Original article 851
Addition of prothrombin to plasma can result in a paradoxical increase in activated partial thromboplastin time Kenny M. Hanssona, Jenny Bjo¨rkqvistb and Johanna Deinuma In the activated partial thromboplastin time (APTT) assay, a variety of nonphysiological reagents is used to induce contact activation. The sensitivity of the APTT response for different thrombin inhibitors has previously been found to be dependent on the used reagent. Recently, infusion of prothrombin (FII) has been used in in-vivo coagulopathy models and its effect has been analyzed in different assays. Therefore, we investigated whether the FII plasma concentration might affect APTT using different commercial reagents, applying both turbidimetry and viscometry. We compared both plasma-derived human FII (pd-hFII) and recombinant human FII (r-hFII). Similar results were found for pd-hFII and r-hFII with different APTT reagents. As expected, no effect on APTT was found by increasing the plasma concentration of FII using APTT reagents consisting of ellagic acid (Actin FS or Actin). Although with Pathromtin SL, consisting of SiO2, only a slight increase was found, with most other commercial APTT reagents, consisting of SiO2 or kaolin, APTT dose-dependently increased by increasing concentration of FII. Therefore, both Pathromtin SL and Actin FS were used to compare r-hFII and pd-hFII by determining the KM at 37oC using FII-depleted plasma,
Introduction Coagulation can be induced by two cascades of coagulation factors, the so-called extrinsic and the intrinsic pathway [1–4]. In both pathways, the completion of each step activates another coagulation factor in a chain reaction and meets in the common pathway in the generation of thrombin from prothrombin (FII) in the prothrombinase complex. Finally, already by small amounts of generated thrombin, fibrinogen is converted into fibrin and thereby a clot is initiated and formed. The time until formation of the clot is generally used to compare the coagulation propensity of the blood or plasma. In the intrinsic pathway, or contact activation, the crucial first step [4] is the activation of factor XII (FXII) by contact with negatively charged surfaces. Active FXII (FXIIa) activates prekallikrein in the presence of highmolecular-weight kininogen to kallikrein, which in turn also activates FXII, in a feedback loop. All further steps in the cascade require Ca2þ and take place on phospholipid surfaces. The activated partial thromboplastin time (APTT) assay is used in the clinic as a global hemostatic assay to measure the intrinsic pathway. Different commercial APTT reagents contain different types of artificial activators and phospholipids, vegetable, synthetic and 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
providing values of 6 W 0.3 nmol/l FII for both. Thus, at normal plasma concentrations of FII, the maximal initial thrombin generation rate should be reached and no effect on the coagulation time is expected at higher FII concentrations. To completely avoid the paradoxical effect in the APTT assay at FII concentrations higher than normal, Actin or Actin FS is the preferable reagent. Blood Coagul Fibrinolysis 25:851–855 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Blood Coagulation and Fibrinolysis 2014, 25:851–855 Keywords: activated partial thromboplastin time, coagulation, ellagic acid, kaolin, prothrombin, silica a
Department of Bioscience, iMED CVMD, AstraZeneca, R&D Mo¨lndal, Mo¨lndal and bDepartment of Molecular Medicine and Surgery and Center of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Correspondence to Kenny M. Hansson, PhD, AstraZeneca R&D Mo¨lndal, S-43183 Mo¨lndal, Sweden Tel: +46 31 7065371; fax: +46 31 7763859; e-mail: [email protected] Received 11 February 2014 Revised 16 April 2014 Accepted 2 May 2014
tissue extracts [5]. Although the APTT assay evaluates the intrinsic pathway of coagulation [6,7], it is sensitive for the plasma concentration of many coagulation factors with the exception of FVII and FXIII. The APTT response with all reagents has been found to be sensitive for the FVIII level [8]. However, the response for mild deficiencies was found to be different with different reagents [9–12]. Moreover, it has previously been found that the response to thrombin, respectively, FXa inhibitors with different APTT reagents varied [13,14], although it is not known why. Large differences have also been found in the presence of heparin found to be dependent on the type of phospholipids [15,16]. Different FII concentrates have been investigated as an antidote for thrombin inhibitors [17,18] investigated in animal models but also with different coagulation assays. Altogether this created an interest to compare the sensitivity of different APTT reagents for the plasma concentration of FII, comparing both plasma-derived (pdhFII) and recombinant human FII (r-hFII). This is also of clinical interest, as r-hFII may be useful in a future treatment of coagulopathy because of bleeding [19]. In this report, the effect of FII on the intrinsic pathway has been studied in vitro using these artificial contact activation systems. Two techniques have been used to DOI:10.1097/MBC.0000000000000161
