The Silence Speaks, but We Do Not Listen: Synonymous c.1824C>T Gene Variant in the Last Exon of the Prothrombin Gene as a New Prothrombotic Risk Factor.

BACKGROUND: Thrombosis is a major global disease burden with almost 60% of cases related to underlying heredity and most cases still idiopathic. Synonymous single nucleotide polymorphisms (sSNPs) are considered silent and phenotypically neutral. Our previous study revealed a novel synonymous FII c.1824C>T variant as a potential risk factor for pregnancy loss, but it has not yet been associated with thrombotic diseases. METHODS: To determine the frequency of the FII c.1824C>T variant we have sequenced patients' DNA. Prothrombin RNA expression was measured by quantitative PCR. Functional analyses included routine hemostasis tests, western blotting and ELISA to determine prothrombin levels in plasma, and global hemostasis assays for thrombin and fibrin generation in carriers of the FII c.1824C>T variant. Scanning electron microscopy was used to examine the structure of fibrin clots. RESULTS: Frequency of the FII c.1824C>T variant was significantly increased in patients with venous thromboembolism and cerebrovascular insult. Examination in vitro demonstrated increased expression of prothrombin mRNA in FII c.1824T transfected cells. Our ex vivo study of FII c.1824C>T carriers showed that the presence of this variant was associated with hyperprothrombinemia, hypofibrinolysis, and formation of densely packed fibrin clots resistant to fibrinolysis. CONCLUSION: Our data indicate that FII c.1824C>T, although a synonymous variant, leads to the development of a prothrombotic phenotype and could represent a new prothrombotic risk factor. As a silent variant, FII c.1824C>T would probably be overlooked during genetic screening, and our results show that it could not be detected in routine laboratory tests.

Prothrombin 3'end gene variants in isolated pulmonary embolism--the first report of FIIc.*64_*66del and FIIc.*303T>C variants.

OBJECTIVE: Pulmonary embolism is usually considered as a complication of deep vein thrombosis, but there are still a number of cases of isolated pulmonary embolism. We aimed to investigate whether prothrombin 3'end gene variants might play a significant role in the pathogenesis of isolated pulmonary embolism. METHODS AND RESULTS: In this study 100 patients with isolated pulmonary embolism and 100 controls were screened by DNA sequencing. Screening included last intron, last exon, 3'UTR and part of the 3'FR region of the prothrombin gene. Our results have shown that heterozygous carriers of the FIi G2021 OA variant have a significantly higher risk of isolated pulmonary embolism (OR 4.83; 95% CI 1.33-17.52; P=0.02). Carriers of the Ili 19911GG genotype (OR 1.41; 95% CI 0.72-2.73; P=0.31) and FII 20068CT genotype (OR 3.06; 95% CI 0.31-29.95; P=0.34) were more frequent in patients with isolated pulmonary embolism compared to controls. We also detected the novel gene variants, FIIc.*64_*66del and FII c.*303T>C, in two patients. CONCLUSIONS: Our results suggest that FII G20210A represents a significant risk factor for isolated pulmonary embolism. The FII G19911A and FII C20068T are potentially associated with an increased risk for the occurrence of isolated pulmonary embolism, but the results did not reach statistical significance. This is the first study in which the two novel 3'end prothrombin gene variants, FIIc.*64_*66del and FlI c.*303T>C, were reported.

Determination of transgene copy number in stably transfected mammalian cells by PCR-capillary electrophoresis assay.

The quantitative determination of transgene copy number in stably transfected mammalian cells has been traditionally estimated by Southern blot analysis. Recently, other methods have become available for appraisal of gene copy number, such as real-time PCR. Herein we describe a new method based on a fluorescently labeled PCR, followed by capillary electrophoresis. We amplified our target gene (prothrombin) and the internal control originating from genomic DNA (18S rRNA) in the same PCR tube and calculated the mean peak height ratio of the target:control gene for every cell clone sample. With this approach we identified stably transfected cell clones bearing the same transgene copy number. The results of our assay were confirmed by real-time PCR. Our method proves to be fast, low-cost, and reproducible compared with traditionally used methods. This assay can be used as a rapid screening tool for the determination of gene copy number in gene expression experiments.

