International Journal of Laboratory Hematology The Official journal of the International Society for Laboratory Hematology
ORIGINAL ARTICLE
INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY
The effect of recombinant and plasma-derived prothrombin on prothrombin time in human plasma 1 € K. M. HANSSON, J. BJ ORKQVIST , J. DEINUM
Department of Bioscience, CVMD iMED, AstraZeneca R&D M€ olndal, M€ olndal, Sweden Correspondence: Kenny M. Hansson, Department of Bioscience, AstraZeneca R&D M€ olndal, S-43183 M€ olndal, Sweden. Tel.: +46 31 7065371; Fax: +47 31 7763859; E-mail: kenny.m.hansson @astrazeneca.com 1
Present address: Department of Molecular Medicine and Surgery and Center of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
doi:10.1111/ijlh.12293
Received 12 May 2014; accepted for publication 11 August 2014 Keywords Prothrombin time, prothrombin, tissue factor, plasma coagulation, calcium
S U M M A RY Introduction: When investigating coagulation assays to measure the effect of infused prothrombin (FII) in in vivo coagulopathy models, we found that addition of FII, plasma-derived human FII (pd-hFII) or recombinant human FII (r-hFII), to normal plasma resulted in a concentration-dependent increase in prothrombin time (PT) initiated with Innovinâ. Methods: The effect on PT by addition to plasma of either pd-hFII or r-hFII, using different commercial PT reagents, was studied both by turbidimetry and viscometry. Result: Addition of FII to plasma resulted in increased PT when initiated with Innovinâ: PT increased with 20% by doubling the concentration. The prolongation of PT became more pronounced with 2–6000 times diluted Innovinâ. However, by adjustment of the final free Ca2+ concentration in the assay with diluted Innovinâ from 8.3 to 1.3 mmol/L, no FII-dependent increase in PT was found. In contrast, no prolongation of PT was found with other commercial PT reagents. A KM= 3 nmol/L was obtained with pd-hFII, respectively, r-hFII with FII-depleted plasma using Thromborelâ to initiate PT. Conclusion: At normal plasma concentration of FII, addition of FII should not have an effect on PT. The prolonged PT with Innovinâ, but not with other PT reagents, at supranormal FII concentration is an artefact.
INTRODUCTION Two parallel pathways of coagulation factors, the so-called extrinsic and intrinsic pathway, can initiate plasma coagulation [1, 2]. In both pathways, the completion of each step activates another coagulation factor in a chain reaction and meet in the common pathway generating thrombin that subsequently © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 357–364
converts fibrinogen to fibrin [3]. In the coagulation process, all the crucial steps are dependent on the presence of free Ca2+ so that the vitamin K-dependent coagulation factors, containing gamma-carboxylated glutamic acid residues, can anchor on the negatively charged procoagulant membrane surfaces, exposing phospholipids (PL), as found on cell surfaces such as on damaged endothelial cells and activated platelets. 357
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The most common haemostatic assays used in the clinical practice are coagulation assays. To initiate fibrin formation, id est coagulation, only a nanomolar thrombin concentration is needed. One of the commonly used coagulation assays is the Prothrombin Time (PT) assay, alternatively called the Quick Prothrombin time [4]. All commercial PT reagents contain a mixture of an activator, tissue factor (TF) together with PL or membrane fragments, and can vary widely in their sensitivity to reductions in the levels of different coagulation factors. PT is the most common laboratory assay used to monitor oral anticoagulant therapy. PT is also used to screen for abnormalities in the extrinsic system and to perform quantitative assays of one or more factors of the system. However, relative sensitivities to changes in the plasma levels of factors FV, FVII, FX and prothrombin (FII) have been found to be differentially influenced by the content and composition of the PL [5] and the NaCl concentration [6]. Thus, the determination of the prothrombin time (PT) is based on an artificial in vitro system with major limitations. Nevertheless, these tests are quite useful as global screening tests for abnormalities in the pathways of coagulation [7]. In vivo, the free Ca2+ concentration in plasma is 1–1.5 mmol/L [8]. Most PT reagents contain an excess of CaCl2 to compensate for the presence of citrate present as an anticoagulant in the plasma samples. Moreover, as the citrate concentration in plasma obtained in blood samples from patients is primarily determined by the individual haematocrit and variability in filling level in sample tubes, these assays in general use a Ca2+ concentration that in the final assay results in a free Ca2+ plasma concentration that is far higher than in vivo levels to ascertain that Ca2+ levels below 1 mmol/L should be avoided. This results in that coagulation analyses using blood that has been exposed to citrate and is recalcified do not yield reliable depictions of the natural dynamics of blood coagulation processes [9]. Different FII concentrations have previously been investigated as an antidote for thrombin inhibitors [10, 11] in animal models and have also been evaluated with different coagulation assays. Moreover, reliable commercial coagulation assays are needed to measure the effect of infusion of FII in in vivo coagulopathy models. Preliminary results had shown that the PT of normal plasma was slightly increased instead
of decreased or unchanged in the presence of higher FII concentrations, applying the Innovinâ PT reagent. Therefore, we studied the effect of addition of FII in different commercial PT reagents; using both plasmaderived human FII (pd-hFII) or recombinant human FII (r-hFII) both on PT and diluted PT.