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852 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 8
Materials and methods Lyophilized pd-hFII was from Enzyme Research Laboratories, (South Bend, Indiana, USA) and correctly gamma-carboxylated r-hFII was produced by AstraZeneca R&D, Mo¨lndal, Sweden. The commercial pd-hFII preparations did all contain low amounts of contaminating proteins, for example FXa (0.2 mg/mg, PC (0.1 mg/mg) and traces of FVIIa and FIXa and varying low amounts of FIIa. In the r-hFII preparations, no other proteins were present and less than 0.4 mmol FIIa/mol FII. 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 (Mo¨lndal, Sweden). All solutions were prepared with deionized water that was further purified by reversed osmosis on an Elgastadt UHP (Elga Ltd., High Wycombe Bucks, England). Platelet-poor plasma, from two in-house prepared normal human plasma pools, was used. These pools were prepared after approval of the local ethics committee (No ADS 180-01 T048-05) by collecting blood from 30 healthy volunteers employed by AstraZeneca R&D Mo¨lndal, Sweden (nine volumes blood with one volume 129 mmol/l trisodium citrate). Normal plasma was prepared by centrifugation of the citrated blood at 2000g in a swing-out rotor for 20 min at 208C. The plasma supernatant was put on ice, pooled, aliquoted and stored at 808C. The APTT assay was performed according to the instructions for the reagents but with 45 ml of citrated plasma incubated with 5-ml FII before the APTT reagents were added to a total of 150 ml. APTT was determined by turbidimetry, from the apparent change in optical absorbance at 405 nm (A405), by a Behring Coagulation System instrument from Siemens (Siemens Healthcare Diagnostics Inc., Marburg, Germany) applying an absorbance difference threshold. In addition, viscometry was used by a KC 10 A Micro from Heinrich Amelung GmbH (Lemgo, Germany).
formation rate, id est coagulation. The initial coagulation rate is thus in principle proportional to the inverse coagulation time, assuming that the time of coagulation, Dt, the same concentration of fibrin, the product DP is formed: v ¼ DP/Dt. The titration curves can therefore be analyzed applying Michaelis-Menten kinetics, by plotting 1/v ¼ APTT versus the concentration of FII added to FII-depleted plasma. Thus, by using the reverse Michaelis-Menten equation, 1/v ¼ 1 þ ([FII]/KM))/Vmax, the KM for FII can be estimated by nonlinear regression analysis.
Results and discussion Previously, in the comparison of different thrombin inhibitors on plasma coagulation, the PTT automate 5 reagent from Stago (Diagnostica Stago, Asnie`res, France), has been proven to be a sensitive assay [13,20]. However, in the present study we have found an unexpected increase in APTT with increasing concentrations of FII added to normal plasma employing the PTT automate 5 reagent, see Figs 1 and 2. An increase in APTT, as shown by the parallel shifts in the coagulation curves (Fig. 1), suggests a paradoxical inhibition of coagulation by FII. The plot of APTT versus the total FII concentration (Fig. 2a and b) showed both with pd-hFII and r-hFII a similar effect on APTT with the different reagents, indicating that the effect was not dependent on a contaminant in the plasma-derived FII. Moreover, neither adjustment of the final free Ca2þ concentration to 1.5 mmol/l nor addition of the PL-TGT phospholipids emulsion up to 16 mmol/l had an effect on the increase in APTT by added FII in the assay with PTT automate 5. Different commercial reagents contain different initiators; Actin and Actin FS from Siemens contain ellagic Fig. 1
2000
A450 with PTT automate 5
measure plasma coagulation, namely turbidimetry and viscometry. The effect of addition of FII, both r-hFII and pd-hFII, on APTT in both normal platelet-poor plasma and FII immune-depleted plasma has been studied.
