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Prothrombin Activity and Concentration in Healthy Subjects with and without the Prothrombin G20210A Mutation
Thrombosis Research 99 (2000) 549–556
ORIGINAL ARTICLE
Prothrombin Activity and Concentration in Healthy Subjects with and without the Prothrombin G20210A Mutation Nicolas von Ahsen1, Piotr Lewczuk2,3, Ekkehard Schu¨tz1, Michael Oellerich1 and Hannelore Ehrenreich3 Department of Clinical Chemistry, Georg-August-University, Robert Koch Str. 40, 37075 Goettingen, Germany, 2Department of Pediatric Neurology, Medical Academy of Bialystok, Bialystok, Poland, and 3Departments of Neurology and Psychiatry, Georg-August-University, Robert Koch Str. 40, 37075 Goettingen and Max-Planck Institute for Experimental Medicine, Goettingen, Germany 1
(Received 17 September 1999 by Editor D.L. Heene; revised/accepted 25 April 2000)
Abstract A common mutation in the prothrombin gene (G20210A) is associated with elevated prothrombin levels and thrombosis. The pathomechanism related to the G20210A mutation is currently not understood and the interdependence of prothrombin activity and prothrombin concentration in plasma is still poorly defined. Six of 191 blood donors examined in the present study carried the prothrombin allele G20210A. Despite the small number of cases, plasma samples from these individuals had significantly higher prothrombin activities than wild type carriers (131⫾7.1% vs. 114⫾18.3%, P⫽0.017), whereas their prothrombin concentrations—although elevated—did not differ significantly from wild type (122⫾30.7 mg/L vs. 107⫾20.6 mg/L, P⫽0.245). In subjects with the G20210A mutation there was also no significant correlation between prothrombin activity and concentration (n⫽6, r⫽0.200, P⫽0.704). Analyzing data from healthy blood donors without the prothrombin G20210A mutation (n⫽185), we found only weak correlations between prothrombin activAbbreviations: Prothrombin fragment 1⫹2, F1⫹2. Corresponding author: Nicolas von Ahsen, Georg-August University, Dept. Clinical Chemistry, Robert-Koch-Str. 40, 37075 Goettingen, Germany. Fax: ⫹49 (551) 39 8551; E-mail:⬍nahsen@ gwdg.de⬎.
ity and concentration of immunoreactive prothrombin (r⫽0.287; p⬍0.001). Samples with a relatively high prothrombin concentration but low activity were observed as well as samples with a relatively high activity for a given concentration (hyperactive prothrombin). F1⫹2 concentrations as indicators of activated coagulation were only elevated in 13 of 125 investigated samples and could not explain any of these findings. Dysfunctional variants of prothrombin, a well known phenomenon, may be responsible for the former, and we speculate that posttranscriptionally modified prothrombin species may explain the observed functional diversity of this factor including hyperactivity. The genotype–phenotype association of the non-coding G20210A mutation is not clear cut. Therefore, further studies are needed to determine which factors apart from the known G20210A polymorphism regulate prothrombin concentration and/or activity and may trigger the manifestation of thrombosis. 2000 Elsevier Science Ltd. All rights reserved. Key Words: Prothrombin activity; Immunoreactive prothrombin concentration; Prothrombin mutation; Prothrombin fragment 1⫹2; Reference interval; Pathomechanism
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rothrombin, the precursor of thrombin, plays a central role in blood coagulation, participating in the process of clotting and platelet activation. In addition, prothrombin is in-
0049-3848/00 $–see front matter 2000 Elsevier Science Ltd. All rights reserved. PII S0049-3848(00)00281-4
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volved in a number of other physiologic mechanisms, e.g., in the central nervous system where it acts on cell proliferation and differentiation [1,2]. Prothrombin abnormalities have traditionally been associated with rare bleeding disorders caused by hypo- or dysprothrombinemia. The former are characterized by reduced prothrombin concentrations, the latter by reduced activity and concentration [3]. In 1996, the first mutation (G20210A) in the 3⬘ untranslated region of the prothrombin gene associated with elevated levels of prothrombin immunoreactivity and activity [4] was reported. Several studies have been able to confirm an association of this common mutation with arterial [5] or venous [4,6–10] thrombotic events. The mutation has been found to lead to significantly elevated prothrombin activity [4,7,9,10] and prothrombin plasma concentrations [4,11]. There is no ongoing thrombin generation even in patients homozygous for this mutation as judged by prothrombin fragment 1⫹2 (F1⫹2) concentration, a sensitive marker of coagulation activation [12]. Except for patients under warfarin therapy [13,14] and patients with thrombophilia [4], the interdependence of prothrombin activity and concentration in plasma of healthy subjects is still poorly defined. The present study was designed to determine reference intervals for immunoreactive prothrombin levels in a large number of healthy individuals, using a sensitive and highly specific ELISA, and to compare prothrombin antigen to prothrombin activity in this collective, under consideration of the G20210A genotype.
