Ir J Med Sci DOI 10.1007/s11845-013-1056-1
BRIEF REPORT
Placental prothrombin mRNA levels in APC resistance (APCR) women with increased placental fibrin deposition S. Sedano-Balbas • M. Lyons • B. Cleary M. Murray • G. Gaffney • M. Maher
•
Received: 13 October 2013 / Accepted: 3 December 2013 Ó Royal Academy of Medicine in Ireland 2013
Abstract We investigated the link between the mRNA of the procoagulant prothrombin in the placental tissue with the increased placental fibrin deposition associated with activated protein C resistance (APCR). Women with APCR were not found to produce higher levels of prothrombin transcript compared to women with a normal APC ratio. This indicates that accumulated fibrin in the placenta is not the consequence of too much production of the procoagulant prothrombin transcript, but may be associated with altered function of other haemostatic factors interacting with APC in the placenta.
S. Sedano-Balbas (&) M. Maher Molecular Diagnostics Research Group, National Centre for Biomedical Engineering Science, National Diagnostics Centre, National University of Ireland, Galway, Ireland e-mail: [email protected] M. Maher e-mail: [email protected] M. Lyons B. Cleary M. Murray Department of Haematology, University College Hospital Galway, Galway, Ireland e-mail: [email protected]
Background The interaction of the coagulation and fibrinolytic systems is particularly important for maintenance of the placenta and to ensure rapid and effective control of bleeding from the placental site during delivery and the puerperium [1]. Prothrombin glycoprotein, also known as Factor II (F2), precursor of thrombin is synthesised mainly in the liver, but its expression has also been observed in other organs including rat placenta [2]. Thrombin is a key component in haemostasis functioning as a procoagulant [3], an anticoagulant [4], and an anti-fibrinolytic [5] (Fig. 1). A poor anti-coagulant response of APC is known as APC resistance which is recognised as one of the commonest causes of excessive generation of fibrin resulting in thrombosis. An increased risk of thrombosis, referred to as thrombophilia, results from a hyper-coagulation or prothrombotic state. Maternal thrombophilia can result in placental vascular disorders and a subsequent variety of adverse pregnancy outcomes [6, 7]. Previous studies in our laboratory showed that higher levels of placental fibrin deposition were associated with APCR [8]. In this study, we investigated whether the higher fibrin deposition identified in APCR was related to abnormally high levels of prothrombin mRNA transcript in the placenta.
B. Cleary e-mail: [email protected]
Materials and methods
M. Murray e-mail: [email protected]
Subject samples
G. Gaffney Department of Obstetrics and Gynaecology, University College Hospital Galway, Galway, Ireland e-mail: [email protected]
This study was conducted on 45 pregnant women which were a sub-set out of our total study cohort which included 907 pregnant women attending the antenatal clinic at
123
Ir J Med Sci Fig. 1 Haemostatic factors and activated protein C (PCa)
UCHG. All subjects gave consent to be included in the study and ethical approval for the study was obtained from the Research Ethics Committee at UCHG. From the 45 women included in this study (33 of whom were previously investigated for placental fibrin deposition [8]) 29 were APCR and 16 women were APC normal. Subjects were not age-matched and placental samples were taken without knowledge of the pregnancy progress. Blood samples were taken and the APC ratio was determined using the CoatestÒ test as described in our previous paper [8]. Extraction of placental RNA Placental samples were collected at the Department of Obstetrics and Gynaecology, UCHG. Sections of placental tissue (approx. 15 g) were collected within 30 min postdelivery and rinsed in saline solution before being placed into 50-ml Falcon tubes, snap-frozen in liquid nitrogen and subsequently stored at -80 °C for 15 days prior to RNA extraction. Extraction of RNA was performed in a ribonuclease-free environment applying RNase away (Labkem Ltd, Ireland) and 0.1 % diethylpyrocarbonate (DEPC) (BDH Laboratory Supplies, UK) to treat H2O and buffers. RNA extractions were performed in a Class I Microflow Biological Safety Cabinet (MDH Ltd, UK) using dedicated pipettes and aerosol barrier tips. The application of a combined method involving initial homogenisation of approximately 15 g of placental tissue using an automatic homogeniser and TRIzolÒ reagent followed by a second extraction using the RiboPureTM Kit
123