1800 1600 1400 Control normal plasma
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Added 0.6 µmol/l r-hFIl Added 1.2 µmol/l r-hFIl
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For all nonlinear regression analyses, Grafit software version 5 (Erithacus Software Ltd., Horley, UK) was used. Data are expressed as mean values with the standard error. Thrombin is generated from the substrate, FII, as the result of the overall enzyme activity of the prothrombinase complex. The thrombin concentration is proportional with its activity as estimated from the fibrin
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Time (s) Effect of FII on partial thromboplastin time (PTT) automate 5-induced activated partial thromboplastin time in normal plasma, incubated with added r-hFII and recorded by turbidimetry. Effect of addition of r-hFII on coagulation curves of plasma: from a total concentration of FII in plasma from 1.4 (control) to 2 and 2.6 mmol/l.
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Effect of human prothrombin on APTT Hansson et al. 853
Fig. 2
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(b) PTT automate 5
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Effect on activated partial thromboplastin time (APTT) measured by turbidimetry, with different concentration of (a) plasma-derived human FII (pd-hFII) (open symbols) or (b) recombinant human (r-hFII) (closed symbols) added to normal plasma, mean values W SD (n ¼ 4) with different APTT reagents (squares: Actin, circles: Actin FS, triangles: Pathromtin SL and rev. triangles: PTT automate 5). The dashed lines are from linear fit to the low plasma concentration range of added FII, 0–0.4 mmol/l.
acid and rabbit brain extract or soy bean phospholipids, respectively, whereas Pathromtin SL from Siemens and PTT automate 5 from Stago contain silica with soy bean phospholipids, respectively cephalin. An increase in APTT was not unique for PTT automate 5 and was found with other commercial APTT reagents, consisting of SiO2 and all with kaolin, not shown. Not only APTT but also the maximal turbidity was dependent on the used reagents (Fig. 3), indicating a direct effect of the type of reagent on fibrin fiber size and structure. However, the slope of the coagulation curves remained
Already with a slight increase in FII from the normal plasma concentration (1.4 mmol/l [21]), the APTT obtained with PTT automate 5 increased. We therefore decided to study the effect of different APTT reagents, Pathromtin SL, Actin FS and PTT automate 5 by titrating FII-depleted plasma with FII to explore the effect with low plasma concentrations of FII. Both Pathromtin SL and Actin FS have previously been found to exhibit the best sensitivity for deficiencies of other coagulation factors [9,12].
Fig. 3
Normal plasma
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approximately the same with the different reagents (Fig. 3). Thus, nearly no effect on APTT by addition of FII was found with two reagents containing ellagic acid, Actin and Actin FS (see Fig. 2a and b). The coagulation curves with Actin and Actin FS could be superimposed for different concentrations of added FII. However, only a very moderate increase was observed with Pathromtin SL at FII plasma concentrations of up to approximately 0.4 mmol/l (dashed line Fig. 2a and b) added to normal plasma (thus a total plasma concentration of 1.8 mmol/l) compared with the strong effect with PTT automate 5.
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Time (s) Effect of different activated partial thromboplastin time (APTT) reagents on coagulation of normal plasma. The coagulation curves, after subtraction of baseline, recorded by the change in turbidimetry DA405 versus time.
