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The Use of Fluorogenic Substrates to Monitor Thrombin Generation for the Analysis of Plasma and Whole Blood Coagulation
Analytical Biochemistry 277, 11–18 (2000) Article ID abio.1999.4380, available online at http://www.idealibrary.com on
The Use of Fluorogenic Substrates to Monitor Thrombin Generation for the Analysis of Plasma and Whole Blood Coagulation Manoj K. Ramjee Peptide Therapeutics Group plc., Peterhouse Technology Park, 100 Fulbourn Road, Cambridge, CB1 9PT, United Kingdom
Received March 30, 1999
Thrombin is central to the process of coagulation and monitoring its activity is a reliable indicator of the rate and extent of coagulation. I have employed a range of fluorogenic peptide substrates as indicators of coagulation via the formation of active thrombin. This system enabled coagulation to be monitored in a kinetic fashion, and the use of fluorescence enabled a wide range of samples to be analyzed including lyophilized plasma containing fibrin, fresh platelet-poor plasma, platelet-rich plasma, and even whole blood. Coagulation could be monitored following triggering by tissue factor, ellagic acid, or each of the proteases preceding thrombin in the coagulation network. Using this assay procedure I have investigated the anticoagulant activities of a number of compounds and the results indicate that this assay would be useful for the kinetic analysis of coagulation in various plasma preparations, or even whole blood. © 2000 Academic Press
Central to the study of plasma coagulation is the accurate measurement of its rate. As such, various methods have been developed to monitor coagulation, and the generally accepted standardized methods include the prothrombin time (PT) 1 (i.e., the time taken for clot formation via tissue factor-dependent stimulation of the extrinsic pathway) and the activated partial thrombin time (APTT, contact stimulation of the intrinsic pathway) (1). These measurements, together 1
Abbreviations used: AMC, 7-amido-4-methylcoumarin; APTT, activated partial thromboplastin time; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; FPR-CMK, H-D-phenylprolylarginylchloromethylketone; HSA, human serum albumin; INR, international normalized ratio; PBS, phosphate-buffered saline; PT, prothrombin time; TGC, thrombin generation curve; ZGGR-AMC, benzoylglycylglycylarginyl-7-amido-4-methylcoumarin; Z-GPR-AMC, benzoylglycylprolylarginyl-7-amido-4-methylcoumarin. 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
with others, have been the basis by which anticoagulant therapy has been monitored (2, 3). However, the use of nonstandardized protocols and materials has resulted in variations in the measurements of the coagulation parameters (4). Hence, various studies have been carried out to try to standardize the methodology with a view to obtaining a universal standard (3, 5, 6). Nevertheless, the current methods still rely on the static measurements of single end-point values and it has been suggested that no single protocol works with all forms of hypercoagulability and/or hypocoagulability (7); hence, slight modifications are still required to dissect the underlying mechanism(s) of coagulation. The central role of thrombin in coagulation has meant that monitoring its activity in stimulated plasma provides a real-time measure of the coagulability of the sample (8). On this basis, the measurement of the endogenous thrombin potential was developed as the basis of a sensitive universal test (9). The method employed the use of specific colourimetric para-nitroanilide substrates to monitor the thrombin generation curve (TGC) in tissue factor-triggered defibrinated plasma (10). These kinetic data are then mathematically reduced to determine the thrombin potential (9, 11). Since the assay is based on chromogenic detection, blood samples must be cleared of cells and then defibrinated to reduced the background absorbtion. Thus, the necessary manipulation of a blood sample inevitably detracts from the physiological situation. In this report I describe the use of commercially available fluorogenic substrates to monitor thrombin generation. The results show that fluorogenic substrates overcome the limitations of chromogenic substrates and they can be used to analyze thrombin generation in fibrin-containing lyophilized plasma, platelet-poor plasma, platelet-rich plasma, and even whole blood. The method was applicable when triggered by a variety of initiators including ellagic acid, 11
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tissue factor, and the full range of proteases preceding thrombin in the coagulation network. I have also investigated the activities of a number of compounds and the data indicate that this method is useful for analyzing the profiles of anticoagulants in blood samples requiring little or no sample preparation. MATERIALS AND METHODS
General Materials and Methods Unless otherwise stated, all chemicals and biochemicals were purchased from the Sigma Chemical Co. (Poole, UK). Human factor a-XIIa, kallikrein, a-thrombin, factor IXab, factor Xa, factor XIa, and factor VIIa were purchased from Enzyme Research Laboratories (Swansea, UK).
Assays were routinely carried out by mixing 10 ml 100 nM tissue factor with 2.5 ml of 10 mM fluorogenic substrate in a plate well incubated at 37°C. To this, 90 ml of plasma was added and the sample was mixed by aspirating and dispensing 90 ml in the well at least three times, being careful to avoid air bubbles in the sample. The plate was transferred to the plate reader (Fluoroskan Ascent equipped with a 390-nm excitation filter, a 460-nm emission filter, and a thermostatted temperature option; Life Sciences International, Basingstoke, UK) and fluorescence kinetic data were collected at 30-s intervals over a period of 1 h at 37°C. All the assay component volumes remained the same for all the experiments. Where the effects of concentration were investigated, the sample volumes were adjusted appropriately prior to addition of 90 ml of plasma to the assay.
Active Site Titration of Thrombin For the accurate determination of the turnover number, an active site titration of human a-thrombin was carried out using a freshly prepared solution of FPRCMK (12) (Bachem UK, Saffron Walden, UK). Aliquots of thrombin (10 ml of 1 mM enzyme) were mixed with various amounts (0 to 3 ml) of 10 mM FPR-CMK and the samples were incubated at room temperature for 60 min. The residual activities (i.e., the change in fluorescence per second) were obtained from the initial rates of substrate hydrolysis upon addition of 90 ml of 10 mM Z-GPR-AMC. The active site concentration was calculated from the abscissa intercept of a linear regression analysis (Prism; GraphPad, San Diego, CA) of a plot of the residual activity versus FPR-CMK concentration. Control Level I Plasma Sample Preparation The coagulation control level I plasma (Sigma Chemical Co.) was reconstituted with water according to the supplier’s instructions just prior to use and preincubated in a 37°C water bath for 5 min before addition to the assay. All the fluorogenic peptide substrates (Bachem UK) were made up as 10 mM stock solutions in 100% DMSO (Rathburns, Glasgow, UK). In all cases the plates (96-well Microfluor W, U-bottomed microtiter plates; Dynex, Billingshurst, UK) were maintained at 37°C (Techne Model DB 1M plate incubator; Scientific Laboratory Supplies, Nottingham, UK) during assay preparation and liquid dispensing. Tissue Factor-Triggered Plasma Coagulation Recombinant human tissue factor (Calbiochem, Nottingham, UK) was reconstituted to 1 mM with water and HSA was added to 0.1% (w/v). Aliquots (100 ml) were snap-frozen in liquid nitrogen and stored at 280°C. For use in the assays, samples were thawed and diluted to 100 nM using 0.5 M calcium chloride.
