Prothrombin Gene Research Articles

Structure-function studies of prothrombin Amrita, a dysfunctional prothrombin characterized by point mutation at Arg553 → Gln

A dysfunctional prothrombin gene characterized by novel point mutation at Arg553 to Gln residue in Deep vein thrombosis (DVT) patient which we designated as “Prothrombin Amrita” was previously reported from our lab. The mutation occurred at nucleotide 20030 in exon 14 and was confirmed by restriction enzyme digestion. Arg553 has been reported as one of the key residues for the binding of cofactor Na+ ion in the thrombin protein. Structural analysis revealed the molecular mechanism behind the coagulant form of thrombin due to point Arg553Gln mutation near the cofactor Na+ ion region. Molecular electrostatic potential maps and molecular dynamics (MD) simulation of the wild type and mutated thrombin showed the key role played by the Na+ ion for its coagulant mechanism by analysing the charge distribution and nature of the hydrogen bonding at the mutated region of interest. We observed maintenance of the fast or procoagulant form of dysfunctional prothrombin due to changes in the charge distribution by this mutation and thereby also keeping strong hydrogen bonding network revealed by MD simulation between prothrombin and Na+ ion. This molecular mechanism might be the main cause for DVT in patients with this dysfunctional prothrombin gene.

Abstract TP160: May-Thurner Syndrome is Associated With Stroke Due to Paradoxical Embolism

Background: May-Thurner Syndrome (MTS) is a potential risk factor for cryptogenic stroke (CS). While retrospective reports have found important association with MTS in CS, prospective data has been lacking. In this study, we prospectively explored the epidemiology and clinical correlates of MTS patients with cryptogenic stroke. Method: Eligible patients were prospectively recruited in accordance with an IRB-approved protocol. All patients underwent full evaluation of etiopathogenesis of stroke such as MRI/MRA, long-term cardiac telemetry (>30 days), echocardiography, and extensive thrombophilia screen (AT3, FV Leiden, Proteins C and S; prothrombin gene mutations, APLAbs, lipoprotein(a), homocysteine) and 1.5T pelvic MRV. Results: We prospectively recruited 1,131 acute ischemic stroke patients, with 59 patients with PFO-related CS; 15 (40.5%) had May-Thurner anatomy (MTA), and 6 (16.2%) had MTS. MTS pts are younger (36.33±9.95 vs. 53.35±14.78, p=0.011), with a lower trend for BMI (24.40±4.87 V.S. 31.13±8.75, p=0.051, table 1). Lower BMI is predictive of MTS in CS (<=22) with high specificity 93.3% (AUC=0.756, p=0.007). Conclusion: In this prospective study, we found MTA and MTS to be very common in PFO CS patients - especially in younger patients with lower BMI and without traditional stroke risk factors. MTS is a novel risk factor that may aide in the diagnosis of PFO CS, and needs further study.

Impact of baseline clinical and laboratory features on the risk of thrombosis in children with acute lymphoblastic leukemia: A prospective evaluation.

Children with acute lymphoblastic leukemia (ALL) have increased risk of thromboembolism (TE). However, the predictors of ALL-associated TE are as yet uncertain. This exploratory, prospective cohort study evaluated the effects of clinical (age, gender, ALL risk group) and laboratory variables (hematological parameters, ABO blood group, inherited and acquired prothrombotic defects [PDs]) at diagnosis on the development of symptomatic TE (sTE) in children (aged 1 to ≤18) treated on the Dana-Farber Cancer Institute ALL 05-001 study. Samples collected prior to the start of ALL therapy were evaluated for genetic and acquired PDs (proteins C and S, antithrombin, procoagulant factors VIII (FVIII:C), IX, XI and von Willebrand factor antigen levels, gene polymorphisms of factor V G1691A, prothrombin gene G20210A and methylene tetrahydrofolate reductase C677T, anticardiolipin antibodies, fasting lipoprotein(a), and homocysteine). Of 131 enrolled patients (mean age [range] 6.4 [1-17] years) 70 were male patients and 20 patients (15%) developed sTE. Acquired or inherited PD had no impact on the risk of sTE. Multivariable analyses identified older age (odds ratio [OR] 1.13; 95% confidence interval [CI]: 1.01, 1.26) and non-O blood group (OR 3.64, 95% CI: 1.06, 12.51) as independent predictors for development of sTE. Patients with circulating blasts had higher odds of developing sTE (OR 6.66; 95% CI: 0.82, 53.85). Older age, non-O blood group, and presence of circulating blasts, but not PDs, predicted the risk of sTE during ALL therapy. We recommend evaluation of these novel risk factors in the development of ALL-associated TE. If confirmed, these easily accessible variables at diagnosis can help develop a risk-prediction model for ALL-associated TE.