As fresh frozen FII-depleted plasma from Haematologic Technologies Inc. (Essex Junction, Vermont, USA) contained less than 1% FII none of the different APTT reagents could initiate coagulation without added FII. However, as expected, addition of FII to FII-depleted plasma induced gradually shorter APTT when initiated with either Pathromtin SL (Fig. 4) or Actin FS reagent (not shown) until an approximate steady state was reached at a plasma concentration of 0.4 mmol/l FII. Note that at plasma concentrations of FII in normal plasma (1.4 mmol/l, 0 added) and FII-depleted plasma
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854 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 8
Fig. 4
As illustrated by Figs 1 and 3, continuous monitoring of the coagulation by turbidimetry provides more information than a single APTT value, and an improved reproducibility by the semiautomatic handling of the reagents, allowing absolutely identical assay conditions in the comparison of the different reagents. However, because of the opaque solutions with APTT reagents containing kaolin as activator, as in C.K Presto 5 from Stago, turbidimetry could not be used. Viscometry showed that also C. K Presto 5 gave rise to a concentration-dependent increase in APTT by added pd-hFII or r-hFII as did additional similar experiments with PTT automate 5 with viscometry (not shown).
100
FII-depleted plasma APTT mean (s)
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60 PTT automate 5 40
Pathromtin SL 20 0.3
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pd-hFII added to FII-depleted plasma (µmol/l) FII-depleted plasma incubated with different FII concentration at 378C before activation with Pathromtin SL (triangles) or partial thromboplastin time (PTT automate 5 (circles). Activated partial thromboplastin time (APTT) [mean values from two series (n ¼ 3) standard error mean] plotted versus the plasma concentration of added plasma-derived human FII (pd-hFII) (0.0175–1.2 mmol/l). The solid line was drawn by nonlinear regression assuming MichaelisMenten kinetics (KM ¼ 5.9 0.1 nmol/l pd-hFII).
(1.2 mmol/l added), different APTT values were obtained (compare Figs 2 and 4), illustrating the difficulty in comparing immune-depleted plasma with normal plasma. From the data with Pathromtin SL in Fig. 4, applying Michaelis-Menten kinetics, nearly identical values for the KM could be estimated for pd-hFII (Fig. 4), 5.9 respectively for r-hFII 5.5 nmol/l (not shown). However, the apparent turnover with r-hFII was 15% lower, than with pd-hFII, probably caused by contamination with other plasma coagulation factors in pd-hFII. The same value for KM was also found with Actin FS as the reagent, illustrating that the slight increase in APTT with Pathromtin SL at high FII concentrations in plasma (Fig. 2a) did not interfere with the regression analysis for the used concentration interval at total FII concentrations below that in normal plasma. A KM of 6 nmol/l is consistent with the values previously found using isolated coagulation factors, FII, FXa and FVa in buffer with added phospholipids [22–25]. In contrast to the effect on APTT with Pathromtin SL, by FII in the used concentrations range added to FII-deficient plasma, no approximate steady state was reached with PTT automate 5 (Fig. 4). With PTT automate 5, the titration exhibited a banana shape and could thus not be used for direct kinetic analysis. Moreover, already at FII concentrations far below the normal plasma concentration of 1.4 mmol/l FII, the APTT with PTT automate 5 increased with increasing FII concentrations.
The reason for this apparent inhibition of plasma coagulation by FII with most commercial APTT reagents consisting of SiO2 and all with kaolin still is an open question. However, this finding is important for clinical measurement of APTT 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 with normal levels [26]. 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 [27], as it may also happen in a future treatment with r-hFII for coagulopathy [19]. In such situations, Actin or Actin FS should preferentially be used, as these APTT reagents did not give rise to a FII concentration-dependent paradoxical increase in APTT.
