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The contribution of Tissue Factor Pathway Inhibitor to Thrombin Generation in children receiving Unfractionated Heparin
Thrombosis Research 127 (2011) 275–276
Contents lists available at ScienceDirect
Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Letter to the Editors-in-Chief The contribution of Tissue Factor Pathway Inhibitor to Thrombin Generation in children receiving Unfractionated Heparin
Dear Editors, Unfractionated Heparin (UFH) is an anticoagulant that is widely used in paediatric healthcare and current recommendations for therapy in this population are primarily extrapolated from adult evidence. However, recent studies have demonstrated that due to the physiological maturation of the haemostatic system, which occurs throughout childhood, adult evidence may not be appropriate to guide anticoagulant management in children. [1–3] UFH predominantly acts by binding to and increasing the natural anticoagulant activity of Antithrombin (AT) and Tissue Factor Pathway Inhibitor (TFPI). In adults, the amount of free TFPI (TFPI not associated with lipoproteins) released into the circulation is dependent on the dose and concentration of UFH administered. [4] A number of in vitro and in vivo studies have demonstrated clear differences in the AT-dependent activity of UFH in children compared with adults, as measured by the APTT, anti-FXa and anti-FIIa assays. [5–7] However, there are no studies to date, directly observing UFH-dependent TFPI release in children receiving UFH via a continuous intravenous (IV) infusion as therapy or prophylaxis. Our study aimed to investigate the contribution of TFPI to the inhibition of thrombin generation by UFH in children receiving a continuous IV infusion.
Materials and Methods This was a prospective, single centre, cohort study. Samples were collected from children up to 16 years of age admitted to inpatient units of the Royal Children's Hospital (RCH), Melbourne, Australia, receiving a continuous infusion of at least 10 IU/kg/hr of UFH. Patients admitted to the paediatric and neonatal intensive care units were not included in this study. Informed consent was obtained from the parents of the children approached for participation. This study was approved by the Royal Children's Hospital Ethics in Human Research Committee. (EHRC #28042A) Blood samples were collected in tubes containing 0.105 mol/L (ie. 3.2%) trisodium citrate anticoagulant in a ratio of 9 volumes of whole blood to 1 volume anticoagulant. Citrated samples were centrifuged at 3000 rpm for 10 minutes at 10 °C (Megafuge 1.0R; Heraeus, Hanau, Germany) and platelet-poor plasma (PPP) was frozen at -80 °C for batch testing. TFPI release was determined as a function of free TFPI, measured antigenically, using a commercially available free TFPI assay (Asserachrom free TFPI ELISA®, Diagnostica STAGO, France). The Endogenous Thrombin Potential (ETP) was quantified using a non-automated microplate method, based on a modification of a previously described technique. [8] Plasma samples with 15 μl Pefabloc FG were incubated with recombinant tissue factor (Thromborel S) and 0049-3848/$ – see front matter © 2010 Published by Elsevier Ltd. doi:10.1016/j.thromres.2010.11.011
CaCl2. At timed intervals, following the addition of CaCl2, a sub-sample of this reaction mixture was mixed with 0.025 M Na2EDTA on ice, to stop further reaction. A sub-sample of this solution was then taken out and mixed with the chromogenic substrate S-2238 (Chromogenix, Milan, Italy). Following 10 minutes of incubation at 37 °C, acetic acid was added to each sample to stop any further reaction from taking place. The amount of thrombin generated was quantified by measuring absorbance at 405 nm, and comparing it to the absorbance produced by reaction of S-2238 with known amount of thrombin. As thrombin bound to alpha-2-macroglobulin retains activity against small substrates, formation of thrombin-alpha-2-macroglobulin complexes was quantified by neutralising the thrombin not bound to alpha-2-macroglobulin with anti-thrombin and