5-2.5 rig/ml had an average linearity of 0.9987 (n = 10). Recoveries for 0.5 and 1.O rig/ml were 103.9 (RSD = 3.9%, 12 = 30) and 97.9% (RSD = 2.4%, n = 40), respectively. Under the given conditions, the factor that limits sensitivity is the lysis time. Greater sensitivity is possible if longer lysis times are tolera-
TABLE 5 PERCENTAGEACTIVI~REMAININGAFTERACCELERATEDDEGRADATIONOF~~-PA Time Sample”
Temperature
1 &Y
1 week
2 weeks
3 weeks
4 weeks
Thermal Acidic Slightly basic Oxidative Carboxypeptidase B Plasmin Trypsin Pronase
37°C 3o”c 30°C Ambient 30°C 30°C 30°C 30°C
92.7 97.6 93.9 89.6 94.0 82.6 27.5 <306
59.0 94.0 89.3 <306 56.0 39.5 <306 <3d
44.3 92.0 75.5 <3Ob 36.7 <3Ob <30b <30b
36.6 88.8 69.8 <3d <3Ob <30b <30b <30b
29.6 78.5 54.4 <30b <3Ob 1306 <3Ob 130”
Note. Control activity was 1.03 U/ml. LIr&PA degraded by various chemical, physical, and enzymatic modes. ’ Sample had less than 30% activity remaining.
TIJRBIDIMETRIC
ASSAY FOR TISSUE PLASMINGGEN
ble and if care is taken to choose interval times yielding the best resolution. In conclusion, modification of the bubble release clot lysis assay to a turbidimetric method enabled the assay to be performed using an MCA. The time-absorbance profile generated in the turbidimetric assay provided a means of quantitation of clot lysis and a source of diagnostic information (i.e., being able to relate the fibrinolytic phenomena to the profile shape). The automation with the MCA simplified, accelerated, and better controlled the manipulations (e.g., pipetting and mixing) normally performed manually, thus minimizing dependence on technician technique. Computer control of the MCA made possible automatic data acquisition, data analysis, and calculations of results. Computer control thus eliminated the need for constant visual monitoring and subjectivity of the endpoint occurrence (lysis time) associated with the bubble release method ( 11,12). In addition, the turbidimetric MCA method was observed to have an accuracy of 99.5% with a precision of 5% (RSD). Although the focus of this paper was development of an automated assay to measure rt-PA activity, it should be noted that, like the bubble release assay, this turbidimetric methodology is applicable to the study of other fibrinolytic components. ACKNOWLEDGMENTS The authors thank Allen Lam for performing many of the experiments, Fred Miller for writing the data analysis computer program, and Mary Jean Pramik and Patricia A. Papa for their assistance in preparing and editing the manuscript.
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4. 5.
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9. 10. 11. 12. 13.
14.
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Goeddel, D. V., and Collen, D. (1983) Nature (London) 301,2 14-22 1. Collen, D. (1980) Thromb. Haemostasis 43,77-89. Wiman, B., and Collen, D. (1978) Nature (London) 272,549-550. Thorsen, S., Glas-Greenwalt, P., and Astrup, T. (1972) Thromb. Diath. Haemorrh. 28, 65-74. Angles-Cane, E. ( 1986) Anal. B&hem. 153.201-2 10. Van De Werf, F., Ludbrook, P. A., Bergmann, S. R., Tiefenbrunn, A. J., Fox, K. A. A., Hilaire de Geest, H., Verstraete, M., Collen, D., and Sobel, B. E. (1984) N. Engl. J. Med. 310, 609-613. Gaffney, P. J., and Curtis, A. D. (1985) Thromb. Haemostasis 53, 134- 136. Philo, R. D., and Gaffney, P. J. (1981) Thromb. Haemostasis 45, 107- 109. Wilkens, H. J., and Back, N. (1976) Amer. J. Clin. Pathol. 66, 124- 13 1. Collen, D., Tytgat, G., and Verstraete, M. (1968) J. Clin. Pathol. 21, 705-107. Johnson, A. J. (1964) in Blood Coagulation, Hemorrhage and Thrombosis (Tocantins, L. M., and Kazal, L. A., Eds.), pp. 458-459, Grune & Stratton, New York. Mosher, D. F., and Johnson, R. B. (1983) Ann. N. Y. Acad.
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15. Deutsch, D. G., and Mertz, E. T. (1970) Science 170, 1095-1096. 16. Marsh, N. ( 198 1) Fibrinolysis, pp. 206-24 1, Wiley, New York. 17. Marsh, N. A., and GaIfney, P. J. (1977) Thromb. Haemostasis 38, 545-55 1. 18. Astrup, T., and Jespersen, J. (1983) in Progress in Fibrinolysis (Davidson, J. F., Bachmann, F., Bouvier, C. A., and Kruithof, E. K. O., Eds.), Vol. 6, pp. 197-201, Churchill Livingstone, New York. 19. Jespersen, J., and Astrup, T. (1983) Haemostasis 13,301-315. 20. Ploug, J., and Kjeldgaard, N. 0. (1957) Biochim. Biophys.
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21. Egberg, N. (1984) Clin. Chem. Newsl. 4, 88-90. 22. Lijnen, H. R., and Cohen, D. (1982) Acta Chir. Stand. Suppl. 509, 129-132. 23. Wijngaards, G., and Nieuwenhuizen, W. (1980) in Synthetic Substrates in Clinical Blood Coagulation Assays (Lijnen, H. R., Collen, D., and Verstraete, M., Eds.), pp. 123- 13 1, Martinus Nijhoff Publishers, Boston. 24. Smith, D. S., Harmon, J., and Owen, W. G. (1985) Thromb. Rex 37, 533-541. 25. Beebe, D. P., and Aronson, D. L. (1987) Thromb. Rex 47, 123-128.