Effect of prothrombin Belgrade mutation, causing antithrombin resistance, on fibrin clot properties.

INTRODUCTION: Prothrombin Belgrade mutation is the result of the c.1787G>A substitution in the prothrombin gene. It is located in the antithrombin and sodium binding site and leads to impaired inactivation of thrombin by antithrombin, resulting in antithrombin resistance and thrombotic disorders. However, it negatively affects sodium binding and may have hypocoagulant effects. Considering that prothrombin Belgrade mutation mechanism is still not fully elucidated and that sodium binding is important for thrombin affinity towards fibrinogen, our aim was to determine whether this mutation affects fibrin clot formation and lysis. METHODS: Using HEK293T cell line, recombinant wild type and mutated prothrombin were generated by transient transfection. Samples that correspond to plasma of a non-carrier, heterozygous and homozygous carriers were reconstituted using prothrombin deficient plasma and recombinant proteins. Reconstituted samples were used in OHP assay (Overall Hemostasis Potential) to determine kinetic profiles of coagulation and fibrinolysis. Clot turbidity assay was performed to observe kinetics of clot formation and lysis more closely. Fibrin clots formed in reconstituted plasma samples were analyzed by confocal microscopy to determine density of fibrin network. Fibrin clots were additionally observed using electron microscopy to determine thickness of individual fibrin fibers. RESULTS: No significant difference found in OHP, OCP, OFP, and fibrin network density between wild type, heterozygous, and homozygous carrier reconstituted plasma samples. There were significant differences between samples for slope and slope time parameters in kinetic profiles and fibrin fiber thickness. CONCLUSIONS: Results indicate that prothrombin Belgrade mutation has no significant impact on fibrinolysis, however it may affect kinetics of clot formation and its architecture.

Prothrombin Knockdown Protects Podocytes and Reduces Proteinuria in Glomerular Disease.

Chronic kidney disease (CKD) is a leading cause of death, and its progression is driven by glomerular podocyte injury and loss, manifesting as proteinuria. Proteinuria includes urinary loss of coagulation zymogens, cofactors, and inhibitors. Importantly, both CKD and proteinuria significantly increase the risk of thromboembolic disease. Prior studies demonstrated that anticoagulants reduced proteinuria in rats and that thrombin injured cultured podocytes. Herein we aimed to directly determine the influence of circulating prothrombin on glomerular pathobiology. We hypothesized that (pro)thrombin drives podocytopathy, podocytopenia, and proteinuria. Glomerular proteinuria was induced with puromycin aminonucleoside (PAN) in Wistar rats. Circulating prothrombin was either knocked down using a rat-specific antisense oligonucleotide or elevated by serial intravenous infusions of prothrombin protein, which are previously established methods to model hypo- (LoPT) and hyper-prothrombinemia (HiPT), respectively. After 10 days (peak proteinuria in this model) plasma prothrombin levels were determined, kidneys were examined for (pro)thrombin co-localization to podocytes, histology, and electron microscopy. Podocytopathy and podocytopenia were determined and proteinuria, and plasma albumin were measured. LoPT significantly reduced prothrombin colocalization to podocytes, podocytopathy, and proteinuria with improved plasma albumin. In contrast, HiPT significantly increased podocytopathy and proteinuria. Podocytopenia was significantly reduced in LoPT vs. HiPT rats. In summary, prothrombin knockdown ameliorated PAN-induced glomerular disease whereas hyper-prothrombinemia exacerbated disease. Thus, (pro)thrombin antagonism may be a viable strategy to simultaneously provide thromboprophylaxis and prevent podocytopathy-mediated CKD progression.

Impaired Fibrinolysis Is Linked With Digital Vasculopathy and Onset of New Digital Ulcers in Systemic Sclerosis.