1. Materials and Methods The study was performed in accordance with the ethics guidelines of the Georg-August-University, Goettingen. Blood samples from 191 healthy blood donors without history of prior thrombosis were provided anonymously by the local blood bank. Samples were drawn in the afternoon from an antecubital vein in non-fasting state before the blood donation. Citrated plasma (0.106 mmol/L trisodium citrate) was immediately separated by 10 minutes of centrifugation (2500⫻g) in a precooled centrifuge (15⬚C), and the prothrombin time was
measured. Aliquots of the plasma were frozen at –80⬚C until further analysis.
1.1. Prothrombin Time Measurement The prothrombin time (Quick) was measured with an automated coagulation system analyser (BCS, Dade Behring GmbH, Marburg, Germany) using Thromborel S reagent (Dade Behring GmbH, Marburg, Germany). Calibration was performed using a standard-human plasma dilution series as per the manufacturer’s instructions (Dade Behring GmbH, Marburg, Germany).
1.2. Prothrombin Activity Measurement Prothrombin activity was measured using prothrombin deficient plasma with a remaining prothrombin activity⬍1% (Dade Behring GmbH, Marburg, Germany) on an automated coagulation system analyser (BCS, Dade Behring GmbH, Marburg, Germany). Calibration was performed with a standard human normal plasma dilution series according to the manufacturer’s instructions (Dade Behring GmbH, Marburg, Germany). The standard was diluted automatically by the instrument. For the assay itself, 30 L of prothrombin deficient plasma and 5 L of test plasma were mixed with 150 L prothrombin reagent (Thromborel S, Dade Behring GmbH, Marburg, Germany) and the time until clotting occurred was measured by the analyser. The coefficient of variation was⬍6% for intra- and inter-series. We assayed the dilution recovery of several samples with both high and low prothrombin activity and high and low prothrombin concentration, and found no significant deviation from linearity with this method (data not shown).
1.3. Determination of Prothrombin in Plasma Using ELISA (Enzyme-linked Immuno Sorbent Assay) Prothrombin concentration was measured according to a published method [15,16]. Briefly, 100 L of goat anti-human prothrombin antibody (ICN Biomedicals, USA) diluted 1:400 in carbonate buffer (pH 9.6) was added to 96 wells of an ELISA plate (MaxiSorp, Nunc, Germany) and incubated overnight in⫹4⬚C on a shaker. On the next day
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wells were washed 3 times with washing buffer [17]. Plasma samples were diluted 1:8000 in assay buffer [17]. To obtain data considering a possible crossreactivity, human thrombin (Sigma, USA) was applied undiluted and diluted 1:2 in the assay buffer. Standard (ICN Biomedicals, USA) was diluted in this same buffer to obtain the following concentrations: 35 g/L, 17.5 g/L, 9.0 g/L, 4.5 g/L, 2.25 g/L, and 1.12 g/L. 100 L of the diluted plasma or the standard were added to wells in duplicate and the plate was incubated for 1 hour at room temperature, shaking. Wells were then washed 3 times in washing buffer, and 100 L of sheep antihuman prothrombin antibody conjugated with HRP peroxidase (Biogenesis, UK) diluted 1:750 in the assay buffer was added to all wells. The plate was incubated for 1 hour at room temperature on a shaker, washed 6 times in the washing buffer, and 100 L of HRP substrate [17] was added to all wells. After 15 minutes of incubation at room temperature, shaking, the reaction was stopped with 100 L of 2N sulphuric acid. The optical density was measured with an automatic ELISA reader (SLT, Germany) set at 450 nm with a correction wavelength of 620 nm. The detection limit of the assay is 0.7 g/L, the coefficient of variation is⬍9% for intra- and inter-series.
1.4. Determination of F1⫹2 in Plasma F1⫹2 plasma concentrations were determined using a commercial ELISA (Enzygnost威 F1⫹2 micro, Dade Behring GmbH, Marburg, Germany) according to the manufacturer’s instructions. Plates were read at 490 nm on a micro plate reader (Millenia Kinetic Analyzer, DPC, Germany) and results were analyzed using a four parameter fit for the standard curve. The reference interval of this assay is 0.4–1.1 nmol/L (5–95th percentile) according to the manufacturer.
1.5. Genotyping the Prothrombin G20210A Mutation For genotyping of the G20210A mutation we used a recently described method [18]. Briefly, genomic DNA was isolated from whole blood using standard procedures. PCR reactions were performed on a LightCycler (Roche Molecular Diagnostics, Mannheim, Germany). The method uses hybridisa-
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tion of labeled oligonucleotide probes and yields a single melting peak at ~57⬚C for the wildtype DNA. In cases with homozygous mutations there is a mismatch under the wildtype DNA compatible probe which leads to strand instability and consecutive earlier melting with a single melting peak at ~50⬚C. Accordingly, patients with heterozygous mutations show two melting peaks. All genotypes can be clearly distinguished.