Protease-Triggered Plasma Coagulation All the proteases were made up using water according to the manufacturer’s instructions. The protease solutions were diluted to the appropriate concentrations using PBS. Typically, assays were carried out using between 1 and 5 ml of protease, 1 ml of 0.5 M calcium chloride and the solution was made up 10 ml with PBS. To this 2.5 ml of 10 mM Z-GGR-AMC and 90 ml of control level I plasma were added. The samples were mixed by pipetting and assayed as described above. Ellagic Acid-Triggered Plasma Coagulation A 10 mM stock solution of ellagic acid was made up in 100% DMSO. The effect of ellagic acid concentration on triggering plasma coagulation was investigated by adding 1 ml 10 mM ellagic acid to 20 ml 50 mM calcium chloride. This was mixed and the ellagic acid was double diluted (i.e., 10 ml plus 10 ml) in calcium chloride to produce a working concentration range. To this, 2.5 ml of 10 mM Z-GGR-AMC and 90 ml of coagulation control level I plasma were added, and the samples were mixed and assayed as described above. Heparin, Hirudin, and Dermatan Sulfate Anticoagulant Analysis Stock solutions of heparin, hirudin (Calbiochem), and dermatan sulfate were made up and subsequently diluted appropriately in PBS. A molecular weight of 50,000 was used for calculating the concentration of dermatan sulfate (13). Assays contained 5 ml of anticoagulant, 5 ml of 200 nM tissue factor in 0.5 M calcium chloride, 2.5 ml 10 mM Z-GGR-AMC, and 90 ml of control level I plasma. The samples were mixed and assayed as described above.
FLUOROGENIC SUBSTRATES AS MONITORS OF COAGULATION
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the rate curves. The files were exported to Prism for further analysis, curve fitting, and presentation. RESULTS AND DISCUSSION
Tissue Factor-Triggered Coagulation
FIG. 1. Tissue factor- and calcium chloride-dependent coagulation of plasma. All samples contained 10 ml 100 nM tissue factor and/or 0.5 M calcium chloride; 2.5 ml 10 mM Z-GGR-AMC, and 90 ml control level I plasma. The samples are (a) 50 mM calcium chloride alone, (b) 10 nM tissue factor plus calcium chloride, (c) 10 nM tissue factor alone, and (d) 10 nM tissue factor, calcium chloride plus 10 mM hirudin.
Figure 1 shows typical results obtained when fluorogenic peptide substrates were used to monitor thrombin generation in plasma containing fibrin. Hirudin is a specific and extremely potent inhibitor of thrombin (14, 15). The sensitivity of the fluorescence response to hirudin would indicate that the rapid increase in fluorescence was due to thrombin (Fig. 1d) (15). This hypothesis was supported by observations with different substrates for thrombin; i.e., the better the substrate, the greater the fluorescence response generated during the coagulation of plasma (Fig. 2; Table 1). In addition, the plasma concentration of thrombin (;1 mM; (16)) is at least twofold higher than fXII and prekallikrein, and significantly higher than all of the remaining coagulation proteases (although the plasminogen concentration is ;2 mM; it is not strictly a procoagulation protease). Triggering plasma coagulation with the proteases preceding thrombin (see later) and the sensitivity of the fluorescence signals to hirudin provided further evidence that the curves were due to thrombin activation. In all cases the exogenous addition of calcium was essential for the initiation of coagulation within the time span of the assay (i.e., 60 min), irrespective of
Thrombin Generation Curve Using Fresh Plasma and Whole Blood Venous blood was collected from a consenting 24year-old, nonsmoking healthy male who had been fasting for 12 h prior to sample collection. Immediately upon sample collection, a 1/10th volume (500 ml) of 0.13 M sodium citrate was added and the sample was kept on ice. For the whole blood assay, 10 ml of 100 nM tissue factor in 0.5 M calcium chloride and 2.5 ml of 10 mM Z-GGR-AMC were mixed with 90 ml of blood. The sample was assayed as described above. To obtain platelet-poor plasma and platelet-rich plasma, separate blood aliquots (1 ml each) were centrifuged at 350g and 100g for 2 min each. The upper opaque phases were collected and assayed as described above. All assays were initiated within 10 min of collecting the blood samples. Data Handling and Manipulation The data from the Ascent microplate reader were saved in Excel (Microsoft Corporation, Seattle, WA) format and converted to the first derivative by subtraction (t n130s value 2 t n value). From this, the maximal rate of fluorescence production (t max) and the sum of the derivatives were calculated to obtain the area under
FIG. 2. Effect of substrate utilized on the fluorescence intensity in tissue factor-triggered plasma. All assays contained 10 nM tissue factor, 50 mM calcium chloride, and 200 mM of each substrate. The substrates were (■) CBZ-arg-AMC, (ƒ) Bz-phe-val-arg-AMC, (Œ) Zphe-arg-AMC, (h) Z-gly-pro-arg-AMC, ({) Z-pro-arg-AMC, and (E) Z-GGR-AMC. The open symbols are represented by the left ordinate and the filled symbols are represented by the right; the data points are connected by straight lines.
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MANOJ K. RAMJEE TABLE 1
Kinetic Constants for Various Fluorogenic Peptides Tested with Thrombin Substrate
K M (mM)
k cat (s 21)
k cat/K M (M 21 s 21)
Z-Gly-Gly-Arg-AMC Z-Phe-Arg-AMC Z-Pro-Arg-AMC Z-Gly-Pro-Arg-AMC CBZ-Arg-AMC Bz-Phe-Val-Arg-AMC
22.4 6 3.3 5.0 6 0.6 13.2 6 1.3 21.7 6 1.1 18.0 6 2.5 27.4 6 3.1
0.53 6 0.03 0.0082 6 0.00029 2.10 6 0.067 18.6 6 0.35 0.022 6 0.0013 5.58 6 0.25
2.4 3 10 4 1.6 3 10 2 1.6 3 10 5 8.6 3 10 5 1.2 3 10 2 2.0 3 10 5
Note. All assays were carried out in 10 mM Hepes, pH 8.0, containing 5 mM calcium chloride and 0.02% sodium azide.