heparin prior to addition of S-2238. This value was subtracted from the total thrombin activity to calculate the free thrombin activity. The amidolytic activities were plotted against time to produce thrombin generation curves and the ETP represents the area under these curves. To demonstrate the specific contribution of TFPI to UFH anticoagulant activity, the ETP was re-measured following the inhibition of TFPI using a solution of 10 mg/ml sheep-anti human TFPI antibody. (Affinity Biologicals, Ontario, Canada) This antibody was combined with PPP and incubated at 37 °C for 5 minutes. The measurement of the ETP was then conducted as previously described. Results Samples were collected from 17 children admitted to the RCH between February and July 2009. Demographic and clinical information of the patients is summarised in Table 1. Across the overall population, following a continuous IV infusion of UFH, the mean free TFPI concentration was 30.32 ± 7.08 ng/ml and the ETP before adding anti- TFPI antibodies was 122 ± 10 pM.min. Following the inhibition of TFPI, the ETPincreased by 12.0 ± 8.0 percent (p = 0.01) from the ETP recorded before addition of anti-TFPI antibodies. Discussion Although the anticoagulant activity of UFH is significantly influenced by TFPI, no studies to date have investigated the UFH-induced TFPI release in paediatric populations. By measuring free TFPI release in children receiving continuous UFH infusion, this is the first study to demonstrate an AT-independent activity of UFH in the haemostatic system of children. As expected, due to the anticoagulant activity of UFH, a reduction in the ETP before adding anti- TFPI antibodies, and an increase in TFPI plasma concentration was observed across the total population included in our study when compared to reference ranges of children in the same age groups, not receiving UFH. [1] Following the inhibition of TFPI, the thrombin generating capacity of plasma was observed to be greater than the ETP measured before adding anti-TFPI antibodies . This is in accordance with studies conducted in adults which suggest
276
Letter to the Editors-in-Chief
Conflict of interest statement
Table 1 Patient Demographics. No. Of Patients (male/female) Age-range Weight-range (kg) Indications for UFH (n) Post-Fontan Thromboembolic event Cardiomyopathy Infected pacemaker Prosthetic Heart Valve Duration of UFH before sample collection (days) [range] UFH dose (IU/kg/hr) [range]
17 (10/7) 2 months – 16.7 years 3.8 – 82.0 8 5 2 1 1
None. Acknowledgements Mandira Hiremath was supported by the Royal College of Pathologists of Australasia (RCPA) Medical School Scholarship. References
4.2 ± 0.9 [1-16] 21.5 ± 1.3 [14-30]
that TFPI significantly contributes to the inhibition of thrombin generation by UFH. [4,9] Adult studies also suggest that free TFPI release correlates with the dose and concentration of UFH administered. [4,9] However, due to the population size and limited sample volume collected from each patient in our study, we were unable to accurately analyse this relationship. Our study indicates that TFPI provides a significant influence on the activity of UFH on thrombin generation in children receiving UFH for therapy or prophylaxis. Further studies are required to analyse any age and dose-dependent response of UFH induced free TFPI in the paediatric population.
Summary UFH predominantly acts by binding to and increasing the natural anticoagulant activity of Antithrombin (AT) and Tissue Factor Pathway Inhibitor (TFPI). A number of in vitro and in vivo studies have demonstrated clear differences in the AT-dependent activity of UFH in children compared with adults. However, there are no studies to date, directly observing UFH-dependent TFPI release in children receiving UFH via a continuous intravenous (IV) infusion. The results of our study are in accordance with studies conducted in adults which suggest that TFPI significantly contributes to the inhibition of thrombin generation by UFH in the haemostatic system of children. However future studies are required to further investigate these findings.