1.6. Statistics Statistics were computed using SPSS 7.5.2 for Windows and Analyze-It for Excel. Normal distribution was assessed by the Shapiro–Wilk test. Group comparisons were done with the Mann–Whitney U-test. Correlations were calculated using the Spearman rank–sum correlation. The S.D. is used as a measure of variation throughout the study.
2. Results Data on prothrombin time (Quick), activity, and concentration in plasma of 191 healthy blood donors are summarised in Table 1. Weak correlations were detected between prothrombin activity and concentration in 127 males (r⫽0.252, r2⫽0.06, P⫽0.004) and 64 females (r⫽0.362, r2⫽0,13, P⫽0.003) (Figure 1). There was a statistically significant gender difference for prothrombin activity (p⫽0.034) but not for prothrombin concentration (p⫽0.980) or for prothrombin ratio (p⫽0.950). Since in the present study men were on average significantly older than women (34⫾11.5 vs. 30⫾11.8 years, P⫽0.001), we repeated the calculation after age-matching 64 men with the 64 women in our collective. Nevertheless, men still had lower prothrombin activity than females (108⫾18.8% vs. 119⫾17.9%, P⫽0.001) but no differences in prothrombin antigen concentration (107⫾22.5 mg/L vs. 108⫾22.0 mg/L, P⫽0.684) or prothrombin ratio (99⫾8.1% vs. 101⫾8.7%, P⫽0.277). Age was weakly correlated with prothrombin activity in both males and females (r⫽0.304, P⫽0.001 and r⫽0.241, P⫽0.055, respectively). A correlation between age and prothrombin concentration was found in females but not in males (r⫽0.399, P⫽0.001 vs. r⫽0.136, P⫽0.127).
n.c. 82–164 122⫾30.7 121–141 n.c., not computed due to small sample size. a p⫽0.034 for gender difference. b p⫽0.017 for difference to wild type allele carriers.
99⫾8.4 6
92–114
n.c.
131⫾7.1b
n.c.
67–148 68–146 64–151 57–169 65–160 57–169 107⫾20.6 107⫾20.0 107⫾22.1 66–161 66–161 81–157 101⫾8.3 101⫾8.1 101⫾8.7
All 20210 wild type including: females males All G20210A mutation carriers (5 males, 1 female)
185 122 63
85–132 85–129 87–132
85–117 85–117 84–118
114⫾18.3a 112⫾18.1 119⫾18.0a
78–150 77–148 84–154
95% interval Range Mean⫾SD 95% interval Range
Prothrombin activity (%)
Mean⫾SD 95% interval Range Mean⫾SD
The prothrombin concentration is usually given in arbitrary units, where one unit is assigned to the prothrombin antigen present in one millilitre of pooled normal plasma [4,10,11]. We used commercially available prothrombin to calibrate our assay in mg/L. The prothrombin concentrations as determined by ELISA in 191 healthy blood donors are in good agreement with those reported previously measured by ELISA in a smaller group (n⫽22) [19]. Other investigations using RIA [13] or electroimmunodiffusion [20] yielded comparable results. In both the whole group and after age matching, a significant influence of gender on prothrombin activity but not on prothrombin concentration was found. While the former finding may not be surprising considering that women are more susceptible to thrombosis than men [21], the latter finding is somewhat unexpected. However, it is in line with
n
3. Discussion
Quick (%)
In 125 samples with the highest prothrombin activity, we measured the F1⫹2 concentration as indicators of potentially activated coagulation. In 13 of 125 (10.4%) samples we found elevated F1⫹2 concentrations. F1⫹2 concentrations were ⬎5.5 nmol/L, i.e. five times the upper range of normal, in only 4 of these 13 samples. Highlighting these 13 samples within the activity/concentration graph representing all 125 samples revealed that samples with elevated F1⫹2 concentration were scattered all over the graph (data not shown). There was a correlation between F1⫹2 and prothrombin activity (r⫽0.446, p⬍0.001) and prothrombin concentration (r⫽0.177, P⫽0.048). Out of 191 investigated cases, 6 subjects (~3%) were heterozygous for the mutant prothrombin allele G20210A. These cases had significantly higher prothrombin activity (131⫾7.1%) than those individuals homozygous for the wild type (activity 114⫾18.3%, P⫽0.017). Although the mean prothrombin concentration was higher in the cases carrying the mutation (122⫾30.7 vs. 107⫾20.6, P⫽0.245), the difference was not significant (Table 1). There was no significant correlation between prothrombin activity and concentration (n⫽6, r⫽0.200, P⫽0.704) in the subjects with the mutation.