OBJECTIVE: To assess thrombin generation, fibrin formation, and structure together with the fibrinolytic status in patients with systemic sclerosis (SSc) in relation to the occurrence of digital ulcers (DUs) during the course of disease. METHODS: We studied variables of endothelial dysfunction, thrombin generation, overall hemostatic potential, and fibrin clot turbidity in plasma from 58 patients with SSc (39 with DU history and 19 DU-naïve) and 46 matched healthy controls (HCs). Fibrin structure was visualized using scanning electron microscopy (SEM). Finally, 39 patients with a history of DUs were followed for 1.5 years and the predictive value of all investigated markers for new DU onset was explored. RESULTS: Significantly enhanced endogenous thrombin potential (ETP) and prolonged clot lysis time (CLT) were found in patients with DUs compared to HCs. CLT was prolonged in patients with DUs compared to those without, showing good validity in identifying DUs with an area under the curve of 0.7 (95% CI 0.6-0.8). The levels of ETP and intercellular adhesion molecule 1 were independently associated with CLT. Over the follow-up period, 20 patients developed new DUs. CLT was prolonged (P < 0.001) in patients with new DU episodes, especially those with recurrent DUs. Regression analysis showed that the Raynaud phenomenon visual analog scale and CLT were predictors of new DUs (OR 1.1, 95% CI 1.0-1.1 and OR 1.2, 95% CI 1.1-1.3, respectively). SEM confirmed denser fibrin clots in patients with new DUs. CONCLUSION: Our results suggest that impaired fibrinolysis might have an emerging role in underlying digital vasculopathy and its progression in SSc.

Increased Expression of Extracellular Vesicles Is Associated With the Procoagulant State in Patients With Established Rheumatoid Arthritis.

This study sought to identify different subpopulations of extracellular vesicles (EVs) in plasma from female patients with established rheumatoid arthritis (RA) in relation to the activation of coagulation and fibrin formation in these patients. Forty women were included in the study, 20 patients and 20 age-matched healthy controls. The mean disease duration in patients was 13.0 (5.0-25.0) years, with medium to high disease activity despite ongoing treatment with low-dose prednisolone and methotrexate. There were no differences between the investigated groups regarding the presence of traditional cardiovascular risk factors. The concentration of phosphatidylserine-positive (PS+) EVs; platelet (CD42a+), leucocyte (CD45+), monocyte (CD14+), and endothelial (CD144+)-derived EVs; and EVs-expressing tissue factor (CD142+), P-selectin (CD62P+), and E-selectin (CD62E+) were determined by flow cytometry analysis. Overall hemostasis potential (OHP) was assessed to follow the hemostatic disturbances, including the parameters for overall coagulation potential (OCP) and overall fibrinolytic potential (OFP). Fibrin clot turbidity was measured together with clot lysis time, and scanning electron microscopy was performed. Increased concentrations of PS+, CD42a+, CD142+, CD45+, CD14+, and CD62P+ EVs were found in plasma from patients with RA compared to healthy controls, and the concentrations of PS+, CD42a+, CD14+, and CD62P+ EVs were positively correlated with the inflammatory parameters in RA patients. Positive correlations were also found between the levels of PS+ and CD42a+ EVs and OCP as well as between the levels of PS+, CD42a+, and CD62P+EVs and OHP. The levels of PS+, CD42a+, CD14+, CD62P+, and CD62E+ EVs were negatively correlated with OFP. Elevated levels of circulating EVs of different cell origins were found in patients with established RA, in relation to the inflammatory burden and coagulation activation in the disease.

Synergistic Effect of Bypassing Agents and Sequence Identical Analogue of Emicizumab and Fibrin Clot Structure in the In Vitro Model of Hemophilia A.