gave the best quality and highest yield of RNA. The eluted RNA was stored at -80 °C until required. Quantification and visualisation of extracted RNA RNA quality was determined from all samples using the 2100 Biolanalyser (Agilent) using the LabchipTM gels and on a RNA 6000 ladder. DNase treatment of RNA samples RNA samples (10 ll) were treated with DNase to remove contaminating DNA using the DNA-FreeTM DNA Removal Kit (Ambion, UK). DNA free-RNA was stored at -80 °C. cDNA synthesis Reverse transcription of RNA (1 lg) was performed using M-MLV (20 U) Reverse transcriptase (Promega, UK) and RNase Inhibitor (20 U) (Ambion, UK) as detailed by the manufacturer and the cDNA was stored at -20 °C until required. Primer and probe design The gene sequences for b-actin (NM_001101) and prothrombin (NM_000506) were available in the GenBank database. PCR primers and 50 exonuclease probes were designed using the Primer 3 program (http://frodo.wi.mit. edu/cgi-bin/primer3/primer3_www.cgi), in accordance with recommended guidelines (LightCyclerTM Operator’s
Ir J Med Sci
Manual). The sequences below were found to be optimal for amplification and detection of prothrombin and b-actin transcripts; Prothrombin Primer forward 50 CCgCATCACTgACAACATgTTC30 Primer reverse 50 AgCggTTgTTAAAggggCTCT30 Probe 50 6FAM-ACAAgCCTgATgAAgggAAACgAggggAT–DQ30 b-actin Primer forward 50 CAggATgCAgAAggAgATCACT30 Primer reverse 50 ACATCTgCTggAAggTggAC30 Probe 50 6FAM-CgATCCACACggAgTACTTgCgC– 0 DQ3 . The PCR primers were designed across introns. All the 50 exonuclease probes were synthesised by TIB MOLBIOL, Germany with the reporter dye 6-carboxyfluorescein (60 -FAM) at the 50 end, and the dark DABCYL (DQ) quencher at the 30 end. An independent cDNA synthesis step was followed by PCR on the LightCyclerTM using the LightCycler Fast Start DNA Master Hybridisation Probes Kit (Roche Molecular Systems, Germany) and appropriate 50 exonuclease probes. Aliquots (18 ll) of this mixture were added to the LightCyclerTM glass capillaries along with 2 ll of cDNA from the study samples or DNA standards. The optimised thermocycling conditions included: a denaturing step for 1 cycle at 95 °C for 10 min, PCR amplification for 45 cycles at 95 °C for 10 s, annealing and extension at 65 °C for 40 s and a final cooling step for 1 cycle at 40 °C for 30 s. Each study sample cDNA was analysed in duplicate within the same run and in two independent LightCyclerTM runs for prothrombin and bactin gene expression. Generation of standard curves for prothrombin and bactin Standard curves were generated from known concentrations of prothrombin and b-actin PCR products amplified from cDNA on the LightCyclerTM. The PCR products were analysed on a 2 % agarose gel excised from the gel and purified using the Gel extraction Kit (Qiagen, Germany) according to manufacturer’s instructions. After purification, the concentration of PCR products in molecules/ll was determined using the following formula [9]: Copy number ðmol/llÞ concentration of transcript RNAðg=llÞ ¼ size transcript ðbpÞ Y0 ðg/molÞ Serial dilutions of the prothrombin and b-actin PCR
products (1 9 107 to 19 103 molecules) were analysed in quadruplet by real-time PCR on the LightCyclerTM and the mean values obtained for each replicate of the five specific dilutions were used to generate external standard curves for the prothrombin and b-actin assays. The PCR efficiencies of prothrombin and b-actin amplification were determined using the formula E = 10-1/slope, where E is the efficiency value. Efficiency values of 1.94 and 1.99 were determined for b-actin and prothrombin, respectively. Channel setting F1 and the second derivative maximum analysis mode with arithmetic baseline were selected for analysis using the LightCyclerTM Software. DNA standards were generated to be used as the standard curves for determining the expression levels of prothrombin and bactin mRNA in each of the study samples. A mean value for prothrombin and for b-actin copy number was determined for each sample, and the expression data for prothrombin were normalised to b-actin expression data in each run as follows:
Sample Y = run 1 mean concentration of prothrombin/run 1 mean concentration of b-actin = A ‘‘Normalised Copy number of prothrombin transcripts in run 1’’ Sample Y = run 2 mean concentration of prothrombin/run 2 mean concentration of b-actin = B ‘‘Normalised Copy number of prothrombin transcripts in run 2’’ Mean AB values = Normalised value for prothrombin transcript copy number in sample Y
Statistical analysis Independent Student’s t test from the software package SPSS (version 11.5) was used to compare prothrombin gene expression in pregnant women with APCR to expression in pregnant women APC normal. Box plots from the same software package were also applied to the data results to visualise and evaluate how asymmetric the distribution of the data was.