Prothrombin concentration (mg/L)
N. von Ahsen et al./Thrombosis Research 99 (2000) 549–556
Table 1. Prothrombin activity and concentration in a total of 191 healthy blood donors
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Fig. 1. Prothrombin activity vs. concentration in a total of 191 healthy blood donors grouped by gender and prothrombin G20210A genotype.
the results of Simioni and colleagues [11] who found no gender difference in prothrombin concentration even after exclusion of women on oral contraceptive treatment or in pregnancy. In coagulation analyses, a factor activity is usually reported instead of the antigen concentration [22]. However, the correlation between prothrombin activity and concentration was only weak for wild type allele carriers of both genders and absent in G20210A heterozygotes (Figure 1). The coefficient of determination was very low (0.06 and 0.13 in males and females, respectively), so other factors must be responsible for the observed variance in the activity levels at a given prothrombin concentration. Previous reports found correlations between prothrombin activity and concentration, but either evaluated only a small number of individuals (n⫽22) [19] or reported spurious correlations after including both patients under warfarin therapy and healthy subjects in the analysis [13,14]. In the lower right quadrant of Figure 1, samples with relatively high prothrombin concentration but low activity are found. These may be dysfunctional variants of prothrombin, a well-known phenomenon. However, we also observed samples with a relatively high activity for a given concentration (hyperactive prothrombin). This is a new finding which we could not trace back to methodological problems. To rule out significant preactivation of coagulation, we measured F1⫹2. The F1⫹2 con-
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centration is a sensitive marker of thrombin generation, known to increase, e.g., after unsuccessful venepuncture [23]. The F1⫹2 concentration was elevated in only 10% of the samples. These were non-systematically scattered all over the data pool. Interestingly, the F1⫹2 concentrations are known not to be influenced by the presence of the prothrombin G20210A mutation [12]. Since circadian rhythms have been shown to modulate coagulation factor activities including that of prothrombin in rats [24] and, less clearly, in humans [25,26], blood sampling in the present study was performed in the afternoon in all subjects. The biological variation of prothrombin activity itself is small within healthy individuals [27]. Therefore, repeated sampling would not be expected to significantly alter the present findings. Considering the poor correlation of prothrombin activity and concentration in healthy subjects with or without the G20210A mutation, one may speculate on the existence of posttranscriptionally modified immunoreactive prothrombin molecules with different biological activity. The prevalence of the G20210A mutation in our sample was ~3%, which is in the upper range of what is to be expected according to a large multicenter study [28]. We found the G20210A mutation in 5 males and only 1 female. The gender ratio is most likely due to the study size and the greater number of males in the sample population. In the present study, plasma prothrombin activity of healthy subjects with the mutant allele was significantly higher than in those without, as previously reported [4,7,9,10]. Prothrombin antigen concentrations were also elevated in carriers of the mutation compared to those without, but this elevation did not reach statistical significance, possibly due to the small number of mutation carriers evaluated. Elevated antigen concentrations in carriers of the prothrombin G20210A variant have been reported [4,11]. Nevertheless, even homozygous carriers of the G20210A mutation can have normal or only slightly elevated prothrombin concentrations [11] and may not develop a thrombosis during their lives even if exposed to triggering factors [29]. The genotype–phenotype association of the non-coding G20210A mutation is not as clear cut as the association between activated protein C resistance and the factor V Leiden mutation.
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Elevated prothrombin levels themselves are a risk factor for thrombosis; as many as 20% of individuals have elevated prothrombin levels [4,10]. The influence of the G20210A mutation on prothrombin activity is often estimated by comparing patients with thrombosis according to their genotype, but not necessarily as controls and patients [7,9]. This may lead to misinterpretation if factors apart from the known G20210A polymorphism regulate prothrombin concentration and/or activity and may trigger the manifestation of thrombosis. Only 1 study so far has compared prothrombin antigen levels in symptomatic and asymptomatic cases with and without the G20210A mutation [11] It found elevated concentrations in carriers of the G20210A mutation compared with wild type carriers, but no difference among G20210A carriers with or without thrombosis. Prospective outcome studies are needed to determine the value of prothrombin activity and concentration measurements in the thrombotic patient, especially as the pathomechanism related to the G20210A mutation which promotes thrombosis is not understood [30]. In conclusion, the present study provides prothrombin activity and concentration intervals for subjects with the prothrombin 20210 wild type allele. Only a weak correlation between prothrombin concentration and activity in 191 healthy subjects, including 6 individuals with the G20210A mutation, was found. The coefficient of determination for the interdependency of prothrombin activity and concentration was low. We hypothesise that posttranscriptionally modified prothrombin species may explain the observed functional diversity of this factor. We thank Professor Victor W. Armstrong for his helpful comments on the manuscript, Peter Lange for his help in setting up the prothrombin ELISA and Sandra Hartung for excellent technical assistance in genotyping, prothrombin time and activity measurements, and the F1⫹2 determination.