M AT E R I A L S A N D M E T H O D S Chemicals and buffers All chemicals were of reagent grade. A stock solution eres, France). of CaCl2 in water was from Stago (Asni Bovine serum albumin (BSA) 20% (w/v) solution in water and the phospholipid emulsion containing synthetic sources of phosphatidylserine, phosphatidylcholine and sphingomyelin (PL-TGT, 0.5 mmol/L) were from Rossix AB (M€ olndal, Sweden). All solutions were made with deionized water that was further purified on an Elgastadt UHP (Elga Ltd., High Wycombe Bucks, England). Fysalb consisted of 9 g/L NaCl solution in water from Fresenius (Kabi, Halden, Norway) and 1% (w/v) BSA. Ten-time-concentrated HBS-N buffer from GE Healthcare Lifesciences, Uppsala, Sweden) was diluted to HBS-N buffer consisting of 10 mmol/L Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and 0.15 mol/L NaCl at pH 7.4. Platelet-poor plasma Three different pools of platelet-poor plasma were prepared by centrifugation of fresh, citrated blood at 2000 9 g in a swing-out rotor for 20 min at 20 °C. For each plasma pool, blood was collected from 30 healthy volunteers employed by AstraZeneca R&D M€ olndal, Sweden (9 volumes blood with one volume 129 mmol/ L trisodium citrate). The plasma supernatant was immediately put on ice, pooled, aliquoted and stored at 80 °C. Fresh frozen immune-depleted human FII deficient plasma was purchased from Haematologic Technologies Inc., Essex Junction, VT, USA. Plasma from a haemophilia A patient was purchased from George King, Bio-Medical Inc., Overland Park, KS, USA. Just before an experiment, the plasma samples were rapidly thawed in a water bath at 37 °C and kept at 37 °C for at least 30 min. The mean normal plasma concentration of FII is 1.4 lmol/L FII [12, 13], which equals 100 IU/dL. © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 357–364
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Prothrombin Different batches of lyophilized pd-hFII were obtained from Enzyme Research Laboratories, (South Bend, IN, USA). Correctly gamma-carboxylated r-hFII was produced by AstraZeneca. The protein concentration was determined applying e280=102/mM/cm for human FII [14]. FII was kept stored at –80 °C and was rapidly thawed in a 37 °C water bath on the day of the experiment and then immediately transferred to an ice bath and further diluted with ice-cold Fysalb to a final concentration of 24 lmol/L. Further dilutions were also made with Fysalb. Fysalb was used as blank/control in all experiments. Solutions containing BSA were freshly prepared on the day of the experiment. General experimental design for PT and diluted PT The used commercial PT reagents, containing TF and phospholipids, respectively, membrane fragments, were three different lots of Innovinâ and Thromborelâ from Dade Behring (Marburg, Germany) Simplastinâ from Organon Teknika BV (Oss, The Netherlands), RecombiPlasTinâ from Instrumentation Laboratories Co (Lexington, KY, USA) STA-Neoplastineâ from Diagnostic Stago (Asni eres, France) and TrinityCLOT HTFâ from Trinity Biotech (Jamestown, NY, USA). The PT reagents were reconstituted and the assay was performed at 37 °C as recommended by the manufacturer by addition of two volumes reagent to one volume plasma to initiate coagulation. In most commercial PT assay kits, a CaCl2 concentration is used, which results in a final free Ca2+ concentration that differs from the physiological 1–1.5 mmol/L. Therefore, in some experiments with diluted Innovinâ, the CaCl2 content was adjusted so that in the final assay in the serum after fulfilled coagulation the concentration of free Ca2+ became 1.3 mmol/L, alternatively 8.3 mmol/L. The free Ca2+ concentration was measured with a calcium ion-selective electrode included in a Radiometer ABL700 analysis instrument (Radiometer, Copenhagen, Denmark). To study the effect of lower TF concentrations, a bottle with lyophilized Innovinâ PT was reconstituted with 10 mL water to 6 nmol/L TF and if needed, further diluted 2, 10, 100, 1000 and 6000 times with © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 357–364
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HBS-N buffer containing different CaCl2 concentrations and to compensate for the dilution of the reagents, additional PL were added to plasma to a final assay concentration of 4 lmol/L. Before start of the coagulation assays, first 1 volume Fysalb solution, containing FII and extra PL were pre-incubated with 9 volumes plasma at 37 °C for 30 min, before initiation by addition of 20 volumes of the diluted TF reagent with CaCl2. Coagulation measurements Coagulation times were measured applying two different physical phenomena related to the formation of the fibrin clot, viscometry and turbidimetry. Viscometry was used to determine the coagulation times by a ball coagulation timer KC 10 A Micro from Heinrich Amelung GmbH (Lemgo, Germany). Turbidimetry was used to monitor the whole coagulation curves, from the apparent increase in optical absorbance at 405 nm, DA405, using a semi-automatic spectrophotometer, a BCS from Siemens Healthcare Diagnostics Inc. (Marburg, Germany). The coagulation time was determined from the coagulation curves using the BCS software, applying a DA405 threshold if not otherwise indicated.
R E S U LT S Comparison of PT reagents In contrast to viscometry, which is a physical method using manual pipetting for each sample and just provides an end-point measurement of the coagulation time, turbidimetry, measured from the DA405, can monitor the whole coagulation curve. With this method in the normal PT assay with Innovinâ, it was found that the whole coagulation curves of plasma were FII-concentration dependently parallel shifted in time, see Figure 1a, at higher plasma concentration of pd-hFII, indicating a prolonged coagulation time. The shift in the coagulation curves suggested a concentration-dependent inhibition of the Innovinâ initiated coagulation by FII. Similar effect on PT by addition of FII to plasma with Innovinâ was found with either rhFII or pd-hFII, suggesting that the observed effect on PT with the Innovinâ reagent did not depend on contamination in the pd-hFII sample with other plasma
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(a)
(b)
Figure 1. Effect of added FII on PT with Innovinâ by turbidimetry. Innovinâ was used as recommended by the manufacturer. Normal plasma (pool 1) 90 lL was mixed with 10 lL pd-hFII and incubated for 180 s. (a) The coagulation curves are labelled with the FII conc. in plasma before addition of 200 lL PT reagent Innovinâ at time 0 (final free Ca2+ 6 mmol/L). The data from each curve (each 0.5 s) from the BCS were used to calculate PT. (b) Mean PT coagulation times (n = 4) from the coagulation curves versus final assay conc. of added FII, using a threshold at DA405 = 200 mAbs.