[1] Monagle P, Barnes C, Ignjatovic V, Furmedge J, Newall F, Chan A, et al. Developmental Haemostasis: Impact for clinical haemostasis laboratories. Thromb Haemost 2006;95:362–72. [2] Newall F, Ignjatovic V, Johnston L, Summerhayes R, Lane G, Cranswick N, et al. Age is a determinant factor for measures of concentration and effect in children requiring unfractionated heparin. Thromb Haemost 2010;103(5):1085–90. [3] Newall F, Johnston L, Ignjatovic V, Monagle P. Unfractionated Heparin Therapy in Infants and Children. Pediatrics 2009;123(3):e510–8. [4] Alban S, Gastpar R. Plasma Levels of Total and Free Tissue Factor Pathway Inhibitor (TFPI) as Individual Pharmacological Parameters of Various Heparins. Thromb Haemost 2001;85(5):824–9. [5] Ignjatovic V, Furmedge J, Newall F, Chan A, Berry L, Fong C, et al. Age-related differences in Heparin response. Thromb Res 2005;118(6):741–5. [6] Ignjatovic V, Summerhayes R, Than J, Gan A, Monagle P. Therapeutic range for unfractionated heparin therapy: age-related differences in response in children. J Thromb Haemost 2006;4:2280–3. [7] Newall F, Ignjatovic V, Summerhayes R, Gan A, Butt W, Johnston L, et al. In vivo age dependency of unfractionated heparin in infants and children. Thromb Res 2008. [8] Ignjatovic V, Greenway A, Summerhayes R, Monagle P. Thrombin generation: the functional role of alpha-2-macroglobulin and influence of developmental haemostasis. Br J Haematol 2007;138:366–8. [9] Sandset P, Bendz B, Jansen J. Physiological Function of Tissue Factor Pathway Inhibitor and Interactions with Heparin. Haemostasis 2000;30(Suppl 2):48–56.
Mandira Hiremath Fiona Newall Vera Ignjatovic Robyn Summerhayes Paul Monagle⁎ Department of Haematology Research, Murdoch Children's Research Institute, Melbourne, Victoria, Australia Department of Clinical Haematology, Royal Children's Hospital, Melbourne, Victoria, Australia ⁎Corresponding author. Department of Clinical Haematology, Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia. Tel.: +61 03 9345 5161; fax: +61 03 9345 6667. E-mail address: [email protected] (P. Monagle). 17 July 2010
Contents lists available at ScienceDirect
Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Letter to the Editors-in-Chief The contribution of Tissue Factor Pathway Inhibitor to Thrombin Generation in children receiving Unfractionated Heparin
Dear Editors, Unfractionated Heparin (UFH) is an anticoagulant that is widely used in paediatric healthcare and current recommendations for therapy in this population are primarily extrapolated from adult evidence. However, recent studies have demonstrated that due to the physiological maturation of the haemostatic system, which occurs throughout childhood, adult evidence may not be appropriate to guide anticoagulant management in children. [1–3] UFH predominantly acts by binding to and increasing the natural anticoagulant activity of Antithrombin (AT) and Tissue Factor Pathway Inhibitor (TFPI). In adults, the amount of free TFPI (TFPI not associated with lipoproteins) released into the circulation is dependent on the dose and concentration of UFH administered. [4] A number of in vitro and in vivo studies have demonstrated clear differences in the AT-dependent activity of UFH in children compared with adults, as measured by the APTT, anti-FXa and anti-FIIa assays. [5–7] However, there are no studies to date, directly observing UFH-dependent TFPI release in children receiving UFH via a continuous intravenous (IV) infusion as therapy or prophylaxis. Our study aimed to investigate the contribution of TFPI to the inhibition of thrombin generation by UFH in children receiving a continuous IV infusion.