Results The normalised expression values obtained for prothrombin expression were 4.771E-0 for the APCR group and 4.505E-0 for the APC normal group. The b-actin gene remained stable in all samples of this study. Figure 2 shows a side by side box plot for the data indicating also, the outlier samples in the APCR and the APC normal groups. Application of a student t test to the two data sets (APCR versus APC normal) showed no statistically significant
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Ir J Med Sci
linked to other impaired haemostatic factors inhibited by APC under normal circumstances. Therefore, further investigation of other factors that could link APCR with high fibrin deposition may be merited including FV and FVIII in the procoagulant system, and TAFI and PAI-1 in the fibrinolytic system. Acknowledgments The authors gratefully acknowledge the pregnant women who consented to be involved in the study and the midwives from the labour ward at the UCHG. This work was supported by the Irish Health Research Board. I wish to express my gratitude also to Dr. D. Mongan, UCH Galway, for providing the information on adverse outcomes of the subjects included in the study. The Irish Health Research Board supported this work.
References Fig. 2 Prothrombin expression in our APC resistance group versus our APC normal group
differences in prothrombin mRNA levels between the groups.
Discussion Low levels of prothrombin in the blood results in insufficient prothrombin to promote blood clotting and hence leads to abnormal bleeding after delivery in pregnancy [10]. High prothrombin levels in plasma, however, enhance the risk of thrombosis resulting in thrombophilia [11]. Although thrombophilia may lead to increased fibrin deposition, it has been shown that this does not always inevitably end with an adverse outcome [11]. From the maternity records of the present study, we identified that two subjects (1,157 and 1,266 on Fig. 1) within the APCR group had previous EPL, whereas neither of the APC normal subjects with higher levels of mRNA had EPL. Subject 1,266 also had PE during pregnancy. No other adverse outcomes investigated were identified in the subjects who showed outliers values in the expression of prothrombin transcripts. Castoldi et al. [12] shows that elevated prothrombin in plasma is related to APCR measured by the common aPTT-based test. We found that APCR subjects with higher fibrin deposition were not producing more prothrombin transcript than the APC normal cohort. The causes for the previously identified excess of fibrin deposition in the placenta of these APCR women could be
123
1. Holmes VA, Wallace JM (2005) Haemostasis in normal pregnancy: a balancing act? Biochem Soc Trans 33(Pt 2):428–432 2. Jamison CS, Degen SJ (1991) Prenatal and postnatal expression of mRNA coding for rat prothrombin. Biochim Biophys Acta 1088(2):208–216 3. Narayanan S (1999) Multifunctional roles of thrombin. Ann Clin Lab Sci 29(4):275–280 4. Esmon CT, Owen WG (2004) The discovery of thrombomodulin. J Thromb Haemost 2(2):209–213 5. Carrieri C, Galasso R, Semeraro F et al (2010) The role of thrombin activatable fibrinolysis inhibitor and factor XI in platelet-mediated fibrinolysis resistance: a thromboelastographic study in whole blood. J Thromb Haemost 9(1):154–162 6. Verspyck E, Marpeau L (2005) Thrombophilias and vascular placental pathology. A survey of the literature. Rev Med Interne 26(2):103–108 7. Vora S, Shetty S, Salvi V, Satoskar P, Ghosh K (2008) Thrombophilia and unexplained pregnancy loss in Indian patients. Natl Med J India 21(3):116–119 8. Sedano S, Gaffney G, Mortimer G et al (2008) Activated protein C resistance (APCR) and placental fibrin deposition. Placenta 29(9):833–837 9. Fronhoffs S, Totzke G, Stier S et al (2002) A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction. Mol Cell Probes 16(2):99–110 10. Meeks SL, Abshire TC (2008) Abnormalities of prothrombin: a review of the pathophysiology, diagnosis, and treatment. Haemophilia 14(6):1159–1163 11. Kujovich JL (2011) Prothrombin-related thrombophilia. In: Pagon RA, Bird TD, Dolan CR et al (eds) GeneReviews. University of Washington, Seattle. http://www.ncbi.nlm.nih.gov/ books/NBK1148/ 12. Castoldi E, Simioni P, Tormene D et al (2007) Differential effects of high prothrombin levels on thrombin generation depending on the cause of the hyperprothrombinemia. J Thromb Haemost 5(5):971–979
BRIEF REPORT
Placental prothrombin mRNA levels in APC resistance (APCR) women with increased placental fibrin deposition S. Sedano-Balbas • M. Lyons • B. Cleary M. Murray • G. Gaffney • M. Maher
•
Received: 13 October 2013 / Accepted: 3 December 2013 Ó Royal Academy of Medicine in Ireland 2013
Abstract We investigated the link between the mRNA of the procoagulant prothrombin in the placental tissue with the increased placental fibrin deposition associated with activated protein C resistance (APCR). Women with APCR were not found to produce higher levels of prothrombin transcript compared to women with a normal APC ratio. This indicates that accumulated fibrin in the placenta is not the consequence of too much production of the procoagulant prothrombin transcript, but may be associated with altered function of other haemostatic factors interacting with APC in the placenta.