proteins. Because of the very short coagulation time of less than 10 s, id est at approximately the first measuring point of the BCS, with the used plasma (pool 1), a modified evaluation algorithm with a higher cutoff of the DA405 was used to determine the PT, as illustrated in Figure 1b. However, with other plasma pools, a slightly higher PT was obtained with Innovinâ, making it possible to use the BCS algorithm straight forward. Also with other plasma pools, with a PT of above 10 s, similar apparent FII-concentrationdependent inhibition of PT, illustrated by parallel shifts in the coagulation curves, was observed. The apparent inhibition of PT by FII using Innovinâ seems to be unique since the increase of the plasma concentration of FII up to 300% of the normal level, that is addition of 2.8 lmol/L or 200 IU/dL, did not result in a prolongation effect on the coagulation curves with the other PT reagents assays containing either TF from tissue extract from rabbit brain (Simplastinâ), or from cultured human cells (TrinityCLOT HTFâ) or with two other reagents with recombinant TF and phospholipids (STA-Neoplastineâ or RecombiPlasTinâ). For all these other reagents, the PT defined by the BCS algorithm could be used and the coagulation curves did perfectly overlap and were indistinguishable from the control. The prolongation of PT Innovinâ by added FII was confirmed by viscometry. For example, in a typical
experiment with a normal plasma (pool 2) and Innovinâ PT was increased from 9.4 0.10 s to 10.5 0.025 s (mean PT standard error, n = 4) by an increase in the plasma concentration to 2.7 lmol/L by addition of pd-hFII, while no effect was found with the other commercial PT reagents. Also with this manual viscometry method, an increase in PT Innovinâ was found by addition of FII to plasma using different batches of pd-hFII or r-hFII or using other Innovinâ lots. No effect on PT of addition of prothrombin fragment 1 and 2 Prothrombin fragment 1 + 2, id est FII without its thrombin fragment, still has a high affinity for negatively charged PL [15] and as such compete with FII for binding to PL and the other Ca2+-dependent coagulation factors. However, addition to plasma of 4 lmol/L prothrombin fragment 1 + 2 had no effect on PT with Innovinâ although the final free Ca2+ concentration was as high as 6 mmol/L. PT with diluted Innovinâ and the concentration of Ca2+ In contrast to the other PT reagents, the final free serum Ca2+ concentration in the samples with Innovinâ was 6 mmol/L, which is far above the © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 357–364
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physiological plasma concentration. To further explore the effect of a high concentration of free Ca2+ Innovinâ was diluted 2, 10, 100, 1000 and 6000 fold, first in 10 ml water and further in water with 20 mmol/L CaCl2 and 6 lmol/L PL giving rise to final free Ca2+ concentration of 8.3 mmol/L in serum. Under these conditions, there was a strong TF concentrationdependent increase in coagulation time, see Figure 2a. Furthermore, for all TF dilutions a near-linear increase in coagulation time was observed with increasing plasma concentration of FII. Moreover, the same results were obtained when the final PL concentration was increased from 4 to 40 lmol/L. However, by changing the conditions so that the final free Ca2+ concentration was 1.3 mmol/L, similar to the physiological free serum concentration, there was no longer an increase in PT with diluted Innovinâ by increasing the plasma concentration of FII with 2.4 lmol/L, see Figure 2b. The assay with diluted Innovinâ at 1.3 mmol/L Ca2+ was repeated with different batches of pd-hFII and with r-hFII providing the same results. Apparently, the paradoxical effect on PT measured by diluted Innovinâ is not found at a physiological free Ca2+ concentration. The effect of the concentration of r-hFII or pd-hFII on the coagulation time was further compared using FII-depleted plasma and two PT reagents, Innovinâ (a)
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and Thromborelâ, using viscometry under conditions as recommended by the manufacturer of the reagents. As shown in Figure 3a,b with Innovinâ, banana shaped titration curves were found, consistent with apparent inhibition at higher concentrations of FII both for pd-hFII and r-hFII. The titration with Thromborelâ was as theoretically expected for simple Michaelis–Menten kinetics. The coagulation rate can be approximated to be proportional with the inverse coagulation time, assuming that at the time of coagulation, Dt, the same concentration of fibrin, the product, DP, is formed, if there is a linear relationship of the increase in turbidity and the fibrin concentration. Thus, the rate of fibrin formation becomes v = DP/ Dt. Then, the coagulation time PT is proportional with 1/v. Therefore, the KM could be estimated, applying the inverted equation of the Michaelis–Menten kinetics (PT=1/Vmax+(KM/Vmax)/[FII] as the result of the enzyme activity of the prothrombinase complex, with the substrate, FII, by nonlinear regression. From the data with Thromborelâ in Figure 3a,b an apparent KM= 3 and 2.9 nmol/L for pd-hFII, respectively, r-hFII was estimated by nonlinear regression.
DISCUSSION PT, a commonly used coagulation assay, is used as global screening test for abnormalities in the (b)
Figure 2. PT with diluted Innovinâ at different conc. of added pd-hFII and Ca2+. The coagulation time PT (n = 4) was derived from the coagulation curves from the BCS. The 50 lL mixture of 45 lL normal plasma (pool 2) with 6 lmol/L PL and 1.2 and 2.4 lmol/L added pd-hFII was pre-incubated for 30 min before initiation of coagulation by addition of 100 lL Innovinâ (a) The PT reagent Innovin â (6 nmol/L TF) was diluted 2, 10, 100, 1000 and 6000 fold with 20 mmol/L CaCl2 in HBS-N buffer with 6 lmol/L PL to final serum conc. of 8.3 mmol/L Ca2+ and 4 6 lmol/L PL (b) Innovinâ was diluted 10-, 100-, 1000- and 6000-fold with 7 mmol/L CaCl2 in HBS-N to a final free Ca2+ concentration of 1.3 mmol/L. The arrow points to the normal plasma conc. of FII. The error bars representing standard deviation are within the outlines of the symbols.
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(a)
(b)
Figure 3. Effect on PT by added FII by viscometry. PT was determined by first incubation for 180 s of 45 lL FIIdepleted plasma with 5 lL FII and initiated by addition of 100 lL PT reagent, Thromborelâ open circles and Innovinâ closed circles. Without added FII, there was no coagulation: (a) with pd-hFII and (b) r-hFII. Nonlinear regression was used to fit the reversed Michaelis–Menten equation 1/v = 1 + ([FII]/KM))/Vmax to the data with Thromborelâ providing KM= 3 0.2 resp. 2.9 0.1 nmol/L using 72 kD and the final assay conc. The data are mean PT SD values, n = 3.