Materials and Methods This was a prospective, single centre, cohort study. Samples were collected from children up to 16 years of age admitted to inpatient units of the Royal Children's Hospital (RCH), Melbourne, Australia, receiving a continuous infusion of at least 10 IU/kg/hr of UFH. Patients admitted to the paediatric and neonatal intensive care units were not included in this study. Informed consent was obtained from the parents of the children approached for participation. This study was approved by the Royal Children's Hospital Ethics in Human Research Committee. (EHRC #28042A) Blood samples were collected in tubes containing 0.105 mol/L (ie. 3.2%) trisodium citrate anticoagulant in a ratio of 9 volumes of whole blood to 1 volume anticoagulant. Citrated samples were centrifuged at 3000 rpm for 10 minutes at 10 °C (Megafuge 1.0R; Heraeus, Hanau, Germany) and platelet-poor plasma (PPP) was frozen at -80 °C for batch testing. TFPI release was determined as a function of free TFPI, measured antigenically, using a commercially available free TFPI assay (Asserachrom free TFPI ELISA®, Diagnostica STAGO, France). The Endogenous Thrombin Potential (ETP) was quantified using a non-automated microplate method, based on a modification of a previously described technique. [8] Plasma samples with 15 μl Pefabloc FG were incubated with recombinant tissue factor (Thromborel S) and 0049-3848/$ – see front matter © 2010 Published by Elsevier Ltd. doi:10.1016/j.thromres.2010.11.011
CaCl2. At timed intervals, following the addition of CaCl2, a sub-sample of this reaction mixture was mixed with 0.025 M Na2EDTA on ice, to stop further reaction. A sub-sample of this solution was then taken out and mixed with the chromogenic substrate S-2238 (Chromogenix, Milan, Italy). Following 10 minutes of incubation at 37 °C, acetic acid was added to each sample to stop any further reaction from taking place. The amount of thrombin generated was quantified by measuring absorbance at 405 nm, and comparing it to the absorbance produced by reaction of S-2238 with known amount of thrombin. As thrombin bound to alpha-2-macroglobulin retains activity against small substrates, formation of thrombin-alpha-2-macroglobulin complexes was quantified by neutralising the thrombin not bound to alpha-2-macroglobulin with anti-thrombin and heparin prior to addition of S-2238. This value was subtracted from the total thrombin activity to calculate the free thrombin activity. The amidolytic activities were plotted against time to produce thrombin generation curves and the ETP represents the area under these curves. To demonstrate the specific contribution of TFPI to UFH anticoagulant activity, the ETP was re-measured following the inhibition of TFPI using a solution of 10 mg/ml sheep-anti human TFPI antibody. (Affinity Biologicals, Ontario, Canada) This antibody was combined with PPP and incubated at 37 °C for 5 minutes. The measurement of the ETP was then conducted as previously described. Results Samples were collected from 17 children admitted to the RCH between February and July 2009. Demographic and clinical information of the patients is summarised in Table 1. Across the overall population, following a continuous IV infusion of UFH, the mean free TFPI concentration was 30.32 ± 7.08 ng/ml and the ETP before adding anti- TFPI antibodies was 122 ± 10 pM.min. Following the inhibition of TFPI, the ETPincreased by 12.0 ± 8.0 percent (p = 0.01) from the ETP recorded before addition of anti-TFPI antibodies. Discussion Although the anticoagulant activity of UFH is significantly influenced by TFPI, no studies to date have investigated the UFH-induced TFPI release in paediatric populations. By measuring free TFPI release in children receiving continuous UFH infusion, this is the first study to demonstrate an AT-independent activity of UFH in the haemostatic system of children. As expected, due to the anticoagulant activity of UFH, a reduction in the ETP before adding anti- TFPI antibodies, and an increase in TFPI plasma concentration was observed across the total population included in our study when compared to reference ranges of children in the same age groups, not receiving UFH. [1] Following the inhibition of TFPI, the thrombin generating capacity of plasma was observed to be greater than the ETP measured before adding anti-TFPI antibodies . This is in accordance with studies conducted in adults which suggest
276
Letter to the Editors-in-Chief
Conflict of interest statement
Table 1 Patient Demographics. No. Of Patients (male/female) Age-range Weight-range (kg) Indications for UFH (n) Post-Fontan Thromboembolic event Cardiomyopathy Infected pacemaker Prosthetic Heart Valve Duration of UFH before sample collection (days) [range] UFH dose (IU/kg/hr) [range]
17 (10/7) 2 months – 16.7 years 3.8 – 82.0 8 5 2 1 1
None. Acknowledgements Mandira Hiremath was supported by the Royal College of Pathologists of Australasia (RCPA) Medical School Scholarship. References
4.2 ± 0.9 [1-16] 21.5 ± 1.3 [14-30]
that TFPI significantly contributes to the inhibition of thrombin generation by UFH. [4,9] Adult studies also suggest that free TFPI release correlates with the dose and concentration of UFH administered. [4,9] However, due to the population size and limited sample volume collected from each patient in our study, we were unable to accurately analyse this relationship. Our study indicates that TFPI provides a significant influence on the activity of UFH on thrombin generation in children receiving UFH for therapy or prophylaxis. Further studies are required to analyse any age and dose-dependent response of UFH induced free TFPI in the paediatric population.