S. Sedano-Balbas (&) M. Maher Molecular Diagnostics Research Group, National Centre for Biomedical Engineering Science, National Diagnostics Centre, National University of Ireland, Galway, Ireland e-mail: [email protected] M. Maher e-mail: [email protected] M. Lyons B. Cleary M. Murray Department of Haematology, University College Hospital Galway, Galway, Ireland e-mail: [email protected]
Background The interaction of the coagulation and fibrinolytic systems is particularly important for maintenance of the placenta and to ensure rapid and effective control of bleeding from the placental site during delivery and the puerperium [1]. Prothrombin glycoprotein, also known as Factor II (F2), precursor of thrombin is synthesised mainly in the liver, but its expression has also been observed in other organs including rat placenta [2]. Thrombin is a key component in haemostasis functioning as a procoagulant [3], an anticoagulant [4], and an anti-fibrinolytic [5] (Fig. 1). A poor anti-coagulant response of APC is known as APC resistance which is recognised as one of the commonest causes of excessive generation of fibrin resulting in thrombosis. An increased risk of thrombosis, referred to as thrombophilia, results from a hyper-coagulation or prothrombotic state. Maternal thrombophilia can result in placental vascular disorders and a subsequent variety of adverse pregnancy outcomes [6, 7]. Previous studies in our laboratory showed that higher levels of placental fibrin deposition were associated with APCR [8]. In this study, we investigated whether the higher fibrin deposition identified in APCR was related to abnormally high levels of prothrombin mRNA transcript in the placenta.
B. Cleary e-mail: [email protected]
Materials and methods
M. Murray e-mail: [email protected]
Subject samples
G. Gaffney Department of Obstetrics and Gynaecology, University College Hospital Galway, Galway, Ireland e-mail: [email protected]
This study was conducted on 45 pregnant women which were a sub-set out of our total study cohort which included 907 pregnant women attending the antenatal clinic at
123
Ir J Med Sci Fig. 1 Haemostatic factors and activated protein C (PCa)
UCHG. All subjects gave consent to be included in the study and ethical approval for the study was obtained from the Research Ethics Committee at UCHG. From the 45 women included in this study (33 of whom were previously investigated for placental fibrin deposition [8]) 29 were APCR and 16 women were APC normal. Subjects were not age-matched and placental samples were taken without knowledge of the pregnancy progress. Blood samples were taken and the APC ratio was determined using the CoatestÒ test as described in our previous paper [8]. Extraction of placental RNA Placental samples were collected at the Department of Obstetrics and Gynaecology, UCHG. Sections of placental tissue (approx. 15 g) were collected within 30 min postdelivery and rinsed in saline solution before being placed into 50-ml Falcon tubes, snap-frozen in liquid nitrogen and subsequently stored at -80 °C for 15 days prior to RNA extraction. Extraction of RNA was performed in a ribonuclease-free environment applying RNase away (Labkem Ltd, Ireland) and 0.1 % diethylpyrocarbonate (DEPC) (BDH Laboratory Supplies, UK) to treat H2O and buffers. RNA extractions were performed in a Class I Microflow Biological Safety Cabinet (MDH Ltd, UK) using dedicated pipettes and aerosol barrier tips. The application of a combined method involving initial homogenisation of approximately 15 g of placental tissue using an automatic homogeniser and TRIzolÒ reagent followed by a second extraction using the RiboPureTM Kit
123
gave the best quality and highest yield of RNA. The eluted RNA was stored at -80 °C until required. Quantification and visualisation of extracted RNA RNA quality was determined from all samples using the 2100 Biolanalyser (Agilent) using the LabchipTM gels and on a RNA 6000 ladder. DNase treatment of RNA samples RNA samples (10 ll) were treated with DNase to remove contaminating DNA using the DNA-FreeTM DNA Removal Kit (Ambion, UK). DNA free-RNA was stored at -80 °C. cDNA synthesis Reverse transcription of RNA (1 lg) was performed using M-MLV (20 U) Reverse transcriptase (Promega, UK) and RNase Inhibitor (20 U) (Ambion, UK) as detailed by the manufacturer and the cDNA was stored at -20 °C until required. Primer and probe design The gene sequences for b-actin (NM_001101) and prothrombin (NM_000506) were available in the GenBank database. PCR primers and 50 exonuclease probes were designed using the Primer 3 program (http://frodo.wi.mit. edu/cgi-bin/primer3/primer3_www.cgi), in accordance with recommended guidelines (LightCyclerTM Operator’s