Acknowledgements This research was funded by AstraZeneca R&D Mo¨lndal, Sweden. Conflicts of interest
K.H. is employed by AstraZeneca and J.D. is a former employee now consulting for AstraZeneca. This research was part of J.B.’s diploma work at AstraZeneca and she is now a PhD student at Karolinska Institutet Stockholm, Sweden, with no conflicts of interest.
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Addition of prothrombin to plasma can result in a paradoxical increase in activated partial thromboplastin time Kenny M. Hanssona, Jenny Bjo¨rkqvistb and Johanna Deinuma In the activated partial thromboplastin time (APTT) assay, a variety of nonphysiological reagents is used to induce contact activation. The sensitivity of the APTT response for different thrombin inhibitors has previously been found to be dependent on the used reagent. Recently, infusion of prothrombin (FII) has been used in in-vivo coagulopathy models and its effect has been analyzed in different assays. Therefore, we investigated whether the FII plasma concentration might affect APTT using different commercial reagents, applying both turbidimetry and viscometry. We compared both plasma-derived human FII (pd-hFII) and recombinant human FII (r-hFII). Similar results were found for pd-hFII and r-hFII with different APTT reagents. As expected, no effect on APTT was found by increasing the plasma concentration of FII using APTT reagents consisting of ellagic acid (Actin FS or Actin). Although with Pathromtin SL, consisting of SiO2, only a slight increase was found, with most other commercial APTT reagents, consisting of SiO2 or kaolin, APTT dose-dependently increased by increasing concentration of FII. Therefore, both Pathromtin SL and Actin FS were used to compare r-hFII and pd-hFII by determining the KM at 37oC using FII-depleted plasma,
Introduction Coagulation can be induced by two cascades of coagulation factors, the so-called extrinsic and the intrinsic pathway [1–4]. In both pathways, the completion of each step activates another coagulation factor in a chain reaction and meets in the common pathway in the generation of thrombin from prothrombin (FII) in the prothrombinase complex. Finally, already by small amounts of generated thrombin, fibrinogen is converted into fibrin and thereby a clot is initiated and formed. The time until formation of the clot is generally used to compare the coagulation propensity of the blood or plasma. In the intrinsic pathway, or contact activation, the crucial first step [4] is the activation of factor XII (FXII) by contact with negatively charged surfaces. Active FXII (FXIIa) activates prekallikrein in the presence of highmolecular-weight kininogen to kallikrein, which in turn also activates FXII, in a feedback loop. All further steps in the cascade require Ca2þ and take place on phospholipid surfaces. The activated partial thromboplastin time (APTT) assay is used in the clinic as a global hemostatic assay to measure the intrinsic pathway. Different commercial APTT reagents contain different types of artificial activators and phospholipids, vegetable, synthetic and 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
providing values of 6 W 0.3 nmol/l FII for both. Thus, at normal plasma concentrations of FII, the maximal initial thrombin generation rate should be reached and no effect on the coagulation time is expected at higher FII concentrations. To completely avoid the paradoxical effect in the APTT assay at FII concentrations higher than normal, Actin or Actin FS is the preferable reagent. Blood Coagul Fibrinolysis 25:851–855 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Blood Coagulation and Fibrinolysis 2014, 25:851–855 Keywords: activated partial thromboplastin time, coagulation, ellagic acid, kaolin, prothrombin, silica a
Department of Bioscience, iMED CVMD, AstraZeneca, R&D Mo¨lndal, Mo¨lndal and bDepartment of Molecular Medicine and Surgery and Center of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Correspondence to Kenny M. Hansson, PhD, AstraZeneca R&D Mo¨lndal, S-43183 Mo¨lndal, Sweden Tel: +46 31 7065371; fax: +46 31 7763859; e-mail: [email protected] Received 11 February 2014 Revised 16 April 2014 Accepted 2 May 2014