The incidence of FVL and PT G20210A mutations in patients with idiopathic deep venous thrombosis

Background: Factor V Leiden (FVL), prothrombin gene (PT G20210A), and methylenetetrahydrofolate reductase (MTHFR) C677T mutations are known as molecular biomarkers to evaluate the predisposition of deep venous thrombosis (DVT). These hereditary risk factors affect the natural anticoagulant mechanisms and activate the coagulation mechanisms because of an imbalance between procoagulant and anticoagulant factors. Our study aimed to determine the prevalence of these mutations in Turkish patients presenting with idiopathic DVT. Methods: A total of 135 patients with idiopathic DVT admitted to our clinic were investigated for FVL, PT G20210A, and MTHFR (C677T, A1298C) gene mutations. Screening of polymorphisms was carried out by using SNaPshot® multiplex system (Applied Biosystems Inc.). Wild, heterozygous and homozygous genotypic distributions of these mutations were defined as number and percentage frequency. Results: There were 74 male and 61 female patients ranging in age from 18 to 85 years. FVL mutation was found in 66 (41.47%) patients (12 homozygotes 8.88%, 44 heterozygotes 32.59%), PT G20210A was found in 14 (10.36%) patients (1 homozygous 0.74%, 13 heterozygotes 9.62%). MTHFR C677T was found in 53 (39.25%) patients (10 homozygotes 7.40%, 43 heterozygotes 31.85%), and MTHFR A1298C was found in 84 (62.22%) patients (16 homozygotes 11.85%, 68 heterozygotes 50.37%).

ИНФАРКТ МИОКАРДА В ПОСТПАРТАЛЬНОМ ПЕРИОДЕ У МОЛОДОЙ ЖЕНЩИНЫ С ДЕФИЦИТОМ ПРОТЕИНА С

Women of childbearing age without cardiovascular risk factors have low risk of acute myocardial infarction. Pregnancy has been shown to increase the risk of myocardial infarction compared to the risk in non-pregnant women of similar age. Acute myocardial infarction during pregnancy or postpartum period as a rule develops due to coronary spasm or non-atherogenic thrombosis or spontaneous coronary dissection. Thrombosis is most likely related to hypercoagulable state of pregnancy and postpartum period. It is worth considering the importance of defects of coagulation, such as Leiden factor, protein C, protein S, antithrombin III and mutations of propter genes. There is also the significant role of antiphospholipid antibodies in young women. We present the clinical case of acute ST-elevation myocardial infarction in 34-years old woman without traditional cardiovascular risk factors, which was developed after childbirth due to non-atherogenic left anterior coronary artery thrombosis. Her coagulation profile showed normal results for antithrombin III, protein S, prothrombin gene mutation, factor V Leiden and antiphospholipid antibody syndrome. At the same time the protein C activity decreased to 43 % as well as trombophilia genetic markers MTHFRC677T, MTRR 66A>G, ITGA2:807 C>T and ITGB:1565 T>C genetic polymorphisms were revealed.