trigger (Fig. 1c). Visual analysis of the plasma samples at the end of the experiments also confirmed that the amount of the fluorescence response was concomitant with the degree of clot formation. The tissue factor used in this case was supplied in buffer containing relatively large amounts of D-mannitol (200 mM), Tris (10 mM), and trace amounts of Chaps (0.01%). Tests of each of these components indicated that they did not have any observable effect on results generated by the assay system. As discussed above, the rate of increase in fluorescence is associated with the formation of thrombin. This phase is followed by a decrease in the rate, attributed to the physiological inactivation of thrombin by antithrombin III (ATIII) and a 2-macroglobulin (9). The steady-state linear increase in fluorescence, analogous to that observed previously with a chromogenic substrate, has been attributed to the residual activity of the thrombin z a 2-macroglobulin complex against small peptide substrates (9). A number of commercial tissue factor preparations (i.e., thromboplastin plus calcium) were evaluated as coagulation initiators. Significantly, when used in the concentration recommended, coagulation was almost instantaneous. The final tissue factor concentration in one preparation, calculated by ELISA (Enzyme Research Laboratories), was in the region of 1 nM, significantly lower than the concentration of purified recombinant tissue factor used (10 nM final of the Calbiochem material). The major difference between purified tissue factor (Calbiochem) and the diagnosticgrade reagents was the inclusion of phospholipid in the diagnostic samples. Phospholipid has been shown to be a contact activator of coagulation and lyophilized plasma is particularly sensitive to this form of activation (17). This was confirmed by experiments in which only phospholipid was mixed with lyophilized plasma and activation of coagulation was observed. More importantly, in at least one case, a trace amount of protease activity was also detected in the tissue factor preparation and this was confirmed by its sensitivity to a protease inhibitor cocktail (e.g., Complete; Boehr-
inger UK, Lewes, UK). These results indicate that the choice of tissue factor was an important element in the level of triggering of coagulation. These data support previous reports detailing differences in tissue factor preparations (18). The replacement of the coagulation control level I plasma (Sigma) with other control plasmas (e.g., Alpha Laboratories, Dade Behring, etc.) resulted in similar types of sigmoidal-shaped curves (Fig. 1b); however, the profiles were slightly different for each manufacturer. Also, in some cases batch-to-batch variations between plasma samples were also observed. This again was reflected in the shape of the sigmoidal curve and the differences were more pronounced in the derivative curves (Figs. 3c and 3d). Importantly, in all cases the plasma samples were quoted to have PT times within the recommended normal range (18). The Effect of Using Different Substrates in Tissue Factor- and Calcium-Triggered Plasma Figure 1 shows the curves obtained with Z-GGRAMC; the same type of data were obtained with a number of AMC based substrates (Fig. 2). The results clearly indicate that a number of fluorescent substrates can be used to monitor plasma coagulation via the generation of thrombin activity using complete lyophilized plasma, platelet-rich plasma, and whole blood. The better thrombin substrates (i.e., those with a second order rate constant greater than 5 3 10 4 M 21 s 21; Table 1) are consumed too quickly and do not follow the course of the reaction, resulting in a premature plateau of the fluorescence curve due to substrate depletion. This is similar to that observed with chromogenic substrates (19 –21). The AMC fluorescence is significantly quenched in plasma (by approximately 80%) compared to assay buffer, primarily due to the light-brown color attributed to the very high protein concentration. The AMC calibration curve in reconstituted plasma (data not shown) indicates that Z-GGR-AMC is consumed by approximately 50% during the experiment. For CBZarg-AMC and Z-phe-arg-AMC less than 10% of the substrates were utilized during the course of the reaction, an increase of approximately 75 and 130 fluorescence units respectively at the end of the experiment. Although all the substrates had similar initial background rates, most probably due to innate background protease activity (see later), for the better substrates (i.e., those with fluorescence changes greater than 1000 units) this initial fluorescence was less than 0.5% of the final intensity. With the poorer substrates (e.g., CBZarg-AMC and Z-phe-arg-AMC), this initial background rate was in the order of 1 to 2.5% of the total fluorescence. Nevertheless, the initial background activity was minor compared to the overall fluorescence change
FLUOROGENIC SUBSTRATES AS MONITORS OF COAGULATION
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FIG. 3. The effect of substrate concentration and tissue factor concentration on the observed rates. Data set a is the effect of Z-GGR-AMC concentration; all assays contained 10 nM tissue factor and 50 mM calcium chloride. The substrate concentrations beginning with the top curve and ending with the bottom curve were 250, 125, 62.5, 31.2, 15.6, and 7.8 mM. Data set b is the effect of tissue factor concentration. The tissue factor concentrations beginning with the top curve and ending with the bottom curve were 10, 5, 2.5, 1.25, 0.62, and 0.31 nM, respectively. The curves shown in c and d are the first order derivatives of data sets a and b, respectively. All assays contained 250 mM Z-GGR-AMC and 50 mM calcium chloride.
and in all cases the linear rates became negligible once the first order derivatives of the data were calculated. The poorer substrates did not give as good a signal-tonoise ratio as the better substrates (i.e., k cat/K M . 10 5 M 21 s 21, Table 1); however, these were not consumed as rapidly. Although a number of substrates with second order rate constants less than or equal to 10 4 M 21 s 21 were of use, Z-GGR-AMC was considered optimal for this system due to the signal-to-noise ratio. The use of fluorescence also overcomes sample opacity problems associated with absorbance measurements and it is a more sensitive method than the para-nitroanilidebased substrate assays (10, 21). The present study employed AMC-based substrates and there is at least one other reported fluorescence-based method used to monitor thrombin generation in blood (22). The curves obtained with that system were analogous to those with para-nitroanilide substrates (9) and the AMCbased substrates used in this study. The results, although similar to those obtained with the AMC-based substrates, were not as sensitive (as judged by the signal-to-noise ratio), primarily because AMC substrates have the advantage of being used at much higher concentrations (.200 mM) without loss of sensitivity due to quenching.
The Dependence on Substrate Concentration and Tissue Factor Concentration The substrate concentration used in the assay had a significant effect on the area under the curve (Figs. 3a and 3c), but there was a minor effect of the time to reach maximal rate (t max) (Fig. 4a). The effect of tissue factor concentration on the other hand had a marked effect both on t max and the area under the curve (Figs. 3b and 3d). The effect of tissue factor concentration indicated that above 2.5 nM, there was no longer an increase in area but there was a significant decrease in t max. Below a certain concentration (less than 0.3 nM) tissue factor no longer triggered plasma coagulation within the time scale of the experiment (i.e., 60 min). A plot of the t max and area data showed that above 25 mM Z-GGR-AMC the area increased linearly with concentration (Fig. 4a). The dependence of the response on tissue factor concentration (Fig. 4b) suggested that an initial threshold concentration was required prior to any observed response, as indicated by the curvature of the data on approaching the origin. This was also reflected in the area under the TGC curve. Between 0.5 and 2 nM, tissue factor concentration has a marked effect on the rate and amount of thrombin generation. Above 2 nM tissue factor, the area response saturates
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FIG. 4. A plot of the area under the curve (closed symbols) and time to maximal rate (t max) (open symbols) as a function of substrate and tissue factor concentrations. The assay conditions are described in the legend to Fig. 3.
such that a small effect is observed; whereas there is still a significant decrease in t max (Fig. 4b). Although these data were significantly different from those previously reported (21), a valid comparison cannot be made since the experiments were carried out by very different methods. However, as previously discussed, the inclusion of phospholipid in the trigger, together with the use of lyophilized plasma, would also make the system more sensitive to induction via contact activation (16, 23). An issue not addressed by the present study was whether the fluorogenic peptides behaved simply as alternative substrates or as partial inhibitors (19). Although not fully investigated here, cleavage of the AMC substrates plus the formation of a clot would indicate that both the AMC peptides and fibrinogen were substrates for thrombin. These results plus the relative second order rate constants would suggest that the AMC peptides do not compete significantly with fibrinogen (Table 1 (24)). Experiments in which the Z-GGR-AMC concentration was varied (Fig. 3a) indicate that the fluorogenic substrate did have an effect on the rate and amount of thrombin production (as judged by the area and t max data); however, this observation may have been a reflection of the substrate kinetics. In addition, very high concentrations of Z-GGR-AMC (up to 1 mM final concentration) do not prevent clot formation. When the substrate and tissue factor concentrations were kept constant (200 mM and 10 nM, respectively), but the plasma was diluted, a series of comparable curves’ results similar to those observed upon tissue factor dilution were obtained (e.g., Figs. 3b and 3d). In this case there was also an effect on t max and the area under the curve. Dilutions of plasma to greater than 1 in 4, i.e., 11.25 ml plasma diluted to 90 ml, did not result in plasma coagulation (as judged by substrate cleavage and clot formation) during the time course of the experiment (60 min).