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ORIGINAL ARTICLE
Prothrombin Activity and Concentration in Healthy Subjects with and without the Prothrombin G20210A Mutation Nicolas von Ahsen1, Piotr Lewczuk2,3, Ekkehard Schu¨tz1, Michael Oellerich1 and Hannelore Ehrenreich3 Department of Clinical Chemistry, Georg-August-University, Robert Koch Str. 40, 37075 Goettingen, Germany, 2Department of Pediatric Neurology, Medical Academy of Bialystok, Bialystok, Poland, and 3Departments of Neurology and Psychiatry, Georg-August-University, Robert Koch Str. 40, 37075 Goettingen and Max-Planck Institute for Experimental Medicine, Goettingen, Germany 1
(Received 17 September 1999 by Editor D.L. Heene; revised/accepted 25 April 2000)
Abstract A common mutation in the prothrombin gene (G20210A) is associated with elevated prothrombin levels and thrombosis. The pathomechanism related to the G20210A mutation is currently not understood and the interdependence of prothrombin activity and prothrombin concentration in plasma is still poorly defined. Six of 191 blood donors examined in the present study carried the prothrombin allele G20210A. Despite the small number of cases, plasma samples from these individuals had significantly higher prothrombin activities than wild type carriers (131⫾7.1% vs. 114⫾18.3%, P⫽0.017), whereas their prothrombin concentrations—although elevated—did not differ significantly from wild type (122⫾30.7 mg/L vs. 107⫾20.6 mg/L, P⫽0.245). In subjects with the G20210A mutation there was also no significant correlation between prothrombin activity and concentration (n⫽6, r⫽0.200, P⫽0.704). Analyzing data from healthy blood donors without the prothrombin G20210A mutation (n⫽185), we found only weak correlations between prothrombin activAbbreviations: Prothrombin fragment 1⫹2, F1⫹2. Corresponding author: Nicolas von Ahsen, Georg-August University, Dept. Clinical Chemistry, Robert-Koch-Str. 40, 37075 Goettingen, Germany. Fax: ⫹49 (551) 39 8551; E-mail:⬍nahsen@ gwdg.de⬎.
ity and concentration of immunoreactive prothrombin (r⫽0.287; p⬍0.001). Samples with a relatively high prothrombin concentration but low activity were observed as well as samples with a relatively high activity for a given concentration (hyperactive prothrombin). F1⫹2 concentrations as indicators of activated coagulation were only elevated in 13 of 125 investigated samples and could not explain any of these findings. Dysfunctional variants of prothrombin, a well known phenomenon, may be responsible for the former, and we speculate that posttranscriptionally modified prothrombin species may explain the observed functional diversity of this factor including hyperactivity. The genotype–phenotype association of the non-coding G20210A mutation is not clear cut. Therefore, further studies are needed to determine which factors apart from the known G20210A polymorphism regulate prothrombin concentration and/or activity and may trigger the manifestation of thrombosis. 2000 Elsevier Science Ltd. All rights reserved. Key Words: Prothrombin activity; Immunoreactive prothrombin concentration; Prothrombin mutation; Prothrombin fragment 1⫹2; Reference interval; Pathomechanism
P
rothrombin, the precursor of thrombin, plays a central role in blood coagulation, participating in the process of clotting and platelet activation. In addition, prothrombin is in-
0049-3848/00 $–see front matter 2000 Elsevier Science Ltd. All rights reserved. PII S0049-3848(00)00281-4
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volved in a number of other physiologic mechanisms, e.g., in the central nervous system where it acts on cell proliferation and differentiation [1,2]. Prothrombin abnormalities have traditionally been associated with rare bleeding disorders caused by hypo- or dysprothrombinemia. The former are characterized by reduced prothrombin concentrations, the latter by reduced activity and concentration [3]. In 1996, the first mutation (G20210A) in the 3⬘ untranslated region of the prothrombin gene associated with elevated levels of prothrombin immunoreactivity and activity [4] was reported. Several studies have been able to confirm an association of this common mutation with arterial [5] or venous [4,6–10] thrombotic events. The mutation has been found to lead to significantly elevated prothrombin activity [4,7,9,10] and prothrombin plasma concentrations [4,11]. There is no ongoing thrombin generation even in patients homozygous for this mutation as judged by prothrombin fragment 1⫹2 (F1⫹2) concentration, a sensitive marker of coagulation activation [12]. Except for patients under warfarin therapy [13,14] and patients with thrombophilia [4], the interdependence of prothrombin activity and concentration in plasma of healthy subjects is still poorly defined. The present study was designed to determine reference intervals for immunoreactive prothrombin levels in a large number of healthy individuals, using a sensitive and highly specific ELISA, and to compare prothrombin antigen to prothrombin activity in this collective, under consideration of the G20210A genotype.