tissue extracts [5]. Although the APTT assay evaluates the intrinsic pathway of coagulation [6,7], it is sensitive for the plasma concentration of many coagulation factors with the exception of FVII and FXIII. The APTT response with all reagents has been found to be sensitive for the FVIII level [8]. However, the response for mild deficiencies was found to be different with different reagents [9–12]. Moreover, it has previously been found that the response to thrombin, respectively, FXa inhibitors with different APTT reagents varied [13,14], although it is not known why. Large differences have also been found in the presence of heparin found to be dependent on the type of phospholipids [15,16]. Different FII concentrates have been investigated as an antidote for thrombin inhibitors [17,18] investigated in animal models but also with different coagulation assays. Altogether this created an interest to compare the sensitivity of different APTT reagents for the plasma concentration of FII, comparing both plasma-derived (pdhFII) and recombinant human FII (r-hFII). This is also of clinical interest, as r-hFII may be useful in a future treatment of coagulopathy because of bleeding [19]. In this report, the effect of FII on the intrinsic pathway has been studied in vitro using these artificial contact activation systems. Two techniques have been used to DOI:10.1097/MBC.0000000000000161
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
852 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 8
Materials and methods Lyophilized pd-hFII was from Enzyme Research Laboratories, (South Bend, Indiana, USA) and correctly gamma-carboxylated r-hFII was produced by AstraZeneca R&D, Mo¨lndal, Sweden. The commercial pd-hFII preparations did all contain low amounts of contaminating proteins, for example FXa (0.2 mg/mg, PC (0.1 mg/mg) and traces of FVIIa and FIXa and varying low amounts of FIIa. In the r-hFII preparations, no other proteins were present and less than 0.4 mmol FIIa/mol FII. 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 (Mo¨lndal, Sweden). All solutions were prepared with deionized water that was further purified by reversed osmosis on an Elgastadt UHP (Elga Ltd., High Wycombe Bucks, England). Platelet-poor plasma, from two in-house prepared normal human plasma pools, was used. These pools were prepared after approval of the local ethics committee (No ADS 180-01 T048-05) by collecting blood from 30 healthy volunteers employed by AstraZeneca R&D Mo¨lndal, Sweden (nine volumes blood with one volume 129 mmol/l trisodium citrate). Normal plasma was prepared by centrifugation of the citrated blood at 2000g in a swing-out rotor for 20 min at 208C. The plasma supernatant was put on ice, pooled, aliquoted and stored at 808C. The APTT assay was performed according to the instructions for the reagents but with 45 ml of citrated plasma incubated with 5-ml FII before the APTT reagents were added to a total of 150 ml. APTT was determined by turbidimetry, from the apparent change in optical absorbance at 405 nm (A405), by a Behring Coagulation System instrument from Siemens (Siemens Healthcare Diagnostics Inc., Marburg, Germany) applying an absorbance difference threshold. In addition, viscometry was used by a KC 10 A Micro from Heinrich Amelung GmbH (Lemgo, Germany).
formation rate, id est coagulation. The initial coagulation rate is thus in principle proportional to the inverse coagulation time, assuming that the time of coagulation, Dt, the same concentration of fibrin, the product DP is formed: v ¼ DP/Dt. The titration curves can therefore be analyzed applying Michaelis-Menten kinetics, by plotting 1/v ¼ APTT versus the concentration of FII added to FII-depleted plasma. Thus, by using the reverse Michaelis-Menten equation, 1/v ¼ 1 þ ([FII]/KM))/Vmax, the KM for FII can be estimated by nonlinear regression analysis.