The Analysis of Heparin, Hirudin, and Dermatan Sulfate Anticoagulants The current assay was used to evaluate a number of anticoagulant compounds. The data showed that all the compounds tested were potent anticoagulants effective at submicromolar concentrations in an in vitro assay (Fig. 5). Hirudin potently decreased the rate and amount of thrombin formation, being effective at concentrations $0.5 nM; heparin and dermatan sulfate were most effective at concentrations $50 nM. Above 500 nM, heparin prevented thrombin formation, and therefore clot generation, within the time course of the experiment. Heparin did not have a significant effect on t max or area up to 50 nM; above this concentration it
FIG. 5. The effect of heparin (circle), hirudin (square), and dermatan sulfate (triangle) on the area of the curve and the time to maximal rate (t max). All assays were carried out in triplicate and contained 5 ml 200 nM tissue factor in 500 mM calcium chloride, 5 ml of anticoagulant diluted in PBS, 2.5 ml of 10 mM Z-GGR-AMC, and 90 ml control level I plasma. The open symbols represent the area data (left abscissa) and the closed symbols represent the t max data (right abscissa), the asterisk (*) symbolizes assays that did not coagulate (DNC) and the connecting lines are projected from the preceding lower concentration data point.
FLUOROGENIC SUBSTRATES AS MONITORS OF COAGULATION
dramatically increased t max and decreased the area such that no observable coagulation occurs. The effects of dermatan sulfate were similar to those for hirudin except that a 100-fold higher concentration was required for the same effect. Hirudin and dermatan sulfate displayed an anticoagulant effect over a 100-fold concentration range, whereas the heparin was effective over only a 10-fold range. From these data, hirudin and dermatan sulfate exhibit a larger dose–response curve than heparin. The steep dose–response curve obtained for heparin would also therefore indicate a narrower therapeutic window as previously suggested (25). These results demonstrate that this method would be useful in the assessment of potential anticoagulants in a secondary assay system with a view toward assessing their therapeutic potential. Since the current assay system employed microtiter plates, this would also provide a high-throughput secondary assay system for compound anticoagulant profiling. Initiation of Coagulation Using Other Triggers Contact activation of plasma was initiated by simultaneous addition of ellagic acid and calcium chloride (23). As in all the protocols described herein, calcium was essential for triggering coagulation. It was noted that fresh solutions of ellagic acid were more effective at triggering coagulation than aged solutions. Dilution of ellagic acid also had the effect of reducing the potency of ellagic acid to trigger coagulation. Interestingly, high concentrations (greater than 1 mM final concentration) of ellagic acid also delayed the rate and amount of thrombin production. These data would indicate that the ellagic acid had multiple effects on the coagulation network and that various effects were manifested at different concentrations. This inference was supported by a previous report that demonstrated significant variation in the effect of 10 different APTT reagents (26). The initiation of coagulation using the various proteases preceding thrombin enabled careful analysis of the prethrombin coagulation network (Fig. 6). Attempts to trigger coagulation using these proteases in the presence of hirudin (1.4 mM final concentration) did not result in any significant signal (,50 units). Again this suggested that the increase in fluorescence was due to the activation of prothrombin. The background rates observed in the presence of protease plus hirudin were similar to those obtained in the absence of trigger (e.g., Fig. 1d). Also, as the protease concentration increased, this background rate also increased. Again this supported earlier observations that the background rate was due to nonthrombin activity (Fig. 1). Analogous effects to the dependence of thrombin generation on tissue factor were observed with the protease triggers. That is to say, above a certain con-
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FIG. 6. The initiation of plasma coagulation using various triggers. All assays contained 50 mM calcium chloride, 250 mM Z-GGR-AMC, and 90 ml plasma. Note: the raw data curves have been positively translated, for visual clarity, in their ordinate values by the numbers in the parentheses. The curves represent data obtained using a, 6 nM factor XIa (1300); b, 37 nM factor IXab (1300); c, 250 nM ellagic acid (1100); d, 17 nM factor Xa (700); e, 10 nM thrombin (650); f, 10 nM tissue factor (400); g, 78 nM factor XIIa (100); h, 12.5 nM factor VIIa (100); and i, 58 nM kallikrein (0).
centration clot formation was extremely rapid and the clot formed before any significant substrate cleavage occurred, whereas below a given concentration, no coagulation occurred. Attempts to use each protease at its physiological concentration were problematic, since in many cases clots formed almost instantaneously in the absence of a fluorescence signal. For each protease, the working concentration for observing substrate cleavage was therefore determined empirically. Interestingly, the closer a particular triggering protease was to thrombin in the network, the more rapid was clot formation over the appearance of fluorescence intensity. This again required the trigger concentration to be biased in favour of monitoring peptide cleavage. CONCLUSIONS
Various chromogenic and fluorogenic substrates have been reported to be utilized for monitoring plasma coagulation. These methods have required that the samples be pretreated to make them suitable for analysis. The current method employed commercially available fluorogenic peptide substrates to monitor thrombin generation in platelet-poor plasma containing fibrinogen (fresh or lyophilized), platelet-rich plasma, and even whole blood (Fig. 7). The current assay was used to analyze the dose–response curves of a number of anticoagulants. Coupled to this, the selectivity of anticoagulants could also be analyzed, in a secondary test system, by observing the effect on the TGC using the proteases preceding thrombin as triggers. The results show that potential anticoagulants can be evaluated by their effect on both the rate and the amount of thrombin production.
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FIG. 7. Thrombin generation curves of fresh blood and plasma showing the raw data (open symbols) and first order derivative data (closed symbols). (a) Thrombin generation curve for plasma derived from a 100g spin of blood; (b) thrombin generation curve for whole blood.
Using the assay described herein, the effectiveness of such compounds can routinely be analyzed using serum, plasma, or even whole blood, thus enabling their effectiveness to be evaluated in physiologically relevant samples. ACKNOWLEDGMENTS The author thanks Drs. Lawrence Garland, Sheila McLoughlin, Ian Henderson, and William Turnell for their advice and previewing the manuscript.
REFERENCES 1. http://www.dadeinternational.com/hemo/tutorial/tutorial.htm. 2. Poller, L. (1996) Haemostasis 26(Suppl. 4), 266 –268. 3. Poller, L., van den Besselaar, A. M., Jespersen, J., Tripodi, A., and Houghton, D. (1998) Thromb. Haemost. 80, 615– 623. 4. Houbouyan, L. L., and Goguel, A. F. (1997) Am. J. Clin. Pathol. 108, 83– 89. 5. Poller, L., van den Besselaar, A. M., Jespersen, J., Tripodi, A., and Houghton, D. (1998) Br. J. Haematol. 102, 910 –917. 6. Poller, L., Barrowcliffe, T. W., van den Besselaar, A. M., Jespersen, J., Tripodi, A., and Houghton, D. (1998) Thromb. Haemost. 79, 122–128. 7. Hemker, H. C., Wielders, S., and Beguin, S. (1990) in Fraxiparine (Bounameaux, H., Samama, M., and ten Cate, J. W., Eds.), pp. 89 –101, Schattauer, Stuttgart/New York. 8. Hemker, H. C. (1994) in Haemostasis and Thrombosis (Bloom, A. L., Forbes, C. D., Thomas, D. P., and Tuddenham, E. G. D., Eds.), Churchill Livingstone, London. 9. Hemker, H. C., Wielders, S., Kessels, H., and Beguin, S. (1993) Thromb. Haemost. 70, 617– 624. 10. Rijkers, D. T., Wielders, S. J., Tesser, G. I., and Hemker, H. C. (1995) Thromb. Res. 79, 491– 499.