1. Materials and Methods The study was performed in accordance with the ethics guidelines of the Georg-August-University, Goettingen. Blood samples from 191 healthy blood donors without history of prior thrombosis were provided anonymously by the local blood bank. Samples were drawn in the afternoon from an antecubital vein in non-fasting state before the blood donation. Citrated plasma (0.106 mmol/L trisodium citrate) was immediately separated by 10 minutes of centrifugation (2500⫻g) in a precooled centrifuge (15⬚C), and the prothrombin time was
measured. Aliquots of the plasma were frozen at –80⬚C until further analysis.
1.1. Prothrombin Time Measurement The prothrombin time (Quick) was measured with an automated coagulation system analyser (BCS, Dade Behring GmbH, Marburg, Germany) using Thromborel S reagent (Dade Behring GmbH, Marburg, Germany). Calibration was performed using a standard-human plasma dilution series as per the manufacturer’s instructions (Dade Behring GmbH, Marburg, Germany).
1.2. Prothrombin Activity Measurement Prothrombin activity was measured using prothrombin deficient plasma with a remaining prothrombin activity⬍1% (Dade Behring GmbH, Marburg, Germany) on an automated coagulation system analyser (BCS, Dade Behring GmbH, Marburg, Germany). Calibration was performed with a standard human normal plasma dilution series according to the manufacturer’s instructions (Dade Behring GmbH, Marburg, Germany). The standard was diluted automatically by the instrument. For the assay itself, 30 L of prothrombin deficient plasma and 5 L of test plasma were mixed with 150 L prothrombin reagent (Thromborel S, Dade Behring GmbH, Marburg, Germany) and the time until clotting occurred was measured by the analyser. The coefficient of variation was⬍6% for intra- and inter-series. We assayed the dilution recovery of several samples with both high and low prothrombin activity and high and low prothrombin concentration, and found no significant deviation from linearity with this method (data not shown).
1.3. Determination of Prothrombin in Plasma Using ELISA (Enzyme-linked Immuno Sorbent Assay) Prothrombin concentration was measured according to a published method [15,16]. Briefly, 100 L of goat anti-human prothrombin antibody (ICN Biomedicals, USA) diluted 1:400 in carbonate buffer (pH 9.6) was added to 96 wells of an ELISA plate (MaxiSorp, Nunc, Germany) and incubated overnight in⫹4⬚C on a shaker. On the next day
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wells were washed 3 times with washing buffer [17]. Plasma samples were diluted 1:8000 in assay buffer [17]. To obtain data considering a possible crossreactivity, human thrombin (Sigma, USA) was applied undiluted and diluted 1:2 in the assay buffer. Standard (ICN Biomedicals, USA) was diluted in this same buffer to obtain the following concentrations: 35 g/L, 17.5 g/L, 9.0 g/L, 4.5 g/L, 2.25 g/L, and 1.12 g/L. 100 L of the diluted plasma or the standard were added to wells in duplicate and the plate was incubated for 1 hour at room temperature, shaking. Wells were then washed 3 times in washing buffer, and 100 L of sheep antihuman prothrombin antibody conjugated with HRP peroxidase (Biogenesis, UK) diluted 1:750 in the assay buffer was added to all wells. The plate was incubated for 1 hour at room temperature on a shaker, washed 6 times in the washing buffer, and 100 L of HRP substrate [17] was added to all wells. After 15 minutes of incubation at room temperature, shaking, the reaction was stopped with 100 L of 2N sulphuric acid. The optical density was measured with an automatic ELISA reader (SLT, Germany) set at 450 nm with a correction wavelength of 620 nm. The detection limit of the assay is 0.7 g/L, the coefficient of variation is⬍9% for intra- and inter-series.
1.4. Determination of F1⫹2 in Plasma F1⫹2 plasma concentrations were determined using a commercial ELISA (Enzygnost威 F1⫹2 micro, Dade Behring GmbH, Marburg, Germany) according to the manufacturer’s instructions. Plates were read at 490 nm on a micro plate reader (Millenia Kinetic Analyzer, DPC, Germany) and results were analyzed using a four parameter fit for the standard curve. The reference interval of this assay is 0.4–1.1 nmol/L (5–95th percentile) according to the manufacturer.
1.5. Genotyping the Prothrombin G20210A Mutation For genotyping of the G20210A mutation we used a recently described method [18]. Briefly, genomic DNA was isolated from whole blood using standard procedures. PCR reactions were performed on a LightCycler (Roche Molecular Diagnostics, Mannheim, Germany). The method uses hybridisa-
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tion of labeled oligonucleotide probes and yields a single melting peak at ~57⬚C for the wildtype DNA. In cases with homozygous mutations there is a mismatch under the wildtype DNA compatible probe which leads to strand instability and consecutive earlier melting with a single melting peak at ~50⬚C. Accordingly, patients with heterozygous mutations show two melting peaks. All genotypes can be clearly distinguished.