Association Between Hypercoagulable Conditions and Calciphylaxis in Patients With Renal Disease

Calciphylaxis is a rare skin disease with high morbidity and mortality that frequently affects patients with renal disease. Hypercoagulable conditions are frequently observed in both patients with calciphylaxis and those with chronic kidney disease (CKD), complicating our understanding of which hypercoagulable conditions are specific to calciphylaxis. To identify hypercoagulable conditions that are risk factors for developing calciphylaxis while controlling for CKD. This was a case-control study, comparing the hypercoagulability status of patients with calciphylaxis and with renal disease with that of a matched control population at 2 large urban academic hospitals in Boston, Massachusetts. Retrospective medical record review of laboratory values was performed to identify patients with hypercoagulable conditions. Case and control patients were further stratified based on both severity of CKD and warfarin. Patients with a dermatologic diagnosis of calciphylaxis between 2006 and 2014 and concomitant CKD were included as cases (n = 38). Three controls (n = 114) per case patient with CKD were included, and were matched by age, sex, and race. The rate of various hypercoagulable states (ie, antithrombin III [ATIII] deficiency, protein C and S deficiency, factor V Leiden mutation, prothrombin gene mutation [G20210A], elevated factor VIII level, lupus anticoagulant, anti-IgG or IgM cardiolipin antibodies, heparin-induced thrombocytopenia antibodies, and elevation of homocysteine) in patients with calciphylaxis compared with their matched controls. Of the calciphylaxis cohort, 28 (58%) were female and 18 (55%) were non-Hispanic white. Among all patients, lupus anticoagulant (13 [48%] positive in cases vs 1 [5%] in controls; P = .001), protein C deficiency (9 [50%] vs 1 [8%]; P = .02), and combined thrombophilias (18 [62%] vs 10 [31%]; P = .02) were found to be significantly associated with calciphylaxis. In a subanalysis of patients with stage 5 CKD, only lupus anticoagulant (12 [53%] vs 9 [0%]; P = .01) and combined thrombophilia (15 [63%] vs 1 [8%]; P = .004) remained significantly associated with calciphylaxis. In a separate subanalysis of warfarin-unexposed patients, only lupus anticoagulant (7 [50%] vs 1 [6%]; P = .01) and protein C deficiency (5 [46%] vs 10 [0%]; P = .04) remained significantly associated with calciphylaxis. Presence of lupus anticoagulant and combined thrombophilias are risk factors for the development of calciphylaxis in patients with late-stage renal disease. Clinicians should be aware of these associations in patients with impaired kidney function and may consider increased screening and appropriate anticoagulation treatment to reduce the risk of calciphylaxis development.

Evaluation and management of acute and chronic portal vein thrombosis in patients with cirrhosis.

Evaluation and management of acute and chronic portal vein thrombosis in patients with cirrhosis.

Common Polymorphisms in MTHFR and Prothrombin Gene in Iranian Women with Abortions at Different Ages

Common Polymorphisms in MTHFR and Prothrombin Gene in Iranian Women with Abortions at Different Ages

Prothrombin G20210A (rs1799963) polymorphism increases myocardial infarction risk in an age-related manner: A systematic review and meta-analysis

G20210A polymorphism (rs1799963) within the prothrombin gene is associated with a higher circulation level of prothrombin, thus increasing the likelihood of developing myocardial infarction (MI). Opinions differ regarding the correlation between prothrombin G20210A genotype and MI risk, which prompted us to conduct a meta-analysis to determine this association. PubMed, EMBASE, Web of Science and CNKI were searched for pertinent reports. A total of 34 studies involving 14 611 MI cases and 84 358 controls were analyzed in this quantitative analysis. We found a statistically significant association between prothrombin G20210A polymorphism and MI in the allele model (A vs. G, OR = 1.43, 95%CI: 1.18–1.72), heterozygote model (GA vs. GG, OR = 1.41, 95%CI: 1.16–1.72) and dominant model (GA + AA vs. GG, OR = 1.41, 95%CI: 1.15–1.72). The association remains significant in Caucasians but not in non-Caucasians. Moreover, prothrombin G20210A polymorphism increases MI risk in an age-related manner. A further significant association was found in a subpopulation younger than 55 years (allele model, OR = 1.76, 95%CI: 1.32–2.35; heterozygote model, OR = 1.70, 95%CI: 1.24–2.33; dominant model, OR = 1.70, 95%CI: 1.24–2.34). Sensitivity analysis and publication bias analysis revealed stable and statistically robust results. Our meta-analysis demonstrated that prothrombin G20210A polymorphism may represent a risk factor for MI.