11. Hemker, H. C., Willems, G. M., and Beguin, S. (1986) Thromb. Haemost. 56, 9 –17. 12. Kettner, C., and Shaw, E. (1979) Thromb. Res. 14, 969 –973. 13. Budavari, S., O’Neil, M. J., Smith, A., Heckelman, P. E., and Kinneary, J. F. (Eds.) (1996) The Merck Index, Merck & Co., Inc., Merck & Co., Inc., Whitehouse Station, NJ. 14. Markwardt, F. (1970) Methods Enzymol. 19, 924 –932. 15. Stone, S. R., and Hofsteenge, J. (1986) Biochemistry 25, 4622– 4628. 16. Halkier, T. (1991) Mechanisms in Blood Coagulation, Fibrinolysis and the Complement System, Cambridge Univ. Press, Cambridge, UK. 17. Griep, M. A., Fujikawa, K., and Nelsestuen, G. L. (1986) Biochemistry 25, 6688 – 6694. 18. Poller, L., Barrowcliffe, T. W., van den Besselaar, A. M., Jespersen, J., Tripodi, A., and Houghton, D. (1996) Thromb. Haemost. 76, 977–982. 19. Hemker, H. C., Weilders, S., Kessels, H., and Beguin, S. (1993) Thromb. Haemost. 70, 617– 624. 20. Rijkers, D. T. S., Weilders, S. J. H., Tesse, G. I., and Hemker, H. C. (1995) Thromb. Res. 79, 491– 499. 21. Weilders, S., Mukherjee, M., Michiels, J., Rijkers, D. T. S., Cambus, J-P., Knebel, R. W. C., Kakkar, V., Hemker, H. C., and Beguin, S. (1997) Thromb. Haemost. 77, 629 – 636. 22. Zweig, S. E., Meyer, B. G., Sharma, S., Min, C., Krakower, J. M., and Shohet, S. B. (1996) Biomed. Instrum. Technol. 30, 245– 256. 23. Ratnoff, O. D., and Crum, J. D. (1964) J. Lab. Clin. Med. 63, 359 –377. 24. Claeson, G. (1994) Blood Coagul. Fibrinolysis 5, 411– 436. 25. Cardigan, R. A., Mackie, I. J., and Machin, S. J. (1996) Thromb. Res. 84, 237–242. 26. Tripodi, A., Chantarangkul, V., Arbini, A. A., Moia, M., and Mannucci, P. M. (1993) Thromb. Haemost. 70, 286 –288.
The Use of Fluorogenic Substrates to Monitor Thrombin Generation for the Analysis of Plasma and Whole Blood Coagulation Manoj K. Ramjee Peptide Therapeutics Group plc., Peterhouse Technology Park, 100 Fulbourn Road, Cambridge, CB1 9PT, United Kingdom
Received March 30, 1999
Thrombin is central to the process of coagulation and monitoring its activity is a reliable indicator of the rate and extent of coagulation. I have employed a range of fluorogenic peptide substrates as indicators of coagulation via the formation of active thrombin. This system enabled coagulation to be monitored in a kinetic fashion, and the use of fluorescence enabled a wide range of samples to be analyzed including lyophilized plasma containing fibrin, fresh platelet-poor plasma, platelet-rich plasma, and even whole blood. Coagulation could be monitored following triggering by tissue factor, ellagic acid, or each of the proteases preceding thrombin in the coagulation network. Using this assay procedure I have investigated the anticoagulant activities of a number of compounds and the results indicate that this assay would be useful for the kinetic analysis of coagulation in various plasma preparations, or even whole blood. © 2000 Academic Press
Central to the study of plasma coagulation is the accurate measurement of its rate. As such, various methods have been developed to monitor coagulation, and the generally accepted standardized methods include the prothrombin time (PT) 1 (i.e., the time taken for clot formation via tissue factor-dependent stimulation of the extrinsic pathway) and the activated partial thrombin time (APTT, contact stimulation of the intrinsic pathway) (1). These measurements, together 1
Abbreviations used: AMC, 7-amido-4-methylcoumarin; APTT, activated partial thromboplastin time; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; FPR-CMK, H-D-phenylprolylarginylchloromethylketone; HSA, human serum albumin; INR, international normalized ratio; PBS, phosphate-buffered saline; PT, prothrombin time; TGC, thrombin generation curve; ZGGR-AMC, benzoylglycylglycylarginyl-7-amido-4-methylcoumarin; Z-GPR-AMC, benzoylglycylprolylarginyl-7-amido-4-methylcoumarin. 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
with others, have been the basis by which anticoagulant therapy has been monitored (2, 3). However, the use of nonstandardized protocols and materials has resulted in variations in the measurements of the coagulation parameters (4). Hence, various studies have been carried out to try to standardize the methodology with a view to obtaining a universal standard (3, 5, 6). Nevertheless, the current methods still rely on the static measurements of single end-point values and it has been suggested that no single protocol works with all forms of hypercoagulability and/or hypocoagulability (7); hence, slight modifications are still required to dissect the underlying mechanism(s) of coagulation. The central role of thrombin in coagulation has meant that monitoring its activity in stimulated plasma provides a real-time measure of the coagulability of the sample (8). On this basis, the measurement of the endogenous thrombin potential was developed as the basis of a sensitive universal test (9). The method employed the use of specific colourimetric para-nitroanilide substrates to monitor the thrombin generation curve (TGC) in tissue factor-triggered defibrinated plasma (10). These kinetic data are then mathematically reduced to determine the thrombin potential (9, 11). Since the assay is based on chromogenic detection, blood samples must be cleared of cells and then defibrinated to reduced the background absorbtion. Thus, the necessary manipulation of a blood sample inevitably detracts from the physiological situation. In this report I describe the use of commercially available fluorogenic substrates to monitor thrombin generation. The results show that fluorogenic substrates overcome the limitations of chromogenic substrates and they can be used to analyze thrombin generation in fibrin-containing lyophilized plasma, platelet-poor plasma, platelet-rich plasma, and even whole blood. The method was applicable when triggered by a variety of initiators including ellagic acid, 11
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tissue factor, and the full range of proteases preceding thrombin in the coagulation network. I have also investigated the activities of a number of compounds and the data indicate that this method is useful for analyzing the profiles of anticoagulants in blood samples requiring little or no sample preparation. MATERIALS AND METHODS
General Materials and Methods Unless otherwise stated, all chemicals and biochemicals were purchased from the Sigma Chemical Co. (Poole, UK). Human factor a-XIIa, kallikrein, a-thrombin, factor IXab, factor Xa, factor XIa, and factor VIIa were purchased from Enzyme Research Laboratories (Swansea, UK).
Assays were routinely carried out by mixing 10 ml 100 nM tissue factor with 2.5 ml of 10 mM fluorogenic substrate in a plate well incubated at 37°C. To this, 90 ml of plasma was added and the sample was mixed by aspirating and dispensing 90 ml in the well at least three times, being careful to avoid air bubbles in the sample. The plate was transferred to the plate reader (Fluoroskan Ascent equipped with a 390-nm excitation filter, a 460-nm emission filter, and a thermostatted temperature option; Life Sciences International, Basingstoke, UK) and fluorescence kinetic data were collected at 30-s intervals over a period of 1 h at 37°C. All the assay component volumes remained the same for all the experiments. Where the effects of concentration were investigated, the sample volumes were adjusted appropriately prior to addition of 90 ml of plasma to the assay.