Development of inhibitors to factor VIII (FVIII) occurs in approximately 30% of severe hemophilia A (HA) patients. These patients are treated with bypassing agents (activated prothrombin complex concentrate [aPCC] and recombinant activated FVII-rFVIIa). Recently, a bispecific FIX/FIXa- and FX/FXa-directed antibody (emicizumab) has been approved for the treatment of HA patients with inhibitors. However, the data from clinical studies imply that coadministration of emicizumab and bypassing agents, especially aPCC, could have a thrombotic effect. This study was aimed to address the question of potential hypercoagulability of emicizumab and bypassing agents' coadministration, we have investigated fibrin clot formation and structure in the in vitro model of severe HA after adding sequence-identical analogue (SIA) of emicizumab and bypassing agents. Combined overall hemostasis potential (OHP) and fibrin clot turbidity assay was performed in FVIII-deficient plasma after addition of different concentrations of SIA, rFVIIa, and aPCC. Pooled normal plasma was used as control. The fibrin clots were analyzed by scanning electron microscopy (SEM). OHP and turbidity parameters improved with the addition of aPCC, while therapeutic concentrations of rFVIIa did not show substantial improvement. SIA alone and in combination with rFVIIa or low aPCC concentration improved OHP and turbidity parameters and stabilized fibrin network, while in combination with higher concentrations of aPCC expressed hypercoagulable pattern and generated denser clots. Our in vitro model suggests that combination of SIA and aPCC could potentially be prothrombotic, due to hypercoagulable changes in fibrin clot turbidity and morphology. Additionally, combination of SIA and rFVIIa leads to the formation of stable clots similar to normal fibrin clots.

Diagnostic Accuracy in Acute Venous Thromboembolism: Comparing D-Dimer, Thrombin Generation, Overall Hemostatic Potential, and Fibrin Monomers.

Introduction For acute venous thromboembolism (VTE), a biomarker with higher specificity than D-dimer would be of great clinical use. Thrombin generation and overall hemostatic potential (OHP) reflect the hemostatic balance by globally assessing multiple coagulation factors and inhibitors. These tests discriminate between healthy controls and patients with a prothrombotic tendency but have yet to be established as clinical biomarkers of VTE. Objective This study compares endogenous thrombin potential (ETP) and OHP to D-dimer and fibrin monomers (FM) in outpatients with suspected VTE. Methods A cross-sectional diagnostic study where 954 patients with suspected pulmonary embolism or deep venous thrombosis were recruited consecutively from the medical emergency department at Karolinska University Hospital. D-dimer, FM, OHP, and ETP were analyzed in a subpopulation of 60 patients with VTE and 98 matched controls without VTE. VTE was verified either by ultrasonography or computed tomography and clinical data were collected from medical records. Results Compared with healthy controls, both VTE and non-VTE patients displayed prothrombotic profiles in OHP and ETP. D-dimer, FM, ETP area under the curve (AUC), and ETP T lag were significantly different between patients with VTE and non-VTE. The largest receiver-operating characteristic AUCs for discrimination between VTE and non-VTE, were found in D-dimer with 0.94, FM 0.77, and ETP AUC 0.65. No useful cutoff could be identified for the ETP or the OHP assay. Conclusion Compared with D-dimer, neither ETP nor OHP were clinically viable biomarkers of acute venous thrombosis. The data indicated that a large portion of the emergency patients with suspected VTE were in a prothrombotic state.

Evaluation of global haemostatic assays and fibrin structure in patients with pre-eclampsia.

INTRODUCTION: Haemostatic balance shifted towards hypercoagulability in normal pregnancy is even more pronounced in pre-eclampsia (P-EC). The aim of this study was to analyse haemostatic disturbances and fibrin clot properties in women with pre-eclampsia and to investigate their association with maternal and foetal outcomes. METHODS: Forty-six pregnant women diagnosed with pre-eclampsia were included in the study, with blood sampling done on the morning following admission to hospital, as well as after delivery (mean duration 4.8 days). Two global haemostatic assays-endogenous thrombin potential (ETP) and assay of overall haemostatic potential (OHP)-were employed, including fibrin clot turbidity measurements and scanning electron microscopy (SEM) of representative samples. RESULTS: Three thrombin generation parameters (ETP, t_lag and peak height) and OHP were significantly increased in pre-eclampsia compared with controls, whereas overall fibrinolytic potential (OFP-determined as a parameter of the OHP assay) had significantly lower values. Clot lysis time was significantly prolonged in patients with pre-eclampsia. In the pre-eclamptic group after delivery, we observed a significant elevation in the peak height and a reduction in the time to peak and OFP compared with values before delivery. Pre-eclamptic patients with renal complications had significantly higher values for ETP, peak height and D-dimer. Turbidity measurements and SEM revealed dense fibrin structure in patients with pre-eclampsia. CONCLUSION: Patients with pre-eclampsia have enhanced coagulation and impaired fibrinolysis before, and even after, delivery. In particular, the presence of multi-organ dysfunction, such as renal dysfunction, may be associated with increased thrombin generation in pre-eclampsia.