In vitro exploration of latent prothrombin mutants conveying antithrombin resistance

IntroductionAntithrombin resistance (ATR) prothrombinemia is an inherited thrombophilic disorder caused by missense mutations in prothrombin gene (F2) at Arg596 of the sodium-binding region. Previously, prothrombin mutants Yukuhashi (Arg596Leu), Belgrade (Arg596Gln), and Padua 2 (Arg596Trp) were reported as ATR-prothrombins possessing a risk of familial venous thrombosis. To identify additional F2 mutations causing the ATR-phenotype, we investigated the coagulant properties of recombinant prothrombins mutated at amino acid residues within the sodium-binding region by single nucleotide substitutions (Thr540, Arg541, Glu592, and Lys599). Materials and methodsWe constructed expression vectors of prothrombin mutants, established stably transfected HEK293 cells, and isolated the recombinant prothrombin proteins. We evaluated procoagulant activity and ATR-phenotypes of those mutants in reconstituted plasma by mixing with prothrombin deficient plasma. ResultsThe secreted quantity of all prothrombin mutants was the same as that of the wild-type prothrombin. Procoagulant activity of each mutant varied from 1.7% to 79.5% in a one-stage clotting assay and from 2.0% to 104.5% in a two-stage chromogenic assay. Most prothrombin mutants tested presented with a severe ATR-phenotype. To estimate the thrombosis risk of these mutations, we determined the residual clotting activity (RCA) after 30min inactivation with antithrombin. RCA scores, normalized to the wild-type, revealed that prothrombin mutants Lys599Arg (5.35) and Glu592Gln (4.71) had high scores, which were comparable with prothrombins Yukuhashi (4.36) and Belgrade (5.19). ConclusionsMutation of prothrombin at the sodium-binding site caused ATR-phenotypes. Of those tested, Lys599Arg and Glu592Gln may possess a thrombosis risk as large as the known pathogenic prothrombins Yukuhashi and Belgrade.

Venous thromboembolism and hyperhomocysteinemia as first manifestation of pernicious anemia: a case series

BackgroundHyperhomocysteinemia has been suspected of favoring thrombosis. Several case–control studies and even a meta-analysis have confirmed a link between venous thrombosis and hyperhomocysteinemia. Homocysteine is due to genetic and acquired factors (poor diet in folate and vitamin B12, older age, renal impairment, thyroid diseases, and malignancies) induced by the intake and the concentrations of vitamin B9 or B12 in the majority of cases.Cases presentationWe report the cases of four Moroccan patients who presented with acute vein thrombosis of different sites: a 34-year-old man, a 60-year-old man, a 58-year-old man, and a 47-year-old woman. All patients had a low level of cobalamin with marked hyperhomocysteinemia with normal serum and red cell folic acid. Venous thrombosis revealed pernicious anemia in all patients. Their low levels of cobalamin, atrophic gastritis, and positive results for gastric parietal cell antibodies confirmed the diagnosis of pernicious anemia. There was no evidence of immobilization, recent surgery, malignancy, antiphospholipid antibody, myeloproliferative disorder, or hormone replacement therapy. No deficiencies in protein C and protein S were detected; they had normal antithrombin III function and factor V Leiden; no prothrombin gene mutations were detected. Treatment included orally administered anticoagulation therapy and cobalamin supplementation. The outcome was favorable in all cases.ConclusionsThese reports demonstrate that pernicious anemia, on its own, can lead to hyperhomocysteinemia that is significant enough to lead to thrombosis. Understanding the molecular pathogenesis of the development of thrombosis in patients with hyperhomocysteinemia related to Biermer disease would help us to identify patients at risk and to treat them accordingly. The literature concerning the relationship between homocysteine and venous thrombosis is briefly reviewed.