Active Site Titration of Thrombin For the accurate determination of the turnover number, an active site titration of human a-thrombin was carried out using a freshly prepared solution of FPRCMK (12) (Bachem UK, Saffron Walden, UK). Aliquots of thrombin (10 ml of 1 mM enzyme) were mixed with various amounts (0 to 3 ml) of 10 mM FPR-CMK and the samples were incubated at room temperature for 60 min. The residual activities (i.e., the change in fluorescence per second) were obtained from the initial rates of substrate hydrolysis upon addition of 90 ml of 10 mM Z-GPR-AMC. The active site concentration was calculated from the abscissa intercept of a linear regression analysis (Prism; GraphPad, San Diego, CA) of a plot of the residual activity versus FPR-CMK concentration. Control Level I Plasma Sample Preparation The coagulation control level I plasma (Sigma Chemical Co.) was reconstituted with water according to the supplier’s instructions just prior to use and preincubated in a 37°C water bath for 5 min before addition to the assay. All the fluorogenic peptide substrates (Bachem UK) were made up as 10 mM stock solutions in 100% DMSO (Rathburns, Glasgow, UK). In all cases the plates (96-well Microfluor W, U-bottomed microtiter plates; Dynex, Billingshurst, UK) were maintained at 37°C (Techne Model DB 1M plate incubator; Scientific Laboratory Supplies, Nottingham, UK) during assay preparation and liquid dispensing. Tissue Factor-Triggered Plasma Coagulation Recombinant human tissue factor (Calbiochem, Nottingham, UK) was reconstituted to 1 mM with water and HSA was added to 0.1% (w/v). Aliquots (100 ml) were snap-frozen in liquid nitrogen and stored at 280°C. For use in the assays, samples were thawed and diluted to 100 nM using 0.5 M calcium chloride.
Protease-Triggered Plasma Coagulation All the proteases were made up using water according to the manufacturer’s instructions. The protease solutions were diluted to the appropriate concentrations using PBS. Typically, assays were carried out using between 1 and 5 ml of protease, 1 ml of 0.5 M calcium chloride and the solution was made up 10 ml with PBS. To this 2.5 ml of 10 mM Z-GGR-AMC and 90 ml of control level I plasma were added. The samples were mixed by pipetting and assayed as described above. Ellagic Acid-Triggered Plasma Coagulation A 10 mM stock solution of ellagic acid was made up in 100% DMSO. The effect of ellagic acid concentration on triggering plasma coagulation was investigated by adding 1 ml 10 mM ellagic acid to 20 ml 50 mM calcium chloride. This was mixed and the ellagic acid was double diluted (i.e., 10 ml plus 10 ml) in calcium chloride to produce a working concentration range. To this, 2.5 ml of 10 mM Z-GGR-AMC and 90 ml of coagulation control level I plasma were added, and the samples were mixed and assayed as described above. Heparin, Hirudin, and Dermatan Sulfate Anticoagulant Analysis Stock solutions of heparin, hirudin (Calbiochem), and dermatan sulfate were made up and subsequently diluted appropriately in PBS. A molecular weight of 50,000 was used for calculating the concentration of dermatan sulfate (13). Assays contained 5 ml of anticoagulant, 5 ml of 200 nM tissue factor in 0.5 M calcium chloride, 2.5 ml 10 mM Z-GGR-AMC, and 90 ml of control level I plasma. The samples were mixed and assayed as described above.
FLUOROGENIC SUBSTRATES AS MONITORS OF COAGULATION
13
the rate curves. The files were exported to Prism for further analysis, curve fitting, and presentation. RESULTS AND DISCUSSION
Tissue Factor-Triggered Coagulation
FIG. 1. Tissue factor- and calcium chloride-dependent coagulation of plasma. All samples contained 10 ml 100 nM tissue factor and/or 0.5 M calcium chloride; 2.5 ml 10 mM Z-GGR-AMC, and 90 ml control level I plasma. The samples are (a) 50 mM calcium chloride alone, (b) 10 nM tissue factor plus calcium chloride, (c) 10 nM tissue factor alone, and (d) 10 nM tissue factor, calcium chloride plus 10 mM hirudin.
Figure 1 shows typical results obtained when fluorogenic peptide substrates were used to monitor thrombin generation in plasma containing fibrin. Hirudin is a specific and extremely potent inhibitor of thrombin (14, 15). The sensitivity of the fluorescence response to hirudin would indicate that the rapid increase in fluorescence was due to thrombin (Fig. 1d) (15). This hypothesis was supported by observations with different substrates for thrombin; i.e., the better the substrate, the greater the fluorescence response generated during the coagulation of plasma (Fig. 2; Table 1). In addition, the plasma concentration of thrombin (;1 mM; (16)) is at least twofold higher than fXII and prekallikrein, and significantly higher than all of the remaining coagulation proteases (although the plasminogen concentration is ;2 mM; it is not strictly a procoagulation protease). Triggering plasma coagulation with the proteases preceding thrombin (see later) and the sensitivity of the fluorescence signals to hirudin provided further evidence that the curves were due to thrombin activation. In all cases the exogenous addition of calcium was essential for the initiation of coagulation within the time span of the assay (i.e., 60 min), irrespective of
Thrombin Generation Curve Using Fresh Plasma and Whole Blood Venous blood was collected from a consenting 24year-old, nonsmoking healthy male who had been fasting for 12 h prior to sample collection. Immediately upon sample collection, a 1/10th volume (500 ml) of 0.13 M sodium citrate was added and the sample was kept on ice. For the whole blood assay, 10 ml of 100 nM tissue factor in 0.5 M calcium chloride and 2.5 ml of 10 mM Z-GGR-AMC were mixed with 90 ml of blood. The sample was assayed as described above. To obtain platelet-poor plasma and platelet-rich plasma, separate blood aliquots (1 ml each) were centrifuged at 350g and 100g for 2 min each. The upper opaque phases were collected and assayed as described above. All assays were initiated within 10 min of collecting the blood samples. Data Handling and Manipulation The data from the Ascent microplate reader were saved in Excel (Microsoft Corporation, Seattle, WA) format and converted to the first derivative by subtraction (t n130s value 2 t n value). From this, the maximal rate of fluorescence production (t max) and the sum of the derivatives were calculated to obtain the area under
FIG. 2. Effect of substrate utilized on the fluorescence intensity in tissue factor-triggered plasma. All assays contained 10 nM tissue factor, 50 mM calcium chloride, and 200 mM of each substrate. The substrates were (■) CBZ-arg-AMC, (ƒ) Bz-phe-val-arg-AMC, (Œ) Zphe-arg-AMC, (h) Z-gly-pro-arg-AMC, ({) Z-pro-arg-AMC, and (E) Z-GGR-AMC. The open symbols are represented by the left ordinate and the filled symbols are represented by the right; the data points are connected by straight lines.