1.6. Statistics Statistics were computed using SPSS 7.5.2 for Windows and Analyze-It for Excel. Normal distribution was assessed by the Shapiro–Wilk test. Group comparisons were done with the Mann–Whitney U-test. Correlations were calculated using the Spearman rank–sum correlation. The S.D. is used as a measure of variation throughout the study.
2. Results Data on prothrombin time (Quick), activity, and concentration in plasma of 191 healthy blood donors are summarised in Table 1. Weak correlations were detected between prothrombin activity and concentration in 127 males (r⫽0.252, r2⫽0.06, P⫽0.004) and 64 females (r⫽0.362, r2⫽0,13, P⫽0.003) (Figure 1). There was a statistically significant gender difference for prothrombin activity (p⫽0.034) but not for prothrombin concentration (p⫽0.980) or for prothrombin ratio (p⫽0.950). Since in the present study men were on average significantly older than women (34⫾11.5 vs. 30⫾11.8 years, P⫽0.001), we repeated the calculation after age-matching 64 men with the 64 women in our collective. Nevertheless, men still had lower prothrombin activity than females (108⫾18.8% vs. 119⫾17.9%, P⫽0.001) but no differences in prothrombin antigen concentration (107⫾22.5 mg/L vs. 108⫾22.0 mg/L, P⫽0.684) or prothrombin ratio (99⫾8.1% vs. 101⫾8.7%, P⫽0.277). Age was weakly correlated with prothrombin activity in both males and females (r⫽0.304, P⫽0.001 and r⫽0.241, P⫽0.055, respectively). A correlation between age and prothrombin concentration was found in females but not in males (r⫽0.399, P⫽0.001 vs. r⫽0.136, P⫽0.127).
n.c. 82–164 122⫾30.7 121–141 n.c., not computed due to small sample size. a p⫽0.034 for gender difference. b p⫽0.017 for difference to wild type allele carriers.
99⫾8.4 6
92–114
n.c.
131⫾7.1b
n.c.
67–148 68–146 64–151 57–169 65–160 57–169 107⫾20.6 107⫾20.0 107⫾22.1 66–161 66–161 81–157 101⫾8.3 101⫾8.1 101⫾8.7
All 20210 wild type including: females males All G20210A mutation carriers (5 males, 1 female)
185 122 63
85–132 85–129 87–132
85–117 85–117 84–118
114⫾18.3a 112⫾18.1 119⫾18.0a
78–150 77–148 84–154
95% interval Range Mean⫾SD 95% interval Range
Prothrombin activity (%)
Mean⫾SD 95% interval Range Mean⫾SD
The prothrombin concentration is usually given in arbitrary units, where one unit is assigned to the prothrombin antigen present in one millilitre of pooled normal plasma [4,10,11]. We used commercially available prothrombin to calibrate our assay in mg/L. The prothrombin concentrations as determined by ELISA in 191 healthy blood donors are in good agreement with those reported previously measured by ELISA in a smaller group (n⫽22) [19]. Other investigations using RIA [13] or electroimmunodiffusion [20] yielded comparable results. In both the whole group and after age matching, a significant influence of gender on prothrombin activity but not on prothrombin concentration was found. While the former finding may not be surprising considering that women are more susceptible to thrombosis than men [21], the latter finding is somewhat unexpected. However, it is in line with
n
3. Discussion
Quick (%)
In 125 samples with the highest prothrombin activity, we measured the F1⫹2 concentration as indicators of potentially activated coagulation. In 13 of 125 (10.4%) samples we found elevated F1⫹2 concentrations. F1⫹2 concentrations were ⬎5.5 nmol/L, i.e. five times the upper range of normal, in only 4 of these 13 samples. Highlighting these 13 samples within the activity/concentration graph representing all 125 samples revealed that samples with elevated F1⫹2 concentration were scattered all over the graph (data not shown). There was a correlation between F1⫹2 and prothrombin activity (r⫽0.446, p⬍0.001) and prothrombin concentration (r⫽0.177, P⫽0.048). Out of 191 investigated cases, 6 subjects (~3%) were heterozygous for the mutant prothrombin allele G20210A. These cases had significantly higher prothrombin activity (131⫾7.1%) than those individuals homozygous for the wild type (activity 114⫾18.3%, P⫽0.017). Although the mean prothrombin concentration was higher in the cases carrying the mutation (122⫾30.7 vs. 107⫾20.6, P⫽0.245), the difference was not significant (Table 1). There was no significant correlation between prothrombin activity and concentration (n⫽6, r⫽0.200, P⫽0.704) in the subjects with the mutation.