Results and discussion Previously, in the comparison of different thrombin inhibitors on plasma coagulation, the PTT automate 5 reagent from Stago (Diagnostica Stago, Asnie`res, France), has been proven to be a sensitive assay [13,20]. However, in the present study we have found an unexpected increase in APTT with increasing concentrations of FII added to normal plasma employing the PTT automate 5 reagent, see Figs 1 and 2. An increase in APTT, as shown by the parallel shifts in the coagulation curves (Fig. 1), suggests a paradoxical inhibition of coagulation by FII. The plot of APTT versus the total FII concentration (Fig. 2a and b) showed both with pd-hFII and r-hFII a similar effect on APTT with the different reagents, indicating that the effect was not dependent on a contaminant in the plasma-derived FII. Moreover, neither adjustment of the final free Ca2þ concentration to 1.5 mmol/l nor addition of the PL-TGT phospholipids emulsion up to 16 mmol/l had an effect on the increase in APTT by added FII in the assay with PTT automate 5. Different commercial reagents contain different initiators; Actin and Actin FS from Siemens contain ellagic Fig. 1
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measure plasma coagulation, namely turbidimetry and viscometry. The effect of addition of FII, both r-hFII and pd-hFII, on APTT in both normal platelet-poor plasma and FII immune-depleted plasma has been studied.
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For all nonlinear regression analyses, Grafit software version 5 (Erithacus Software Ltd., Horley, UK) was used. Data are expressed as mean values with the standard error. Thrombin is generated from the substrate, FII, as the result of the overall enzyme activity of the prothrombinase complex. The thrombin concentration is proportional with its activity as estimated from the fibrin
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Time (s) Effect of FII on partial thromboplastin time (PTT) automate 5-induced activated partial thromboplastin time in normal plasma, incubated with added r-hFII and recorded by turbidimetry. Effect of addition of r-hFII on coagulation curves of plasma: from a total concentration of FII in plasma from 1.4 (control) to 2 and 2.6 mmol/l.
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Effect of human prothrombin on APTT Hansson et al. 853
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Effect on activated partial thromboplastin time (APTT) measured by turbidimetry, with different concentration of (a) plasma-derived human FII (pd-hFII) (open symbols) or (b) recombinant human (r-hFII) (closed symbols) added to normal plasma, mean values W SD (n ¼ 4) with different APTT reagents (squares: Actin, circles: Actin FS, triangles: Pathromtin SL and rev. triangles: PTT automate 5). The dashed lines are from linear fit to the low plasma concentration range of added FII, 0–0.4 mmol/l.
acid and rabbit brain extract or soy bean phospholipids, respectively, whereas Pathromtin SL from Siemens and PTT automate 5 from Stago contain silica with soy bean phospholipids, respectively cephalin. An increase in APTT was not unique for PTT automate 5 and was found with other commercial APTT reagents, consisting of SiO2 and all with kaolin, not shown. Not only APTT but also the maximal turbidity was dependent on the used reagents (Fig. 3), indicating a direct effect of the type of reagent on fibrin fiber size and structure. However, the slope of the coagulation curves remained
Already with a slight increase in FII from the normal plasma concentration (1.4 mmol/l [21]), the APTT obtained with PTT automate 5 increased. We therefore decided to study the effect of different APTT reagents, Pathromtin SL, Actin FS and PTT automate 5 by titrating FII-depleted plasma with FII to explore the effect with low plasma concentrations of FII. Both Pathromtin SL and Actin FS have previously been found to exhibit the best sensitivity for deficiencies of other coagulation factors [9,12].
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approximately the same with the different reagents (Fig. 3). Thus, nearly no effect on APTT by addition of FII was found with two reagents containing ellagic acid, Actin and Actin FS (see Fig. 2a and b). The coagulation curves with Actin and Actin FS could be superimposed for different concentrations of added FII. However, only a very moderate increase was observed with Pathromtin SL at FII plasma concentrations of up to approximately 0.4 mmol/l (dashed line Fig. 2a and b) added to normal plasma (thus a total plasma concentration of 1.8 mmol/l) compared with the strong effect with PTT automate 5.
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Time (s) Effect of different activated partial thromboplastin time (APTT) reagents on coagulation of normal plasma. The coagulation curves, after subtraction of baseline, recorded by the change in turbidimetry DA405 versus time.