pathways of coagulation [7]. However, one should be aware that the determination of PT is based on an artificial in vitro system with major limitations. All commercial PT reagents contain a mixture of a very high concentration of TF together with different types of anionic PL or membrane fragments. These different commercial PT tests have also been shown to be differentially influenced by the content of, among others, the composition of the PL [5] and the NaCl concentration [6]. In all PT assays, an excess of calcium is present to compensate for the presence of the Ca2+ chelator anticoagulant in the plasma samples, as free Ca2+ is needed for plasma to coagulate. In the coagulation process, all the crucial steps are dependent on the presence of free Ca2+ so that the vitamin K-dependent coagulation factors, containing gammacarboxylated glutamic acid residues, can anchor on the negatively charged procoagulant membrane surfaces, exposing PL. For example, regarding prothrombin, Ca2+ induces the conformation required for the expression of the PL-binding site in the binary prothrombin-metal complex [16] and requires two sequential metal-dependent conformational transitions to bind PL [16,17]. Previously, it had been shown that the PL composition and content is crucial for the prothrombinase activity [18, 19]. However, in most kinetic studies calcium concentrations of 5 mmol/L or above are used, allowing strong affinity of FII for anionic PL vesicles [15]. The present report
indicates that the role of calcium is more complicated. We found that the apparent inhibition by FII of PT in the assay with Innovinâ, over a large range of TF dilutions, disappeared when the final assay concentration of free Ca2+ was adjusted from 6 or 8 to 1.3 mmol/L. It is therefore surprising that a calcium concentration in this range should be inhibitory. It should also be noted that in this study, 129 nmol/L trisodium citrate was used as anticoagulant and not 109 nmol/L which is more commonly used. The use of 129 nmol/L might introduce a stronger effect on PT when sample tubes accidentally are underfilled compared with the same situation with 0.109 nmol/L. However, as not all commercial PT reagents did show this calcium-dependent effect, a general conclusion cannot be made. An increase in PT is consistent with the reported increase in lag time in the thrombin generation assay with increasing concentration of FII [20] both with 1 and 5 pmol/L TF. However, regarding the PT assay with Innovinâ, we can exclude membrane crowding [20] as an explanation for the apparent inhibition by FII as no effect was found by increasing the PL concentration 10-fold. The finding that added prothrombin fragment 1 and 2 in high concentrations had no effect on PT with Innovinâ further reinforced the conclusion that membrane crowding is not present. Thus, the surface area on the PL is not a limiting factor for the activity of FII in the PT assay with the Innovinâ reagent, as otherwise, also the presence of © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 357–364
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prothrombin fragment 1 + 2 should introduce this apparent inhibition. The prolongation of PT with the Innovinâ reagent was unique and was not observed with other commercial PT assays. Furthermore, similar results were obtained both with viscometry and turbidimetry both for pd-hFII and r-hFII. Moreover, the paradoxal effect was observed with different batches of Innovinâ. So, the Innovinâ effect might be dependent both on the free Ca2+ concentration, the type of phospholipid, the source of TF or a combination effect. It is out of the scope of this study to analyse the different commercial PT reagents more in detail. Preliminary studies have also shown that addition of FII to normal plasma or to platelet-poor plasma from a haemophilia A patient in the thrombin generation assay (Calibrated Automated Thrombogram) [21], using diluted Innovinâ, extended the lag time (not shown). Therefore, for clinical applications, other PT reagents than Innovinâ should be used to analyse plasma samples that might contain deviating FII concentrations. An apparent KM of 3 nmol/L FII in the prothrombinase complex, see Figure 3, is consistent of that obtained with large PL vesicles [22]. The initial rate of thrombin generation under physiological conditions in plasma with 1.4 lmol/L FII, far above the KM value, should thus not be dependent on the concentration of FII according to simple Michaelis–Menten kinetics. At the normal plasma concentration of FII, addition of FII should thus not have an effect. The activity of the prothrombinase complex in normal plasma can thus not be the rate limiting step, implying neither an increase nor decrease by a further increase in FII, as the maximal initial thrombin generation rate [23], and thus shortest coagulation time is already reached at normal FII plasma concentrations. In general, inhibition at high substrate concentrations suggests either the presence of a contaminant in the substrate preparation or secondary binding of FII to the PL surface. The inhibitory effect is unique for the Innovinâ reagent at high concentrations of free Ca2+ as this phenomenon was not found with the other commercial reagents. With different preparations both of pd-FII and r-hFII, moreover, with different plasma pools and several Innovinâ lots, the same phenomenon was found. Thus contamination with other factors can be excluded. However, further studies are needed to reveal the details of the process. © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 357–364
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The overall conclusion is thus that the increase in prothrombin time initiated with Innovinâ in plasma with increasing FII concentrations is an artefact, apparently unique for Innovinâ and among others dependent on the free Ca2+ concentration but not on the PL concentration. As the FII-dependent increase was even more pronounced at lower, for example picomolar TF concentrations, as used in the thrombin generation assays [21], this should be studied more in detail. It might be of interest, if possible, to adjust the final free Ca2+ concentration to a more physiological level closer to 1 to 1.5 mmol/L, not only in the PT assay, but also in other haemostasis assays. To avoid wrong conclusions analysing blood with deviating FII concentrations, clinicians should be aware of the fact that an increased PT measured with Innovinâ in combination with a suspicion of elevated FII concentrations can be an artefact and should apply other reagents. AS most reagents consist of a combination of different types of TF and PL, respectively, membrane fragments, a comparison should be made of different commercial PT reagents for a reliable assay. This finding is important for clinical measurement of PT in plasma samples containing unknown, possibly supranormal, concentrations of FII. This might be the situation in, for example, heterozygous carriers of the G20210 prothrombin gene mutation that confers a procoagulant lifelong increase in FII concentrations of up to 30% compared to normal levels [24]. Moreover, during treatment with FII containing plasma concentrates (prothrombin complex concentrates) for bleeding disorders repeated dosing can result in concentrations of FII well above normal level [25] as it may also happen in a future treatment with r-hFII for coagulopathy [26].
AC K N OW L E D G E M E N T We thank Anne Legnehed for technical assistance with the Amelung coagulometer.
AU T H O R C O N T R I B U T I O N K. Hansson contributed to conceptual and experimental design, interpreted results and participated in manuscript writing. J. Bj€ orkqvist performed experiments, evaluated results and participated in manuscript writing. J. Deinum contributed to conceptual and
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experimental design, interpretation of results and wrote main part of manuscript.
CONFLICT OF INTEREST This research was funded by AstraZeneca R&D M€ olndal, Sweden.
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Kenny Hansson is employed by AstraZeneca and Johanna Deinum is a former employee now consulting for AstraZeneca. This research was part of Jenny Bj€ orkvist’s diploma work at AstraZeneca 2009, and she is presently PhD student at Karolinska Institutet Stockholm, Sweden with no conflicts of interest.