Prothrombin concentration (mg/L)
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Table 1. Prothrombin activity and concentration in a total of 191 healthy blood donors
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Fig. 1. Prothrombin activity vs. concentration in a total of 191 healthy blood donors grouped by gender and prothrombin G20210A genotype.
the results of Simioni and colleagues [11] who found no gender difference in prothrombin concentration even after exclusion of women on oral contraceptive treatment or in pregnancy. In coagulation analyses, a factor activity is usually reported instead of the antigen concentration [22]. However, the correlation between prothrombin activity and concentration was only weak for wild type allele carriers of both genders and absent in G20210A heterozygotes (Figure 1). The coefficient of determination was very low (0.06 and 0.13 in males and females, respectively), so other factors must be responsible for the observed variance in the activity levels at a given prothrombin concentration. Previous reports found correlations between prothrombin activity and concentration, but either evaluated only a small number of individuals (n⫽22) [19] or reported spurious correlations after including both patients under warfarin therapy and healthy subjects in the analysis [13,14]. In the lower right quadrant of Figure 1, samples with relatively high prothrombin concentration but low activity are found. These may be dysfunctional variants of prothrombin, a well-known phenomenon. However, we also observed samples with a relatively high activity for a given concentration (hyperactive prothrombin). This is a new finding which we could not trace back to methodological problems. To rule out significant preactivation of coagulation, we measured F1⫹2. The F1⫹2 con-
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centration is a sensitive marker of thrombin generation, known to increase, e.g., after unsuccessful venepuncture [23]. The F1⫹2 concentration was elevated in only 10% of the samples. These were non-systematically scattered all over the data pool. Interestingly, the F1⫹2 concentrations are known not to be influenced by the presence of the prothrombin G20210A mutation [12]. Since circadian rhythms have been shown to modulate coagulation factor activities including that of prothrombin in rats [24] and, less clearly, in humans [25,26], blood sampling in the present study was performed in the afternoon in all subjects. The biological variation of prothrombin activity itself is small within healthy individuals [27]. Therefore, repeated sampling would not be expected to significantly alter the present findings. Considering the poor correlation of prothrombin activity and concentration in healthy subjects with or without the G20210A mutation, one may speculate on the existence of posttranscriptionally modified immunoreactive prothrombin molecules with different biological activity. The prevalence of the G20210A mutation in our sample was ~3%, which is in the upper range of what is to be expected according to a large multicenter study [28]. We found the G20210A mutation in 5 males and only 1 female. The gender ratio is most likely due to the study size and the greater number of males in the sample population. In the present study, plasma prothrombin activity of healthy subjects with the mutant allele was significantly higher than in those without, as previously reported [4,7,9,10]. Prothrombin antigen concentrations were also elevated in carriers of the mutation compared to those without, but this elevation did not reach statistical significance, possibly due to the small number of mutation carriers evaluated. Elevated antigen concentrations in carriers of the prothrombin G20210A variant have been reported [4,11]. Nevertheless, even homozygous carriers of the G20210A mutation can have normal or only slightly elevated prothrombin concentrations [11] and may not develop a thrombosis during their lives even if exposed to triggering factors [29]. The genotype–phenotype association of the non-coding G20210A mutation is not as clear cut as the association between activated protein C resistance and the factor V Leiden mutation.
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Elevated prothrombin levels themselves are a risk factor for thrombosis; as many as 20% of individuals have elevated prothrombin levels [4,10]. The influence of the G20210A mutation on prothrombin activity is often estimated by comparing patients with thrombosis according to their genotype, but not necessarily as controls and patients [7,9]. This may lead to misinterpretation if factors apart from the known G20210A polymorphism regulate prothrombin concentration and/or activity and may trigger the manifestation of thrombosis. Only 1 study so far has compared prothrombin antigen levels in symptomatic and asymptomatic cases with and without the G20210A mutation [11] It found elevated concentrations in carriers of the G20210A mutation compared with wild type carriers, but no difference among G20210A carriers with or without thrombosis. Prospective outcome studies are needed to determine the value of prothrombin activity and concentration measurements in the thrombotic patient, especially as the pathomechanism related to the G20210A mutation which promotes thrombosis is not understood [30]. In conclusion, the present study provides prothrombin activity and concentration intervals for subjects with the prothrombin 20210 wild type allele. Only a weak correlation between prothrombin concentration and activity in 191 healthy subjects, including 6 individuals with the G20210A mutation, was found. The coefficient of determination for the interdependency of prothrombin activity and concentration was low. We hypothesise that posttranscriptionally modified prothrombin species may explain the observed functional diversity of this factor. We thank Professor Victor W. Armstrong for his helpful comments on the manuscript, Peter Lange for his help in setting up the prothrombin ELISA and Sandra Hartung for excellent technical assistance in genotyping, prothrombin time and activity measurements, and the F1⫹2 determination.
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