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MANOJ K. RAMJEE TABLE 1
Kinetic Constants for Various Fluorogenic Peptides Tested with Thrombin Substrate
K M (mM)
k cat (s 21)
k cat/K M (M 21 s 21)
Z-Gly-Gly-Arg-AMC Z-Phe-Arg-AMC Z-Pro-Arg-AMC Z-Gly-Pro-Arg-AMC CBZ-Arg-AMC Bz-Phe-Val-Arg-AMC
22.4 6 3.3 5.0 6 0.6 13.2 6 1.3 21.7 6 1.1 18.0 6 2.5 27.4 6 3.1
0.53 6 0.03 0.0082 6 0.00029 2.10 6 0.067 18.6 6 0.35 0.022 6 0.0013 5.58 6 0.25
2.4 3 10 4 1.6 3 10 2 1.6 3 10 5 8.6 3 10 5 1.2 3 10 2 2.0 3 10 5
Note. All assays were carried out in 10 mM Hepes, pH 8.0, containing 5 mM calcium chloride and 0.02% sodium azide.
trigger (Fig. 1c). Visual analysis of the plasma samples at the end of the experiments also confirmed that the amount of the fluorescence response was concomitant with the degree of clot formation. The tissue factor used in this case was supplied in buffer containing relatively large amounts of D-mannitol (200 mM), Tris (10 mM), and trace amounts of Chaps (0.01%). Tests of each of these components indicated that they did not have any observable effect on results generated by the assay system. As discussed above, the rate of increase in fluorescence is associated with the formation of thrombin. This phase is followed by a decrease in the rate, attributed to the physiological inactivation of thrombin by antithrombin III (ATIII) and a 2-macroglobulin (9). The steady-state linear increase in fluorescence, analogous to that observed previously with a chromogenic substrate, has been attributed to the residual activity of the thrombin z a 2-macroglobulin complex against small peptide substrates (9). A number of commercial tissue factor preparations (i.e., thromboplastin plus calcium) were evaluated as coagulation initiators. Significantly, when used in the concentration recommended, coagulation was almost instantaneous. The final tissue factor concentration in one preparation, calculated by ELISA (Enzyme Research Laboratories), was in the region of 1 nM, significantly lower than the concentration of purified recombinant tissue factor used (10 nM final of the Calbiochem material). The major difference between purified tissue factor (Calbiochem) and the diagnosticgrade reagents was the inclusion of phospholipid in the diagnostic samples. Phospholipid has been shown to be a contact activator of coagulation and lyophilized plasma is particularly sensitive to this form of activation (17). This was confirmed by experiments in which only phospholipid was mixed with lyophilized plasma and activation of coagulation was observed. More importantly, in at least one case, a trace amount of protease activity was also detected in the tissue factor preparation and this was confirmed by its sensitivity to a protease inhibitor cocktail (e.g., Complete; Boehr-
inger UK, Lewes, UK). These results indicate that the choice of tissue factor was an important element in the level of triggering of coagulation. These data support previous reports detailing differences in tissue factor preparations (18). The replacement of the coagulation control level I plasma (Sigma) with other control plasmas (e.g., Alpha Laboratories, Dade Behring, etc.) resulted in similar types of sigmoidal-shaped curves (Fig. 1b); however, the profiles were slightly different for each manufacturer. Also, in some cases batch-to-batch variations between plasma samples were also observed. This again was reflected in the shape of the sigmoidal curve and the differences were more pronounced in the derivative curves (Figs. 3c and 3d). Importantly, in all cases the plasma samples were quoted to have PT times within the recommended normal range (18). The Effect of Using Different Substrates in Tissue Factor- and Calcium-Triggered Plasma Figure 1 shows the curves obtained with Z-GGRAMC; the same type of data were obtained with a number of AMC based substrates (Fig. 2). The results clearly indicate that a number of fluorescent substrates can be used to monitor plasma coagulation via the generation of thrombin activity using complete lyophilized plasma, platelet-rich plasma, and whole blood. The better thrombin substrates (i.e., those with a second order rate constant greater than 5 3 10 4 M 21 s 21; Table 1) are consumed too quickly and do not follow the course of the reaction, resulting in a premature plateau of the fluorescence curve due to substrate depletion. This is similar to that observed with chromogenic substrates (19 –21). The AMC fluorescence is significantly quenched in plasma (by approximately 80%) compared to assay buffer, primarily due to the light-brown color attributed to the very high protein concentration. The AMC calibration curve in reconstituted plasma (data not shown) indicates that Z-GGR-AMC is consumed by approximately 50% during the experiment. For CBZarg-AMC and Z-phe-arg-AMC less than 10% of the substrates were utilized during the course of the reaction, an increase of approximately 75 and 130 fluorescence units respectively at the end of the experiment. Although all the substrates had similar initial background rates, most probably due to innate background protease activity (see later), for the better substrates (i.e., those with fluorescence changes greater than 1000 units) this initial fluorescence was less than 0.5% of the final intensity. With the poorer substrates (e.g., CBZarg-AMC and Z-phe-arg-AMC), this initial background rate was in the order of 1 to 2.5% of the total fluorescence. Nevertheless, the initial background activity was minor compared to the overall fluorescence change
FLUOROGENIC SUBSTRATES AS MONITORS OF COAGULATION
15
FIG. 3. The effect of substrate concentration and tissue factor concentration on the observed rates. Data set a is the effect of Z-GGR-AMC concentration; all assays contained 10 nM tissue factor and 50 mM calcium chloride. The substrate concentrations beginning with the top curve and ending with the bottom curve were 250, 125, 62.5, 31.2, 15.6, and 7.8 mM. Data set b is the effect of tissue factor concentration. The tissue factor concentrations beginning with the top curve and ending with the bottom curve were 10, 5, 2.5, 1.25, 0.62, and 0.31 nM, respectively. The curves shown in c and d are the first order derivatives of data sets a and b, respectively. All assays contained 250 mM Z-GGR-AMC and 50 mM calcium chloride.
and in all cases the linear rates became negligible once the first order derivatives of the data were calculated. The poorer substrates did not give as good a signal-tonoise ratio as the better substrates (i.e., k cat/K M . 10 5 M 21 s 21, Table 1); however, these were not consumed as rapidly. Although a number of substrates with second order rate constants less than or equal to 10 4 M 21 s 21 were of use, Z-GGR-AMC was considered optimal for this system due to the signal-to-noise ratio. The use of fluorescence also overcomes sample opacity problems associated with absorbance measurements and it is a more sensitive method than the para-nitroanilidebased substrate assays (10, 21). The present study employed AMC-based substrates and there is at least one other reported fluorescence-based method used to monitor thrombin generation in blood (22). The curves obtained with that system were analogous to those with para-nitroanilide substrates (9) and the AMCbased substrates used in this study. The results, although similar to those obtained with the AMC-based substrates, were not as sensitive (as judged by the signal-to-noise ratio), primarily because AMC substrates have the advantage of being used at much higher concentrations (.200 mM) without loss of sensitivity due to quenching.
The Dependence on Substrate Concentration and Tissue Factor Concentration The substrate concentration used in the assay had a significant effect on the area under the curve (Figs. 3a and 3c), but there was a minor effect of the time to reach maximal rate (t max) (Fig. 4a). The effect of tissue factor concentration on the other hand had a marked effect both on t max and the area under the curve (Figs. 3b and 3d). The effect of tissue factor concentration indicated that above 2.5 nM, there was no longer an increase in area but there was a significant decrease in t max. Below a certain concentration (less than 0.3 nM) tissue factor no longer triggered plasma coagulation within the time scale of the experiment (i.e., 60 min). A plot of the t max and area data showed that above 25 mM Z-GGR-AMC the area increased linearly with concentration (Fig. 4a). The dependence of the response on tissue factor concentration (Fig. 4b) suggested that an initial threshold concentration was required prior to any observed response, as indicated by the curvature of the data on approaching the origin. This was also reflected in the area under the TGC curve. Between 0.5 and 2 nM, tissue factor concentration has a marked effect on the rate and amount of thrombin generation. Above 2 nM tissue factor, the area response saturates
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MANOJ K. RAMJEE
FIG. 4. A plot of the area under the curve (closed symbols) and time to maximal rate (t max) (open symbols) as a function of substrate and tissue factor concentrations. The assay conditions are described in the legend to Fig. 3.