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19. Majumder R, Liang X, Quinn-Allen MA, Kane WH, Lentz BR. Modulation of prothrombinase assembly and activity by phosphatidylethanolamine. J Biol Chem 2011;286:35535–42. 20. Machlus KR, Colby EA, Wu JR, Koch GG, Key NS, Wolberg AS. Effects of tissue factor, thrombomodulin and elevated clotting factor levels on thrombin generation in the calibrated automated thrombogram. Thromb Haemost 2009;102:936–44. 21. Hemker HC, Giesen P, Al Dieri R, Regnault V, de Smedt E, Wagenvoord R, Lecompte T, Beguin S. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003;33:4–15. 22. Giesen PL, Willems GM, Hermens WT. Production of thrombin by the prothrombinase complex is regulated by membrane-mediated transport of prothrombin. J Biol Chem 1991;266:1379–82. 23. Hemker HC, Kerdelo S, Kremers RMW. Is there value in kinetic modeling of thrombin generation? No (unless. . .). J Thromb Haemost 2012;10:1470–7. 24. Vicente V, Gonzalez-Conejero R, Rivera J, Corral J. The prothrombin gene variant 20210A in venous and arterial thromboembolism. Haematologica 1999;84:356–62. 25. Sorensen B, Spahn DR, Innerhofer P, Spannagl M, Rossaint R. Clinical review: Prothrombin complex concentrates–evaluation of safety and thrombogenicity. Crit Care 2011;15:201. 26. Mitterlechner T, Innerhofer P, Streif W, L€ odi M, Danninger T, Klima G, Hansson K, Fries D. Prothrombin complex concentrate and recombinant prothrombin alone or in combination with recombinant factor X and FVIIa in dilutional coagulopathy: a porcine model. J Thromb Haemost 2011;9:729–37.
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ORIGINAL ARTICLE
INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY
The effect of recombinant and plasma-derived prothrombin on prothrombin time in human plasma 1 € K. M. HANSSON, J. BJ ORKQVIST , J. DEINUM
Department of Bioscience, CVMD iMED, AstraZeneca R&D M€ olndal, M€ olndal, Sweden Correspondence: Kenny M. Hansson, Department of Bioscience, AstraZeneca R&D M€ olndal, S-43183 M€ olndal, Sweden. Tel.: +46 31 7065371; Fax: +47 31 7763859; E-mail: kenny.m.hansson @astrazeneca.com 1
Present address: Department of Molecular Medicine and Surgery and Center of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
doi:10.1111/ijlh.12293
Received 12 May 2014; accepted for publication 11 August 2014 Keywords Prothrombin time, prothrombin, tissue factor, plasma coagulation, calcium
S U M M A RY Introduction: When investigating coagulation assays to measure the effect of infused prothrombin (FII) in in vivo coagulopathy models, we found that addition of FII, plasma-derived human FII (pd-hFII) or recombinant human FII (r-hFII), to normal plasma resulted in a concentration-dependent increase in prothrombin time (PT) initiated with Innovinâ. Methods: The effect on PT by addition to plasma of either pd-hFII or r-hFII, using different commercial PT reagents, was studied both by turbidimetry and viscometry. Result: Addition of FII to plasma resulted in increased PT when initiated with Innovinâ: PT increased with 20% by doubling the concentration. The prolongation of PT became more pronounced with 2–6000 times diluted Innovinâ. However, by adjustment of the final free Ca2+ concentration in the assay with diluted Innovinâ from 8.3 to 1.3 mmol/L, no FII-dependent increase in PT was found. In contrast, no prolongation of PT was found with other commercial PT reagents. A KM= 3 nmol/L was obtained with pd-hFII, respectively, r-hFII with FII-depleted plasma using Thromborelâ to initiate PT. Conclusion: At normal plasma concentration of FII, addition of FII should not have an effect on PT. The prolonged PT with Innovinâ, but not with other PT reagents, at supranormal FII concentration is an artefact.
INTRODUCTION Two parallel pathways of coagulation factors, the so-called extrinsic and intrinsic pathway, can initiate plasma coagulation [1, 2]. In both pathways, the completion of each step activates another coagulation factor in a chain reaction and meet in the common pathway generating thrombin that subsequently © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 357–364
converts fibrinogen to fibrin [3]. In the coagulation process, all the crucial steps are dependent on the presence of free Ca2+ so that the vitamin K-dependent coagulation factors, containing gamma-carboxylated glutamic acid residues, can anchor on the negatively charged procoagulant membrane surfaces, exposing phospholipids (PL), as found on cell surfaces such as on damaged endothelial cells and activated platelets. 357
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The most common haemostatic assays used in the clinical practice are coagulation assays. To initiate fibrin formation, id est coagulation, only a nanomolar thrombin concentration is needed. One of the commonly used coagulation assays is the Prothrombin Time (PT) assay, alternatively called the Quick Prothrombin time [4]. All commercial PT reagents contain a mixture of an activator, tissue factor (TF) together with PL or membrane fragments, and can vary widely in their sensitivity to reductions in the levels of different coagulation factors. PT is the most common laboratory assay used to monitor oral anticoagulant therapy. PT is also used to screen for abnormalities in the extrinsic system and to perform quantitative assays of one or more factors of the system. However, relative sensitivities to changes in the plasma levels of factors FV, FVII, FX and prothrombin (FII) have been found to be differentially influenced by the content and composition of the PL [5] and the NaCl concentration [6]. Thus, the determination of the prothrombin time (PT) is based on an artificial in vitro system with major limitations. Nevertheless, these tests are quite useful as global screening tests for abnormalities in the pathways of coagulation [7]. In vivo, the free Ca2+ concentration in plasma is 1–1.5 mmol/L [8]. Most PT reagents contain an excess of CaCl2 to compensate for the presence of citrate present as an anticoagulant in the plasma samples. Moreover, as the citrate concentration in plasma obtained in blood samples from patients is primarily determined by the individual haematocrit and variability in filling level in sample tubes, these assays in general use a Ca2+ concentration that in the final assay results in a free Ca2+ plasma concentration that is far higher than in vivo levels to ascertain that Ca2+ levels below 1 mmol/L should be avoided. This results in that coagulation analyses using blood that has been exposed to citrate and is recalcified do not yield reliable depictions of the natural dynamics of blood coagulation processes [9]. Different FII concentrations have previously been investigated as an antidote for thrombin inhibitors [10, 11] in animal models and have also been evaluated with different coagulation assays. Moreover, reliable commercial coagulation assays are needed to measure the effect of infusion of FII in in vivo coagulopathy models. Preliminary results had shown that the PT of normal plasma was slightly increased instead
of decreased or unchanged in the presence of higher FII concentrations, applying the Innovinâ PT reagent. Therefore, we studied the effect of addition of FII in different commercial PT reagents; using both plasmaderived human FII (pd-hFII) or recombinant human FII (r-hFII) both on PT and diluted PT.