Ir J Med Sci
Manual). The sequences below were found to be optimal for amplification and detection of prothrombin and b-actin transcripts; Prothrombin Primer forward 50 CCgCATCACTgACAACATgTTC30 Primer reverse 50 AgCggTTgTTAAAggggCTCT30 Probe 50 6FAM-ACAAgCCTgATgAAgggAAACgAggggAT–DQ30 b-actin Primer forward 50 CAggATgCAgAAggAgATCACT30 Primer reverse 50 ACATCTgCTggAAggTggAC30 Probe 50 6FAM-CgATCCACACggAgTACTTgCgC– 0 DQ3 . The PCR primers were designed across introns. All the 50 exonuclease probes were synthesised by TIB MOLBIOL, Germany with the reporter dye 6-carboxyfluorescein (60 -FAM) at the 50 end, and the dark DABCYL (DQ) quencher at the 30 end. An independent cDNA synthesis step was followed by PCR on the LightCyclerTM using the LightCycler Fast Start DNA Master Hybridisation Probes Kit (Roche Molecular Systems, Germany) and appropriate 50 exonuclease probes. Aliquots (18 ll) of this mixture were added to the LightCyclerTM glass capillaries along with 2 ll of cDNA from the study samples or DNA standards. The optimised thermocycling conditions included: a denaturing step for 1 cycle at 95 °C for 10 min, PCR amplification for 45 cycles at 95 °C for 10 s, annealing and extension at 65 °C for 40 s and a final cooling step for 1 cycle at 40 °C for 30 s. Each study sample cDNA was analysed in duplicate within the same run and in two independent LightCyclerTM runs for prothrombin and bactin gene expression. Generation of standard curves for prothrombin and bactin Standard curves were generated from known concentrations of prothrombin and b-actin PCR products amplified from cDNA on the LightCyclerTM. The PCR products were analysed on a 2 % agarose gel excised from the gel and purified using the Gel extraction Kit (Qiagen, Germany) according to manufacturer’s instructions. After purification, the concentration of PCR products in molecules/ll was determined using the following formula [9]: Copy number ðmol/llÞ concentration of transcript RNAðg=llÞ ¼ size transcript ðbpÞ Y0 ðg/molÞ Serial dilutions of the prothrombin and b-actin PCR
products (1 9 107 to 19 103 molecules) were analysed in quadruplet by real-time PCR on the LightCyclerTM and the mean values obtained for each replicate of the five specific dilutions were used to generate external standard curves for the prothrombin and b-actin assays. The PCR efficiencies of prothrombin and b-actin amplification were determined using the formula E = 10-1/slope, where E is the efficiency value. Efficiency values of 1.94 and 1.99 were determined for b-actin and prothrombin, respectively. Channel setting F1 and the second derivative maximum analysis mode with arithmetic baseline were selected for analysis using the LightCyclerTM Software. DNA standards were generated to be used as the standard curves for determining the expression levels of prothrombin and bactin mRNA in each of the study samples. A mean value for prothrombin and for b-actin copy number was determined for each sample, and the expression data for prothrombin were normalised to b-actin expression data in each run as follows:
Sample Y = run 1 mean concentration of prothrombin/run 1 mean concentration of b-actin = A ‘‘Normalised Copy number of prothrombin transcripts in run 1’’ Sample Y = run 2 mean concentration of prothrombin/run 2 mean concentration of b-actin = B ‘‘Normalised Copy number of prothrombin transcripts in run 2’’ Mean AB values = Normalised value for prothrombin transcript copy number in sample Y
Statistical analysis Independent Student’s t test from the software package SPSS (version 11.5) was used to compare prothrombin gene expression in pregnant women with APCR to expression in pregnant women APC normal. Box plots from the same software package were also applied to the data results to visualise and evaluate how asymmetric the distribution of the data was.