Effects of rivaroxaban and dabigatran on global hemostasis in patients with atrial fibrillation.

: The study was aimed to evaluate the effects of two standard doses of rivaroxaban and dabigatran on global hemostatic assays in patients with atrial fibrillation. The study included 52 patients treated with rivaroxaban (15/20 mg), 50 on dabigatran (110/150 mg) and 20 healthy individuals. Platelet-poor plasma was used for determination of three global hemostatic assays, namely endogenous thrombin potential (ETP), calibrated automated thrombogram (CAT) and overall hemostasis potential (OHP). Rivaroxaban and dabigatran reduced ETP (P < 0.01) although OHP (P < 0.05) was diminished only by dabigatran. Strong correlations were noticed between ETP parameters and the plasma concentrations of rivaroxaban (ETP, r = -0.51; c-max, r = -0.85; t-lag, r = 0.83; t-max, r = 0.66) as well as with plasma concentration of dabigatran (ETP, r = -0.75; c-max, r = -0.74; t-lag, r = 0.73; t-max, r = 0.52). Analysis of dabigatran concentrations under 50 ng/ml showed that ETP parameter has area under the concentration-time curve-receiver operating characteristic value of 0.879 (95% confidence interval 0.776-0.980). Dabigatran treatment paradoxically increased area under the concentration-time curve and peak values although rivaroxaban decreased peak values (P < 0.01). However, significant correlation between CAT parameters and plasma concentration of both direct oral anticoagulants was not observed. We confirmed that the CAT assay is inappropriate for estimation of dabigatran effects and is not fully sensitive as regards rivaroxaban. The ETP assay can potentially be the appropriate method for estimation of global hemostatic capacity as regards both direct oral anticoagulants. The role of OHP needs to be confirmed in additional studies. ETP parameter of chromogenic assay has promising potential in exclusion of high plasma concentrations of dabigatran.

Phosphatidylserine positive microparticles improve hemostasis in in-vitro hemophilia A plasma models.

Circulating microparticles (MPs) are procoagulant due to the surface containing phosphatidylserine (PS), which facilitates coagulation. We investigated if MPs improve hemostasis in HA plasma models. MPs isolated from pooled normal human plasma were added to severe, moderate and mild HA plasma models (0%, 2.5%, 20% FVIII). The MPs' effect on hemostasis was evaluated by calibrated automated thrombogram (CAT) and overall hemostasis potential (OHP) assays, while fibrin structure was imaged by standard confocal, stimulated emission depletion (STED) microscopy and scanning electron microscopy (SEM). MPs partially restored thrombin generation and fibrin formation in all HA plasma models. The procoagulant effect of MPs requires PS exposure, to a less extent of contact pathway activation, but not tissue factor exposure or in vitro stimulation of MPs. MPs partially normalized the fibrin structure, and using super-resolution STED, MPs attached to fibrin were clearly resolved. In summary, our results demonstrate that PS positive MPs could improve hemostasis in HA plasma models.

Assessment of hemostatic disturbances in women with established rheumatoid arthritis.