M AT E R I A L S A N D M E T H O D S Chemicals and buffers All chemicals were of reagent grade. A stock solution eres, France). of CaCl2 in water was from Stago (Asni Bovine serum albumin (BSA) 20% (w/v) solution in water and the phospholipid emulsion containing synthetic sources of phosphatidylserine, phosphatidylcholine and sphingomyelin (PL-TGT, 0.5 mmol/L) were from Rossix AB (M€ olndal, Sweden). All solutions were made with deionized water that was further purified on an Elgastadt UHP (Elga Ltd., High Wycombe Bucks, England). Fysalb consisted of 9 g/L NaCl solution in water from Fresenius (Kabi, Halden, Norway) and 1% (w/v) BSA. Ten-time-concentrated HBS-N buffer from GE Healthcare Lifesciences, Uppsala, Sweden) was diluted to HBS-N buffer consisting of 10 mmol/L Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and 0.15 mol/L NaCl at pH 7.4. Platelet-poor plasma Three different pools of platelet-poor plasma were prepared by centrifugation of fresh, citrated blood at 2000 9 g in a swing-out rotor for 20 min at 20 °C. For each plasma pool, blood was collected from 30 healthy volunteers employed by AstraZeneca R&D M€ olndal, Sweden (9 volumes blood with one volume 129 mmol/ L trisodium citrate). The plasma supernatant was immediately put on ice, pooled, aliquoted and stored at 80 °C. Fresh frozen immune-depleted human FII deficient plasma was purchased from Haematologic Technologies Inc., Essex Junction, VT, USA. Plasma from a haemophilia A patient was purchased from George King, Bio-Medical Inc., Overland Park, KS, USA. Just before an experiment, the plasma samples were rapidly thawed in a water bath at 37 °C and kept at 37 °C for at least 30 min. The mean normal plasma concentration of FII is 1.4 lmol/L FII [12, 13], which equals 100 IU/dL. © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 357–364
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Prothrombin Different batches of lyophilized pd-hFII were obtained from Enzyme Research Laboratories, (South Bend, IN, USA). Correctly gamma-carboxylated r-hFII was produced by AstraZeneca. The protein concentration was determined applying e280=102/mM/cm for human FII [14]. FII was kept stored at –80 °C and was rapidly thawed in a 37 °C water bath on the day of the experiment and then immediately transferred to an ice bath and further diluted with ice-cold Fysalb to a final concentration of 24 lmol/L. Further dilutions were also made with Fysalb. Fysalb was used as blank/control in all experiments. Solutions containing BSA were freshly prepared on the day of the experiment. General experimental design for PT and diluted PT The used commercial PT reagents, containing TF and phospholipids, respectively, membrane fragments, were three different lots of Innovinâ and Thromborelâ from Dade Behring (Marburg, Germany) Simplastinâ from Organon Teknika BV (Oss, The Netherlands), RecombiPlasTinâ from Instrumentation Laboratories Co (Lexington, KY, USA) STA-Neoplastineâ from Diagnostic Stago (Asni eres, France) and TrinityCLOT HTFâ from Trinity Biotech (Jamestown, NY, USA). The PT reagents were reconstituted and the assay was performed at 37 °C as recommended by the manufacturer by addition of two volumes reagent to one volume plasma to initiate coagulation. In most commercial PT assay kits, a CaCl2 concentration is used, which results in a final free Ca2+ concentration that differs from the physiological 1–1.5 mmol/L. Therefore, in some experiments with diluted Innovinâ, the CaCl2 content was adjusted so that in the final assay in the serum after fulfilled coagulation the concentration of free Ca2+ became 1.3 mmol/L, alternatively 8.3 mmol/L. The free Ca2+ concentration was measured with a calcium ion-selective electrode included in a Radiometer ABL700 analysis instrument (Radiometer, Copenhagen, Denmark). To study the effect of lower TF concentrations, a bottle with lyophilized Innovinâ PT was reconstituted with 10 mL water to 6 nmol/L TF and if needed, further diluted 2, 10, 100, 1000 and 6000 times with © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 357–364
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HBS-N buffer containing different CaCl2 concentrations and to compensate for the dilution of the reagents, additional PL were added to plasma to a final assay concentration of 4 lmol/L. Before start of the coagulation assays, first 1 volume Fysalb solution, containing FII and extra PL were pre-incubated with 9 volumes plasma at 37 °C for 30 min, before initiation by addition of 20 volumes of the diluted TF reagent with CaCl2. Coagulation measurements Coagulation times were measured applying two different physical phenomena related to the formation of the fibrin clot, viscometry and turbidimetry. Viscometry was used to determine the coagulation times by a ball coagulation timer KC 10 A Micro from Heinrich Amelung GmbH (Lemgo, Germany). Turbidimetry was used to monitor the whole coagulation curves, from the apparent increase in optical absorbance at 405 nm, DA405, using a semi-automatic spectrophotometer, a BCS from Siemens Healthcare Diagnostics Inc. (Marburg, Germany). The coagulation time was determined from the coagulation curves using the BCS software, applying a DA405 threshold if not otherwise indicated.
R E S U LT S Comparison of PT reagents In contrast to viscometry, which is a physical method using manual pipetting for each sample and just provides an end-point measurement of the coagulation time, turbidimetry, measured from the DA405, can monitor the whole coagulation curve. With this method in the normal PT assay with Innovinâ, it was found that the whole coagulation curves of plasma were FII-concentration dependently parallel shifted in time, see Figure 1a, at higher plasma concentration of pd-hFII, indicating a prolonged coagulation time. The shift in the coagulation curves suggested a concentration-dependent inhibition of the Innovinâ initiated coagulation by FII. Similar effect on PT by addition of FII to plasma with Innovinâ was found with either rhFII or pd-hFII, suggesting that the observed effect on PT with the Innovinâ reagent did not depend on contamination in the pd-hFII sample with other plasma
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(a)
(b)
Figure 1. Effect of added FII on PT with Innovinâ by turbidimetry. Innovinâ was used as recommended by the manufacturer. Normal plasma (pool 1) 90 lL was mixed with 10 lL pd-hFII and incubated for 180 s. (a) The coagulation curves are labelled with the FII conc. in plasma before addition of 200 lL PT reagent Innovinâ at time 0 (final free Ca2+ 6 mmol/L). The data from each curve (each 0.5 s) from the BCS were used to calculate PT. (b) Mean PT coagulation times (n = 4) from the coagulation curves versus final assay conc. of added FII, using a threshold at DA405 = 200 mAbs.