Summary UFH predominantly acts by binding to and increasing the natural anticoagulant activity of Antithrombin (AT) and Tissue Factor Pathway Inhibitor (TFPI). A number of in vitro and in vivo studies have demonstrated clear differences in the AT-dependent activity of UFH in children compared with adults. However, there are no studies to date, directly observing UFH-dependent TFPI release in children receiving UFH via a continuous intravenous (IV) infusion. The results of our study are in accordance with studies conducted in adults which suggest that TFPI significantly contributes to the inhibition of thrombin generation by UFH in the haemostatic system of children. However future studies are required to further investigate these findings.
[1] Monagle P, Barnes C, Ignjatovic V, Furmedge J, Newall F, Chan A, et al. Developmental Haemostasis: Impact for clinical haemostasis laboratories. Thromb Haemost 2006;95:362–72. [2] Newall F, Ignjatovic V, Johnston L, Summerhayes R, Lane G, Cranswick N, et al. Age is a determinant factor for measures of concentration and effect in children requiring unfractionated heparin. Thromb Haemost 2010;103(5):1085–90. [3] Newall F, Johnston L, Ignjatovic V, Monagle P. Unfractionated Heparin Therapy in Infants and Children. Pediatrics 2009;123(3):e510–8. [4] Alban S, Gastpar R. Plasma Levels of Total and Free Tissue Factor Pathway Inhibitor (TFPI) as Individual Pharmacological Parameters of Various Heparins. Thromb Haemost 2001;85(5):824–9. [5] Ignjatovic V, Furmedge J, Newall F, Chan A, Berry L, Fong C, et al. Age-related differences in Heparin response. Thromb Res 2005;118(6):741–5. [6] Ignjatovic V, Summerhayes R, Than J, Gan A, Monagle P. Therapeutic range for unfractionated heparin therapy: age-related differences in response in children. J Thromb Haemost 2006;4:2280–3. [7] Newall F, Ignjatovic V, Summerhayes R, Gan A, Butt W, Johnston L, et al. In vivo age dependency of unfractionated heparin in infants and children. Thromb Res 2008. [8] Ignjatovic V, Greenway A, Summerhayes R, Monagle P. Thrombin generation: the functional role of alpha-2-macroglobulin and influence of developmental haemostasis. Br J Haematol 2007;138:366–8. [9] Sandset P, Bendz B, Jansen J. Physiological Function of Tissue Factor Pathway Inhibitor and Interactions with Heparin. Haemostasis 2000;30(Suppl 2):48–56.
Mandira Hiremath Fiona Newall Vera Ignjatovic Robyn Summerhayes Paul Monagle⁎ Department of Haematology Research, Murdoch Children's Research Institute, Melbourne, Victoria, Australia Department of Clinical Haematology, Royal Children's Hospital, Melbourne, Victoria, Australia ⁎Corresponding author. Department of Clinical Haematology, Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia. Tel.: +61 03 9345 5161; fax: +61 03 9345 6667. E-mail address: [email protected] (P. Monagle). 17 July 2010