OBJECTIVES: This study was aimed to assess hemostatic disturbances in female patients with established rheumatoid arthritis (RA) in relation to menopausal status and disease activity. METHOD: Ninety women were included in the study, 42 patients and 48 age-matched healthy controls. There were no differences between the investigated groups regarding the presence of traditional cardiovascular risk factors. Two global hemostatic assays were employed, namely endogenous thrombin potential (ETP) and overall hemostasis potential (OHP). The parameters of the ETP assay (ETP, C-max, t-lag, t-max) and OHP assay (overall coagulation potential (OCP) and overall fibrinolytic potential (OFP)) were assessed. Moreover, the parameters of the fibrin clot (lag time, Max Abs, and slope) were measured by clot turbidity and scanning electron microscopy (SEM). Both patients and controls were divided into four subgroups according to menopause status. RESULTS: The premenopausal controls differed significantly from all other subgroups in terms of diminished levels of ETP (p = 0.02), C-max (p = 0.01), OCP (p = 0.02), OHP (p = 0.001), and Max Abs (p = 0.008), while OFP (p = 0.0001) was increased. This tendency was not seen in the premenopausal RA patients compared with the postmenopausal RA patients. SEM images showed denser clots composed of thinner fibers in samples from RA patients. The disease activity measured by DAS28 correlated with OCP and OHP (r = 0.54; p = 0.001 and r = 0.44; p = 0.003, respectively) indicating persistent hypercoagulable condition in the whole group of RA patients. CONCLUSIONS: Our results point towards coagulation activation in premenopausal women with established RA. The patients were well characterized, which enabled assessment in a real-life setting. Key Points • Extensive assessment points towards persistent coagulation activation in premenopausal women with established rheumatoid arthritis. • Impaired thrombin generation and fibrin formation are associated with menopause in healthy women, while rheumatoid arthritis closes the gap within patients regarding menopause. • Fibrin morphology is unfavorably altered and fibrinolysis is decreased in patients with established rheumatoid arthritis. • Increased activity of thrombin activatable fibrinolysis inhibitor (TAFI) may contribute to impaired fibrinolysis in patients with rheumatoid arthritis.

Prothrombin 3'end Gene Variants in Patients With Sporadic Colon Adenocarcinoma.

BACKGROUND/AIM: Thrombin plays significant roles in various types of cancer. However, the expression levels of prothrombin, the thrombin precursor, in cancer remain unclear. Variants of the 3'end of the prothrombin gene lead to increased prothrombin expression. This study aimed to analyze prothrombin 3'end gene variants in colon tumor and adjacent normal tissue samples. MATERIALS AND METHODS: The study group consisted of 93 patients suffering from colon adenocarcinoma. The 3'end of the prothrombin gene was analyzed by DNA sequencing. RESULTS: Three variants, all previously associated with increased prothrombin expression were detected. Frequency of the FII 19911G allele was 46.77% and 47.85% in tumor and normal tissue, respectively. For the FII 20210A allele, the detected frequencies were 2.15% and 1.61%, respectively. The frequency of the FII c.1824T allele was 0.54% in both tissues. Four patients showed different genotypes in tumor and normal tissue. CONCLUSION: Prothrombin 3' end gene variants may play a role in colorectal cancer.

Are Prothrombotic Mutations a Time-to-Event Risk Factor?

BACKGROUND: Deep vein thrombosis (DVT) represents a common disorder involving genetic and acquired risk factors. It has been proposed that acquired risk factors are more important with aging than genetic factors, indicating different prevalence of prothrombotic mutations throughout the lifespan. OBJECTIVE: To determine the role of the most frequent prothrombotic genetic risk factors (Factor V [FV] Leiden and Factor II [FII] G20210A mutations) in first-time DVT etiology in patients of different ages. METHOD: This retrospective study included 701 patients living in Serbia with diagnosed DVT as a first-time thrombotic event. RESULTS: Risk assessment for mutations as age-related markers showed no statistical difference (FV Leiden mutation-OR, 1.027; 95% confidence interval [CI], .87-1.22; P = .76 and FII G20210A mutation-OR, 0.940, 95% CI, .74-1.19; P = .61). Our results show similar mutation prevalence regardless of how old the patients were at the time of the first DVT occurrence. CONCLUSION: Our results indicate that these 2 mutations cannot be used as prognostic marker for time-to-event first DVT in the Serbian population; however, further studies are required.

Factor V Leiden mutation and high FVIII are associated with an increased risk of VTE in women with breast cancer during adjuvant tamoxifen - results from a prospective, single center, case control study.