proteins. Because of the very short coagulation time of less than 10 s, id est at approximately the first measuring point of the BCS, with the used plasma (pool 1), a modified evaluation algorithm with a higher cutoff of the DA405 was used to determine the PT, as illustrated in Figure 1b. However, with other plasma pools, a slightly higher PT was obtained with Innovinâ, making it possible to use the BCS algorithm straight forward. Also with other plasma pools, with a PT of above 10 s, similar apparent FII-concentrationdependent inhibition of PT, illustrated by parallel shifts in the coagulation curves, was observed. The apparent inhibition of PT by FII using Innovinâ seems to be unique since the increase of the plasma concentration of FII up to 300% of the normal level, that is addition of 2.8 lmol/L or 200 IU/dL, did not result in a prolongation effect on the coagulation curves with the other PT reagents assays containing either TF from tissue extract from rabbit brain (Simplastinâ), or from cultured human cells (TrinityCLOT HTFâ) or with two other reagents with recombinant TF and phospholipids (STA-Neoplastineâ or RecombiPlasTinâ). For all these other reagents, the PT defined by the BCS algorithm could be used and the coagulation curves did perfectly overlap and were indistinguishable from the control. The prolongation of PT Innovinâ by added FII was confirmed by viscometry. For example, in a typical
experiment with a normal plasma (pool 2) and Innovinâ PT was increased from 9.4 0.10 s to 10.5 0.025 s (mean PT standard error, n = 4) by an increase in the plasma concentration to 2.7 lmol/L by addition of pd-hFII, while no effect was found with the other commercial PT reagents. Also with this manual viscometry method, an increase in PT Innovinâ was found by addition of FII to plasma using different batches of pd-hFII or r-hFII or using other Innovinâ lots. No effect on PT of addition of prothrombin fragment 1 and 2 Prothrombin fragment 1 + 2, id est FII without its thrombin fragment, still has a high affinity for negatively charged PL [15] and as such compete with FII for binding to PL and the other Ca2+-dependent coagulation factors. However, addition to plasma of 4 lmol/L prothrombin fragment 1 + 2 had no effect on PT with Innovinâ although the final free Ca2+ concentration was as high as 6 mmol/L. PT with diluted Innovinâ and the concentration of Ca2+ In contrast to the other PT reagents, the final free serum Ca2+ concentration in the samples with Innovinâ was 6 mmol/L, which is far above the © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 357–364
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physiological plasma concentration. To further explore the effect of a high concentration of free Ca2+ Innovinâ was diluted 2, 10, 100, 1000 and 6000 fold, first in 10 ml water and further in water with 20 mmol/L CaCl2 and 6 lmol/L PL giving rise to final free Ca2+ concentration of 8.3 mmol/L in serum. Under these conditions, there was a strong TF concentrationdependent increase in coagulation time, see Figure 2a. Furthermore, for all TF dilutions a near-linear increase in coagulation time was observed with increasing plasma concentration of FII. Moreover, the same results were obtained when the final PL concentration was increased from 4 to 40 lmol/L. However, by changing the conditions so that the final free Ca2+ concentration was 1.3 mmol/L, similar to the physiological free serum concentration, there was no longer an increase in PT with diluted Innovinâ by increasing the plasma concentration of FII with 2.4 lmol/L, see Figure 2b. The assay with diluted Innovinâ at 1.3 mmol/L Ca2+ was repeated with different batches of pd-hFII and with r-hFII providing the same results. Apparently, the paradoxical effect on PT measured by diluted Innovinâ is not found at a physiological free Ca2+ concentration. The effect of the concentration of r-hFII or pd-hFII on the coagulation time was further compared using FII-depleted plasma and two PT reagents, Innovinâ (a)
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and Thromborelâ, using viscometry under conditions as recommended by the manufacturer of the reagents. As shown in Figure 3a,b with Innovinâ, banana shaped titration curves were found, consistent with apparent inhibition at higher concentrations of FII both for pd-hFII and r-hFII. The titration with Thromborelâ was as theoretically expected for simple Michaelis–Menten kinetics. The coagulation rate can be approximated to be proportional with the inverse coagulation time, assuming that at the time of coagulation, Dt, the same concentration of fibrin, the product, DP, is formed, if there is a linear relationship of the increase in turbidity and the fibrin concentration. Thus, the rate of fibrin formation becomes v = DP/ Dt. Then, the coagulation time PT is proportional with 1/v. Therefore, the KM could be estimated, applying the inverted equation of the Michaelis–Menten kinetics (PT=1/Vmax+(KM/Vmax)/[FII] as the result of the enzyme activity of the prothrombinase complex, with the substrate, FII, by nonlinear regression. From the data with Thromborelâ in Figure 3a,b an apparent KM= 3 and 2.9 nmol/L for pd-hFII, respectively, r-hFII was estimated by nonlinear regression.
DISCUSSION PT, a commonly used coagulation assay, is used as global screening test for abnormalities in the (b)
Figure 2. PT with diluted Innovinâ at different conc. of added pd-hFII and Ca2+. The coagulation time PT (n = 4) was derived from the coagulation curves from the BCS. The 50 lL mixture of 45 lL normal plasma (pool 2) with 6 lmol/L PL and 1.2 and 2.4 lmol/L added pd-hFII was pre-incubated for 30 min before initiation of coagulation by addition of 100 lL Innovinâ (a) The PT reagent Innovin â (6 nmol/L TF) was diluted 2, 10, 100, 1000 and 6000 fold with 20 mmol/L CaCl2 in HBS-N buffer with 6 lmol/L PL to final serum conc. of 8.3 mmol/L Ca2+ and 4 6 lmol/L PL (b) Innovinâ was diluted 10-, 100-, 1000- and 6000-fold with 7 mmol/L CaCl2 in HBS-N to a final free Ca2+ concentration of 1.3 mmol/L. The arrow points to the normal plasma conc. of FII. The error bars representing standard deviation are within the outlines of the symbols.
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(a)
(b)
Figure 3. Effect on PT by added FII by viscometry. PT was determined by first incubation for 180 s of 45 lL FIIdepleted plasma with 5 lL FII and initiated by addition of 100 lL PT reagent, Thromborelâ open circles and Innovinâ closed circles. Without added FII, there was no coagulation: (a) with pd-hFII and (b) r-hFII. Nonlinear regression was used to fit the reversed Michaelis–Menten equation 1/v = 1 + ([FII]/KM))/Vmax to the data with Thromborelâ providing KM= 3 0.2 resp. 2.9 0.1 nmol/L using 72 kD and the final assay conc. The data are mean PT SD values, n = 3.