EP16.18: Pregnancy association of abnormal Doppler indices with inherited thrombophilia

Thrombophilia reflects a hypercoagulable condition with an increased thrombogenic potential during pregnancy. The most frequent causes of inherited thrombophilia are the factor V Leiden mutation and the prothrombin gene mutation. Repetitively, it has been hypothesised that thrombophilic mutations associate an increased risk of thrombosis at the low flow maternal-placental interface resulting in placenta-mediated complications, such as miscarriage, fetal loss, pre-eclampsia, fetal growth impairment, and abruption. Our prospective study aimed to determine the impact of thrombophilic mutations on second and third trimester umbilical and uterine artery Doppler parameters. Four hundred and forty-eight pregnant women were tested for the presence of factor V Leiden mutation, prothrombin gene mutation and also for a series of minor thrombophilic mutations. Each patient included in the study has been examined by ultrasound corresponding to its gestational age with the measurement of umbilical and uterine artery velocity. The measured parameters (pulsatility index and resistivity index) were interpreted consistent with gestational age nomograms. The incidence of factor V Leiden mutation was 10.4% in the studied group and the incidence of the prothrombin gene mutation was 4.46%; 11 cases (2.4%) expressed both major mutation. In 6.6% of cases corresponding to second trimester pregnancies with factor V Leiden mutation an increased pulsatility index of umbilical artery was found and 11% of these cases had a high pulsatility index of the uterine artery. For the cases with both major thrombophilic mutations present, Doppler parameters were increased in 4 from 6 cases. Even if inherited thrombophilia is not considered a major risk factor for most adverse pregnancy outcomes, Doppler assessment of fetal status is imperative for high-risk population. In our study, the impact of thrombophilia on Doppler indices was modest and limited to major mutations.

Magnitude of Potentially Inappropriate Thrombophilia Testing in the Inpatient Hospital Setting.

Laboratory costs of thrombophilia testing exceed an estimated $650 million (in US dollars) annually. Quantifying the prevalence and financial impact of potentially inappropriate testing in the inpatient hospital setting represents an integral component of the effort to reduce healthcare expenditures. We conducted a retrospective analysis of our electronic medical record to evaluate 2 years' worth of inpatient thrombophilia testing measured against preformulated appropriateness criteria. Cost data were obtained from the Centers for Medicare and Medicaid Services 2016 Clinical Laboratory Fee Schedule. Of the 1817 orders analyzed, 777 (42.7%) were potentially inappropriate, with an associated cost of $40,422. The tests most frequently inappropriately ordered were Factor V Leiden, prothrombin gene mutation, protein C and S activity levels, antithrombin activity levels, and the lupus anticoagulant. Potentially inappropriate thrombophilia testing is common and costly. These data demonstrate a need for institution-wide changes in order to reduce unnecessary expenditures and improve patient care.

Risk of colorectal cancer and clotting factor gene polymorphisms in Moroccan Population.

Venous thrombosis has been described as a common complication for cancer patients. The association between clotting factor gene polymorphisms and the risk of colorectal cancer has been evidenced. The aim of the present study was to investigate the association of G20210A factor II prothrombin (FII) and factor V Leiden ( FVL) G1691A with the risk of colorectal cancer(CRC). Genotyping of FVL and G20210A FII was performed using the polymerase chain reaction restriction fragment length polymorphism method on a sample of 76 patients with CRC as well as 182 controls. No significant difference in FVL gene variations was observed between cases and controls. However, with regard to the G20210A FII, the homozygous mutated genotype AA was associated with an increased risk of CRC. A significant association between the G20210A FII mutation and the risk of CRC was identified using recessive (OR=57.63, 95% CI: 3.33-997.26, P=0.0053), dominant (OR=27.87, 95% CI: 12.67 -61.28, P<0, 0001) and additive (OR=21.24, 95% CI: 10.45-43.16, P<0, 0001) models. No statistical difference was observed in parameters such as sex, age and positive family history for cancer. Our results did not support an effect of FVL gene on CRC risk and suggested that the G20210A FII prothrombin gene variant may be a risk factor for CRC in Moroccan population.