such that a small effect is observed; whereas there is still a significant decrease in t max (Fig. 4b). Although these data were significantly different from those previously reported (21), a valid comparison cannot be made since the experiments were carried out by very different methods. However, as previously discussed, the inclusion of phospholipid in the trigger, together with the use of lyophilized plasma, would also make the system more sensitive to induction via contact activation (16, 23). An issue not addressed by the present study was whether the fluorogenic peptides behaved simply as alternative substrates or as partial inhibitors (19). Although not fully investigated here, cleavage of the AMC substrates plus the formation of a clot would indicate that both the AMC peptides and fibrinogen were substrates for thrombin. These results plus the relative second order rate constants would suggest that the AMC peptides do not compete significantly with fibrinogen (Table 1 (24)). Experiments in which the Z-GGR-AMC concentration was varied (Fig. 3a) indicate that the fluorogenic substrate did have an effect on the rate and amount of thrombin production (as judged by the area and t max data); however, this observation may have been a reflection of the substrate kinetics. In addition, very high concentrations of Z-GGR-AMC (up to 1 mM final concentration) do not prevent clot formation. When the substrate and tissue factor concentrations were kept constant (200 mM and 10 nM, respectively), but the plasma was diluted, a series of comparable curves’ results similar to those observed upon tissue factor dilution were obtained (e.g., Figs. 3b and 3d). In this case there was also an effect on t max and the area under the curve. Dilutions of plasma to greater than 1 in 4, i.e., 11.25 ml plasma diluted to 90 ml, did not result in plasma coagulation (as judged by substrate cleavage and clot formation) during the time course of the experiment (60 min).
The Analysis of Heparin, Hirudin, and Dermatan Sulfate Anticoagulants The current assay was used to evaluate a number of anticoagulant compounds. The data showed that all the compounds tested were potent anticoagulants effective at submicromolar concentrations in an in vitro assay (Fig. 5). Hirudin potently decreased the rate and amount of thrombin formation, being effective at concentrations $0.5 nM; heparin and dermatan sulfate were most effective at concentrations $50 nM. Above 500 nM, heparin prevented thrombin formation, and therefore clot generation, within the time course of the experiment. Heparin did not have a significant effect on t max or area up to 50 nM; above this concentration it
FIG. 5. The effect of heparin (circle), hirudin (square), and dermatan sulfate (triangle) on the area of the curve and the time to maximal rate (t max). All assays were carried out in triplicate and contained 5 ml 200 nM tissue factor in 500 mM calcium chloride, 5 ml of anticoagulant diluted in PBS, 2.5 ml of 10 mM Z-GGR-AMC, and 90 ml control level I plasma. The open symbols represent the area data (left abscissa) and the closed symbols represent the t max data (right abscissa), the asterisk (*) symbolizes assays that did not coagulate (DNC) and the connecting lines are projected from the preceding lower concentration data point.
FLUOROGENIC SUBSTRATES AS MONITORS OF COAGULATION
dramatically increased t max and decreased the area such that no observable coagulation occurs. The effects of dermatan sulfate were similar to those for hirudin except that a 100-fold higher concentration was required for the same effect. Hirudin and dermatan sulfate displayed an anticoagulant effect over a 100-fold concentration range, whereas the heparin was effective over only a 10-fold range. From these data, hirudin and dermatan sulfate exhibit a larger dose–response curve than heparin. The steep dose–response curve obtained for heparin would also therefore indicate a narrower therapeutic window as previously suggested (25). These results demonstrate that this method would be useful in the assessment of potential anticoagulants in a secondary assay system with a view toward assessing their therapeutic potential. Since the current assay system employed microtiter plates, this would also provide a high-throughput secondary assay system for compound anticoagulant profiling. Initiation of Coagulation Using Other Triggers Contact activation of plasma was initiated by simultaneous addition of ellagic acid and calcium chloride (23). As in all the protocols described herein, calcium was essential for triggering coagulation. It was noted that fresh solutions of ellagic acid were more effective at triggering coagulation than aged solutions. Dilution of ellagic acid also had the effect of reducing the potency of ellagic acid to trigger coagulation. Interestingly, high concentrations (greater than 1 mM final concentration) of ellagic acid also delayed the rate and amount of thrombin production. These data would indicate that the ellagic acid had multiple effects on the coagulation network and that various effects were manifested at different concentrations. This inference was supported by a previous report that demonstrated significant variation in the effect of 10 different APTT reagents (26). The initiation of coagulation using the various proteases preceding thrombin enabled careful analysis of the prethrombin coagulation network (Fig. 6). Attempts to trigger coagulation using these proteases in the presence of hirudin (1.4 mM final concentration) did not result in any significant signal (,50 units). Again this suggested that the increase in fluorescence was due to the activation of prothrombin. The background rates observed in the presence of protease plus hirudin were similar to those obtained in the absence of trigger (e.g., Fig. 1d). Also, as the protease concentration increased, this background rate also increased. Again this supported earlier observations that the background rate was due to nonthrombin activity (Fig. 1). Analogous effects to the dependence of thrombin generation on tissue factor were observed with the protease triggers. That is to say, above a certain con-
17
FIG. 6. The initiation of plasma coagulation using various triggers. All assays contained 50 mM calcium chloride, 250 mM Z-GGR-AMC, and 90 ml plasma. Note: the raw data curves have been positively translated, for visual clarity, in their ordinate values by the numbers in the parentheses. The curves represent data obtained using a, 6 nM factor XIa (1300); b, 37 nM factor IXab (1300); c, 250 nM ellagic acid (1100); d, 17 nM factor Xa (700); e, 10 nM thrombin (650); f, 10 nM tissue factor (400); g, 78 nM factor XIIa (100); h, 12.5 nM factor VIIa (100); and i, 58 nM kallikrein (0).
centration clot formation was extremely rapid and the clot formed before any significant substrate cleavage occurred, whereas below a given concentration, no coagulation occurred. Attempts to use each protease at its physiological concentration were problematic, since in many cases clots formed almost instantaneously in the absence of a fluorescence signal. For each protease, the working concentration for observing substrate cleavage was therefore determined empirically. Interestingly, the closer a particular triggering protease was to thrombin in the network, the more rapid was clot formation over the appearance of fluorescence intensity. This again required the trigger concentration to be biased in favour of monitoring peptide cleavage. CONCLUSIONS
Various chromogenic and fluorogenic substrates have been reported to be utilized for monitoring plasma coagulation. These methods have required that the samples be pretreated to make them suitable for analysis. The current method employed commercially available fluorogenic peptide substrates to monitor thrombin generation in platelet-poor plasma containing fibrinogen (fresh or lyophilized), platelet-rich plasma, and even whole blood (Fig. 7). The current assay was used to analyze the dose–response curves of a number of anticoagulants. Coupled to this, the selectivity of anticoagulants could also be analyzed, in a secondary test system, by observing the effect on the TGC using the proteases preceding thrombin as triggers. The results show that potential anticoagulants can be evaluated by their effect on both the rate and the amount of thrombin production.
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FIG. 7. Thrombin generation curves of fresh blood and plasma showing the raw data (open symbols) and first order derivative data (closed symbols). (a) Thrombin generation curve for plasma derived from a 100g spin of blood; (b) thrombin generation curve for whole blood.
Using the assay described herein, the effectiveness of such compounds can routinely be analyzed using serum, plasma, or even whole blood, thus enabling their effectiveness to be evaluated in physiologically relevant samples. ACKNOWLEDGMENTS The author thanks Drs. Lawrence Garland, Sheila McLoughlin, Ian Henderson, and William Turnell for their advice and previewing the manuscript.
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