pathways of coagulation [7]. However, one should be aware that the determination of PT is based on an artificial in vitro system with major limitations. All commercial PT reagents contain a mixture of a very high concentration of TF together with different types of anionic PL or membrane fragments. These different commercial PT tests have also been shown to be differentially influenced by the content of, among others, the composition of the PL [5] and the NaCl concentration [6]. In all PT assays, an excess of calcium is present to compensate for the presence of the Ca2+ chelator anticoagulant in the plasma samples, as free Ca2+ is needed for plasma to coagulate. In the coagulation process, all the crucial steps are dependent on the presence of free Ca2+ so that the vitamin K-dependent coagulation factors, containing gammacarboxylated glutamic acid residues, can anchor on the negatively charged procoagulant membrane surfaces, exposing PL. For example, regarding prothrombin, Ca2+ induces the conformation required for the expression of the PL-binding site in the binary prothrombin-metal complex [16] and requires two sequential metal-dependent conformational transitions to bind PL [16,17]. Previously, it had been shown that the PL composition and content is crucial for the prothrombinase activity [18, 19]. However, in most kinetic studies calcium concentrations of 5 mmol/L or above are used, allowing strong affinity of FII for anionic PL vesicles [15]. The present report
indicates that the role of calcium is more complicated. We found that the apparent inhibition by FII of PT in the assay with Innovinâ, over a large range of TF dilutions, disappeared when the final assay concentration of free Ca2+ was adjusted from 6 or 8 to 1.3 mmol/L. It is therefore surprising that a calcium concentration in this range should be inhibitory. It should also be noted that in this study, 129 nmol/L trisodium citrate was used as anticoagulant and not 109 nmol/L which is more commonly used. The use of 129 nmol/L might introduce a stronger effect on PT when sample tubes accidentally are underfilled compared with the same situation with 0.109 nmol/L. However, as not all commercial PT reagents did show this calcium-dependent effect, a general conclusion cannot be made. An increase in PT is consistent with the reported increase in lag time in the thrombin generation assay with increasing concentration of FII [20] both with 1 and 5 pmol/L TF. However, regarding the PT assay with Innovinâ, we can exclude membrane crowding [20] as an explanation for the apparent inhibition by FII as no effect was found by increasing the PL concentration 10-fold. The finding that added prothrombin fragment 1 and 2 in high concentrations had no effect on PT with Innovinâ further reinforced the conclusion that membrane crowding is not present. Thus, the surface area on the PL is not a limiting factor for the activity of FII in the PT assay with the Innovinâ reagent, as otherwise, also the presence of © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 357–364
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prothrombin fragment 1 + 2 should introduce this apparent inhibition. The prolongation of PT with the Innovinâ reagent was unique and was not observed with other commercial PT assays. Furthermore, similar results were obtained both with viscometry and turbidimetry both for pd-hFII and r-hFII. Moreover, the paradoxal effect was observed with different batches of Innovinâ. So, the Innovinâ effect might be dependent both on the free Ca2+ concentration, the type of phospholipid, the source of TF or a combination effect. It is out of the scope of this study to analyse the different commercial PT reagents more in detail. Preliminary studies have also shown that addition of FII to normal plasma or to platelet-poor plasma from a haemophilia A patient in the thrombin generation assay (Calibrated Automated Thrombogram) [21], using diluted Innovinâ, extended the lag time (not shown). Therefore, for clinical applications, other PT reagents than Innovinâ should be used to analyse plasma samples that might contain deviating FII concentrations. An apparent KM of 3 nmol/L FII in the prothrombinase complex, see Figure 3, is consistent of that obtained with large PL vesicles [22]. The initial rate of thrombin generation under physiological conditions in plasma with 1.4 lmol/L FII, far above the KM value, should thus not be dependent on the concentration of FII according to simple Michaelis–Menten kinetics. At the normal plasma concentration of FII, addition of FII should thus not have an effect. The activity of the prothrombinase complex in normal plasma can thus not be the rate limiting step, implying neither an increase nor decrease by a further increase in FII, as the maximal initial thrombin generation rate [23], and thus shortest coagulation time is already reached at normal FII plasma concentrations. In general, inhibition at high substrate concentrations suggests either the presence of a contaminant in the substrate preparation or secondary binding of FII to the PL surface. The inhibitory effect is unique for the Innovinâ reagent at high concentrations of free Ca2+ as this phenomenon was not found with the other commercial reagents. With different preparations both of pd-FII and r-hFII, moreover, with different plasma pools and several Innovinâ lots, the same phenomenon was found. Thus contamination with other factors can be excluded. However, further studies are needed to reveal the details of the process. © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 357–364
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The overall conclusion is thus that the increase in prothrombin time initiated with Innovinâ in plasma with increasing FII concentrations is an artefact, apparently unique for Innovinâ and among others dependent on the free Ca2+ concentration but not on the PL concentration. As the FII-dependent increase was even more pronounced at lower, for example picomolar TF concentrations, as used in the thrombin generation assays [21], this should be studied more in detail. It might be of interest, if possible, to adjust the final free Ca2+ concentration to a more physiological level closer to 1 to 1.5 mmol/L, not only in the PT assay, but also in other haemostasis assays. To avoid wrong conclusions analysing blood with deviating FII concentrations, clinicians should be aware of the fact that an increased PT measured with Innovinâ in combination with a suspicion of elevated FII concentrations can be an artefact and should apply other reagents. AS most reagents consist of a combination of different types of TF and PL, respectively, membrane fragments, a comparison should be made of different commercial PT reagents for a reliable assay. This finding is important for clinical measurement of PT in plasma samples containing unknown, possibly supranormal, concentrations of FII. This might be the situation in, for example, heterozygous carriers of the G20210 prothrombin gene mutation that confers a procoagulant lifelong increase in FII concentrations of up to 30% compared to normal levels [24]. Moreover, during treatment with FII containing plasma concentrates (prothrombin complex concentrates) for bleeding disorders repeated dosing can result in concentrations of FII well above normal level [25] as it may also happen in a future treatment with r-hFII for coagulopathy [26].
AC K N OW L E D G E M E N T We thank Anne Legnehed for technical assistance with the Amelung coagulometer.
AU T H O R C O N T R I B U T I O N K. Hansson contributed to conceptual and experimental design, interpreted results and participated in manuscript writing. J. Bj€ orkqvist performed experiments, evaluated results and participated in manuscript writing. J. Deinum contributed to conceptual and
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experimental design, interpretation of results and wrote main part of manuscript.
CONFLICT OF INTEREST This research was funded by AstraZeneca R&D M€ olndal, Sweden.
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Kenny Hansson is employed by AstraZeneca and Johanna Deinum is a former employee now consulting for AstraZeneca. This research was part of Jenny Bj€ orkvist’s diploma work at AstraZeneca 2009, and she is presently PhD student at Karolinska Institutet Stockholm, Sweden with no conflicts of interest.
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