Double versus single thrombophilias during pregnancy

Objective: The primary objective of this study was to evaluate whether women with double thrombophilias have a greater risk for obstetric complications as compared with women who have single thrombophilias.Study design: This is a retrospective cohort study of all patients in a single practice with a clinically significant inherited thrombophilia and treated with anticoagulation between 2005 and 2013. Thrombophilias evaluated include: factor V Leiden, prothrombin G20210A gene mutation, protein S deficiency, protein C deficiency, and antithrombin III deficiency. Double thrombophilia was defined as the presence of two thrombophilias or homozygosity for factor V Leiden or prothrombin Gene Mutation. Demographic and obstetrical outcome data were collected. Data on all patients with double thrombophilias who met inclusion criteria was reported. Data was then compared between the patients with double thrombophilias and single thrombophilias with singleton gestations. The data was analyzed with Pearson’s chi-squared or Student’s t-test as appropriate with p value <.05 used for significance.Results: Eighteen patients with clinically significant double thrombophilias who met inclusion criteria were identified. Most patients delivered full term (88.9%) and appropriate for gestational age (77.8%) infants. One hundred thirty-two patients with single thrombophilias and 14 patients with double thrombophilias with singleton gestations were then compared. Demographic characteristics were not significantly different between the two groups. There were no significant differences in obstetrical outcomes between patients.Conclusions: There were no significant differences in obstetrical outcomes for patients with clinically significant double thrombophilias versus single thrombophilias when treated with anticoagulation.

Potential inheritance patterns of a prothrombin gene mutation in a 23-year-old female and ethical considerations of a positive diagnosis: a case report

Background: Prothrombin, also called Factor II, is a blood clotting protein found in all individuals that is necessary to form blood clots. In most individuals, a balance between bleeding and blood clot formation occurs. However, in individuals with a mutation in the prothrombin gene, the balance is disrupted due to excess production of prothrombin which leads to an increase in blood clot formation (1). Inherited predispositions to blood clot formation are termed hereditary thrombophilia. Prothrombin G20210A mutations are one of the most common hereditary gene associations. Case Description: This report examines the case of a 23-year-old female who has tested positive for the prothrombin gene mutation. The individual has an extensive history of blood clots including 8 deep vein thromboses (DVTs), 4 pulmonary embolisms with one complete infarction, 4 superficial clots, and a miscarried pregnancy attributed to her thrombophilia. The individual has a significant family history of the mutation and takes Coumadin daily for prevention of further clots. Practical and Ethical Implications: Despite a strong familial history of blood clots and related hospitalizations, the parents of the case individual do not want to get their other children tested for the mutation for fear that the children will be denied insurance coverage in their futures. The case individual will likely continue to be on blood thinners indefinitely. This will affect many aspects of her life, including dental treatment, as she will be at an increased risk for bleeding.

Portomesenteric venous thrombosis complicated by a haemorrhagic shock: a rare complication of laparoscopic gastrectomy

Portomesenteric venous thrombosis is a rare complication reported in only a few cases involving laparoscopic bariatric surgery. We report a case of a 44-year-old woman who presented 14 days after...

Economy class syndrome: what is it and who are the individuals at risk?

The term ‘economy class syndrome’ refers to the occurrence of thrombotic events during long-haul flights that mainly occur in passengers in the economy class of the aircraft. This syndrome results from several factors related to the aircraft cabin (immobilization, hypobaric hypoxia and low humidity) and the passenger (body mass index, thrombophilia, oral contraceptives or hormone replacement therapy, cancer), acting together to predispose to excessive blood coagulation, which can result in venous thromboembolism. Several risk factors, both genetic and acquired, are associated with venous thromboembolism. The most important genetic risk factors are natural anticoagulant deficiencies (antithrombin, protein C and protein S), factor V Leiden, prothrombin and fibrinogen gene mutations and non-O blood group individuals. Acquired risk factors include age, pregnancy, surgery, obesity, cancer, hormonal contraceptives and hormone replacement therapy, antiphospholipid syndrome, infections, immobilization and smoking. People who have these risk factors are predisposed to hypercoagulability and are more susceptible to suffer venous thromboembolism during air travel. For these individuals, a suitable outfit for the trip, frequent walks, calf muscle exercises, elastic compression stockings and hydration are important preventive measures. Hence, it is essential to inform about economic class syndrome in an attempt to encourage Brazilian health and transport authorities to adopt measures, in partnership with the pharmaceutical industry, to prevent venous thromboembolism.