BACKGROUND: Estimates of the risk ratio of tamoxifen-associated venous thromboembolism (VTE) in breast cancer patients range from 2.4 to 7.1. The occurrence of thrombosis in patients with breast cancer complicates the clinical condition and causes a change of treatment. Our study was conducted in order to investigate the influence of patient-related risk factors for thrombosis development in breast cancer patients whose treatment included adjuvant tamoxifen. METHODS: The prospective, single center, case control study included 150 breast cancer women, 50 whom developed venous thrombosis during adjuvant tamoxifen and 100 whom did not have thrombosis, as a control group. Patient-related risk factors such as: age, body mass index, previous VTE, varicose veins, concomitant diseases, the presence of prothrombotic mutations (FV Leiden, FII G20210A) and FVIII activity were evaluated in both groups. RESULTS: In respect of prothrombotic mutations, the FV Leiden mutation was present in a higher number of women from the VTE group (10/50 vs 7/100; P=0.020). Additionally, FVIII activity was significantly higher in the VTE group; median (IQR), of 1.79 (0.69) vs 1.45 (0.55); P<0.001 and more women in this group (24/50 vs 34/100) had increased FVIII activity; P=0.020. In those women with FVIII>1.5IU/ml, who were carriers of prothrombotic mutations, an OR of 3.76 (CI 95% 1.276-11.096; P=0.016) was obtained for VTE. CONCLUSION: The results of our study showed that the factor V Leiden mutation and high FVIII are associated with an increased risk of VTE in women with breast cancer during adjuvant tamoxifen.

The 3'end prothrombin gene variants in patients with different thrombotic events.

BACKGROUND: Prothrombin (FII) A19911G and C20221T gene variants are associated with increased prothrombin levels and potentially represent thrombotic risk factors. OBJECTIVE: To determine the frequency of A19911G and C20221T FII gene variants in patients with thrombotic disorders and in women who have experienced pregnancy loss (PL). METHODS: We determined the frequency of these variants in 133 patients with deep venous thrombosis (DVT), 80 patients with isolated pulmonary embolism (PE), 101 patients with idiopathic PL, and 180 control individuals. RESULTS: The FII A19911G variant was more prevalent in patients with DVT and with PL compared with controls; however, these differences were not statistically significant. The 19911GG genotype was associated with increased risk of PE (odds ratio, 1.91; 95% confidence interval, 1.04-3.51). We did not detect carriers of the FII C20221T gene variant in this study. CONCLUSIONS: This is the first study, to our knowledge, that demonstrates the FII 19911GG genotype may represent a risk factor for isolated PE. Also, our results show that the FII C20221T is a rare variant in this population and therefore, routine thrombophilia screening should not include screening for this genotype.

+1040 C/T polymorphism in coding region of thrombin-activatable fibrinolysis inhibitor gene and the risk of idiopathic recurrent fetal loss.

Recurrent fetal loss (RFL) is common health problem affecting up to 5% of women of reproductive age. It has been shown that plasma thrombin-activatable fibrinolysis inhibitor (TAFI) concentrations increase during pregnancy and return to baseline levels soon after delivery. The +1040C/T single nucleotide polymorphism in coding region of TAFI gene is associated with TAFI blood levels. The aim of our study was to investigate the relationship between +1040C/T polymorphism in TAFI gene and idiopathic RFL. Study was carried out in a group of 120 women (61 controls and 59 women with idiopathic RFL). The +1040C/T polymorphism was detected by restriction fragment length polymorphism PCR. Increased frequency of +1040T/T genotype was observed in a study group, but without statistically significant difference. Carriers of T/T genotype have increased risk of fetal loss by 1.23-fold, compared with carriers of C/C (95% CI 0.462-3.277; P = 0.7) and 1.34-fold compared with carriers of C/T genotype (95% CI 0.501-3.601; P = 0.6). C allele is associated with reduced risk of recurrent fetal loss compared with T allele (OR 0.91; 95% CI 0.545-1.533; P = 0.7). In conclusion, we observed increased frequency of +1040T/T genotype in a patient group, suggesting that this genotype could be potential risk factor for idiopathic RFL. Further investigation should be carried out in order to establish the role of this polymorphism in the etiology of idiopathic recurrent miscarriages.