Results The normalised expression values obtained for prothrombin expression were 4.771E-0 for the APCR group and 4.505E-0 for the APC normal group. The b-actin gene remained stable in all samples of this study. Figure 2 shows a side by side box plot for the data indicating also, the outlier samples in the APCR and the APC normal groups. Application of a student t test to the two data sets (APCR versus APC normal) showed no statistically significant
123
Ir J Med Sci
linked to other impaired haemostatic factors inhibited by APC under normal circumstances. Therefore, further investigation of other factors that could link APCR with high fibrin deposition may be merited including FV and FVIII in the procoagulant system, and TAFI and PAI-1 in the fibrinolytic system. Acknowledgments The authors gratefully acknowledge the pregnant women who consented to be involved in the study and the midwives from the labour ward at the UCHG. This work was supported by the Irish Health Research Board. I wish to express my gratitude also to Dr. D. Mongan, UCH Galway, for providing the information on adverse outcomes of the subjects included in the study. The Irish Health Research Board supported this work.
References Fig. 2 Prothrombin expression in our APC resistance group versus our APC normal group
differences in prothrombin mRNA levels between the groups.
Discussion Low levels of prothrombin in the blood results in insufficient prothrombin to promote blood clotting and hence leads to abnormal bleeding after delivery in pregnancy [10]. High prothrombin levels in plasma, however, enhance the risk of thrombosis resulting in thrombophilia [11]. Although thrombophilia may lead to increased fibrin deposition, it has been shown that this does not always inevitably end with an adverse outcome [11]. From the maternity records of the present study, we identified that two subjects (1,157 and 1,266 on Fig. 1) within the APCR group had previous EPL, whereas neither of the APC normal subjects with higher levels of mRNA had EPL. Subject 1,266 also had PE during pregnancy. No other adverse outcomes investigated were identified in the subjects who showed outliers values in the expression of prothrombin transcripts. Castoldi et al. [12] shows that elevated prothrombin in plasma is related to APCR measured by the common aPTT-based test. We found that APCR subjects with higher fibrin deposition were not producing more prothrombin transcript than the APC normal cohort. The causes for the previously identified excess of fibrin deposition in the placenta of these APCR women could be
123
1. Holmes VA, Wallace JM (2005) Haemostasis in normal pregnancy: a balancing act? Biochem Soc Trans 33(Pt 2):428–432 2. Jamison CS, Degen SJ (1991) Prenatal and postnatal expression of mRNA coding for rat prothrombin. Biochim Biophys Acta 1088(2):208–216 3. Narayanan S (1999) Multifunctional roles of thrombin. Ann Clin Lab Sci 29(4):275–280 4. Esmon CT, Owen WG (2004) The discovery of thrombomodulin. J Thromb Haemost 2(2):209–213 5. Carrieri C, Galasso R, Semeraro F et al (2010) The role of thrombin activatable fibrinolysis inhibitor and factor XI in platelet-mediated fibrinolysis resistance: a thromboelastographic study in whole blood. J Thromb Haemost 9(1):154–162 6. Verspyck E, Marpeau L (2005) Thrombophilias and vascular placental pathology. A survey of the literature. Rev Med Interne 26(2):103–108 7. Vora S, Shetty S, Salvi V, Satoskar P, Ghosh K (2008) Thrombophilia and unexplained pregnancy loss in Indian patients. Natl Med J India 21(3):116–119 8. Sedano S, Gaffney G, Mortimer G et al (2008) Activated protein C resistance (APCR) and placental fibrin deposition. Placenta 29(9):833–837 9. Fronhoffs S, Totzke G, Stier S et al (2002) A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction. Mol Cell Probes 16(2):99–110 10. Meeks SL, Abshire TC (2008) Abnormalities of prothrombin: a review of the pathophysiology, diagnosis, and treatment. Haemophilia 14(6):1159–1163 11. Kujovich JL (2011) Prothrombin-related thrombophilia. In: Pagon RA, Bird TD, Dolan CR et al (eds) GeneReviews. University of Washington, Seattle. http://www.ncbi.nlm.nih.gov/ books/NBK1148/ 12. Castoldi E, Simioni P, Tormene D et al (2007) Differential effects of high prothrombin levels on thrombin generation depending on the cause of the hyperprothrombinemia. J Thromb Haemost 5(5):971–979