Clinical outcomes and a high prevalence of abnormalities on comprehensive arterial and venous thrombophilia screening in TIA or ischaemic stroke patients with a patent foramen ovale, an inter­atrial septal aneurysm or both

IntroductionData are limited on the optimal management of cryptogenic TIA/stroke patients with a patent foramen ovale (PFO)±inter-atrial septal aneurysm (IASA), especially with an inherited thrombophilia. MethodsProspectively-collected data on TIA/ischaemic stroke patients with PFO, IASA or both who received ‘goal-directed secondary-prevention medical treatment’ were analysed. All patients had trans-oesophageal echocardiography, anti-nuclear, anti-cardiolipin, anti-beta 2 glycoprotein I antibodies, rheumatoid factor, lupus anticoagulant, protein C&S, anti-thrombin, factor VIII activity, activated protein C resistance, Factor V Leiden, prothrombin gene and MTHFR-c.677C>T mutation screening. ENA and homocysteine were assessed in the latter study period. ResultsEighty-three patients were recruited. Mean follow-up: 48.1months. Forty-seven patients (56.6%) had an isolated PFO, 32 (38.6%) a PFO and an IASA, and 4 (4.8%) an IASA alone. Eighteen (21.7%) had ≥1 abnormality on thrombophilia screening. The most important abnormalities which lead to treatment changes in 11 patients (13.3%) were primary anti-phospholipid syndrome (N=3; 3.6%), protein S deficiency (N=2; 2.4%) hyper-homocysteinaemia (N=6/72 screened, 8.3%). Four patients (4.8%) opted for PFO closure: two with protein S deficiency, and two with no identified thrombophilia. Seven (8.4%) had recurrent TIA/ischaemic stroke during follow-up (overall annualised incidence: 2.1%), of whom five had a PFO alone and two a PFO and IASA. DiscussionComprehensive arterial and venous thrombophilia screening is warranted in TIA/ischaemic stroke patients with a PFO±IASA, is conclusively abnormal in over a fifth, and informed important decision-making regarding individualised therapy in 13.3% of patients. The incidence of recurrent vascular events in this population is low on optimal, personalised secondary-prevention treatment, even with an underlying thrombophilia.

Poly-(ADP-Ribose) Polymerase-1 Promotes Prothrombin Gene Transcription and Produces Des-Gamma-Carboxy Prothrombin in Hepatocellular Carcinoma

Background and Aim: Although des-gamma-carboxy prothrombin (DCP) is a well-known tumor marker for hepatocellular carcinoma (HCC), the mechanism of DCP production is unclear. This study aimed to investigate the mechanism how DCP is produced in HCC cells. Methods: Levels of mRNA and DCP were analyzed by real-time polymerase chain reaction and electro-chemiluminescence immunoassay respectively. Secreted alkaline phosphatase (SEAP) expression vectors including deletion mutants of the prothrombin gene promoter were constructed for reporter gene assay. The transcription factors bound to DNA fragments were analyzed by mass spectrometry. An electrophoretic mobility shift assay (EMSA) was performed using a biotin end-labeled DNA. Results: The prothrombin mRNA levels in all 5 DCP producing cell lines were appreciably high. However, those in 2 DCP non-producing cell lines were below detectable levels. A SEAP vector with -2985 to +27 showed a very high transcription activity in DCP-producing Huh-1 cells. However, transcription abruptly decreased when the vector with -2955 to +27 was transfected, and then remained at the similar levels with larger deletion mutants, indicating the existence of a cis-element at -2985 to -2955 (31-bp). Mass spectrometry analysis identified the protein that bound to the 31-bp DNA as poly-(ADP-ribose) polymerase-1 (PARP-1). Knockdown of the PARP-1 gene by small interfering RNA in Huh-1 cells induced marked inhibition of prothrombin gene transcription. The EMSA clearly showed that PARP-1 specifically binds to the 31-bp DNA fragment in the prothrombin gene promoter. Conclusions: Our data suggest that PARP-1 activates prothrombin gene transcription and that the excessive prothrombin gene transcription induces DCP production in DCP-producing HCC cells.