As fresh frozen FII-depleted plasma from Haematologic Technologies Inc. (Essex Junction, Vermont, USA) contained less than 1% FII none of the different APTT reagents could initiate coagulation without added FII. However, as expected, addition of FII to FII-depleted plasma induced gradually shorter APTT when initiated with either Pathromtin SL (Fig. 4) or Actin FS reagent (not shown) until an approximate steady state was reached at a plasma concentration of 0.4 mmol/l FII. Note that at plasma concentrations of FII in normal plasma (1.4 mmol/l, 0 added) and FII-depleted plasma
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854 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 8
Fig. 4
As illustrated by Figs 1 and 3, continuous monitoring of the coagulation by turbidimetry provides more information than a single APTT value, and an improved reproducibility by the semiautomatic handling of the reagents, allowing absolutely identical assay conditions in the comparison of the different reagents. However, because of the opaque solutions with APTT reagents containing kaolin as activator, as in C.K Presto 5 from Stago, turbidimetry could not be used. Viscometry showed that also C. K Presto 5 gave rise to a concentration-dependent increase in APTT by added pd-hFII or r-hFII as did additional similar experiments with PTT automate 5 with viscometry (not shown).
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pd-hFII added to FII-depleted plasma (µmol/l) FII-depleted plasma incubated with different FII concentration at 378C before activation with Pathromtin SL (triangles) or partial thromboplastin time (PTT automate 5 (circles). Activated partial thromboplastin time (APTT) [mean values from two series (n ¼ 3) standard error mean] plotted versus the plasma concentration of added plasma-derived human FII (pd-hFII) (0.0175–1.2 mmol/l). The solid line was drawn by nonlinear regression assuming MichaelisMenten kinetics (KM ¼ 5.9 0.1 nmol/l pd-hFII).
(1.2 mmol/l added), different APTT values were obtained (compare Figs 2 and 4), illustrating the difficulty in comparing immune-depleted plasma with normal plasma. From the data with Pathromtin SL in Fig. 4, applying Michaelis-Menten kinetics, nearly identical values for the KM could be estimated for pd-hFII (Fig. 4), 5.9 respectively for r-hFII 5.5 nmol/l (not shown). However, the apparent turnover with r-hFII was 15% lower, than with pd-hFII, probably caused by contamination with other plasma coagulation factors in pd-hFII. The same value for KM was also found with Actin FS as the reagent, illustrating that the slight increase in APTT with Pathromtin SL at high FII concentrations in plasma (Fig. 2a) did not interfere with the regression analysis for the used concentration interval at total FII concentrations below that in normal plasma. A KM of 6 nmol/l is consistent with the values previously found using isolated coagulation factors, FII, FXa and FVa in buffer with added phospholipids [22–25]. In contrast to the effect on APTT with Pathromtin SL, by FII in the used concentrations range added to FII-deficient plasma, no approximate steady state was reached with PTT automate 5 (Fig. 4). With PTT automate 5, the titration exhibited a banana shape and could thus not be used for direct kinetic analysis. Moreover, already at FII concentrations far below the normal plasma concentration of 1.4 mmol/l FII, the APTT with PTT automate 5 increased with increasing FII concentrations.
The reason for this apparent inhibition of plasma coagulation by FII with most commercial APTT reagents consisting of SiO2 and all with kaolin still is an open question. However, this finding is important for clinical measurement of APTT 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 with normal levels [26]. 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 [27], as it may also happen in a future treatment with r-hFII for coagulopathy [19]. In such situations, Actin or Actin FS should preferentially be used, as these APTT reagents did not give rise to a FII concentration-dependent paradoxical increase in APTT.
Acknowledgements This research was funded by AstraZeneca R&D Mo¨lndal, Sweden. Conflicts of interest
K.H. is employed by AstraZeneca and J.D. is a former employee now consulting for AstraZeneca. This research was part of J.B.’s diploma work at AstraZeneca and she is now a PhD student at Karolinska Institutet Stockholm, Sweden, with no conflicts of interest.
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