Higher Prothrombin Levels Research Articles

Interferon-stimulated gene 15 in hepatitis B-related liver diseases

This study investigates the association of Interferon-stimulated gene 15 (ISG15) polymorphisms, ISG15 serum levels and expression with HBV-related liver diseases. The ISG15 promoter and the two exons of the gene were screened for polymorphisms in 766 HBV-infected patients and in 223 controls. Soluble ISG15 levels were measured by ELISA. ISG15 mRNA expression was quantified by qRT-PCR in 36 tumor and adjacent non-tumor tissues. The exon 2 allele rs1921A was found associated with decreased progression of HBV-related liver diseases (LC vs. CHB: OR = 0.6, 95%CI = 0.4-0.8, adjusted P = 0.003; HCC vs. CHB: OR = 0.6, 95%CI = 0.4-0.9, adjusted P = 0.005). The rs1921AA genotype was associated with low levels of AST, ALT and total bilirubin, but with high prothrombin levels (P < 0.05). ISG15 serum levels were higher among HBV patients compared to controls (P < 0.0001) and positively associated with HBV-related liver diseases, with highest levels among LC patients. ISG15 levels were correlated with HBV-DNA loads (P = 0.001). In non-tumor tissues from HCC patients, ISG15 mRNA expression was increased in HBV compared to non-HBV infection (P = 0.016). The ISG15 rs1921 variant and ISG15 expression are associated with HBV-related liver diseases. Taken together, ISG15 appears to be a proviral factor involved in HBV replication and triggering progression of HBV-related liver diseases.

Lack of association between the prothrombin rs1799963 polymorphism and juvenile myoclonic epilepsy.

Juvenile myoclonic epilepsy (JME) accounts for 26% of generalized idiopathic epileptic syndromes. The highest levels of thrombin activity are closely involved in the development of neurological diseases, including epilepsy. The prothrombin c.20210G>A (rs1799963) variation, which alters prothrombin mRNA stability, is associated with high plasma prothrombin levels. The present study was designed to investigate whether the SNP rs1799963 is a risk factor for JME in the northeastern Brazilian population. The polymorphism was genotyped in 207 controls and 123 patients using polymerase chain reaction-restriction fragment length polymorphism method. No significant differences were observed in the genotype and allele frequencies of this polymorphism between cases and controls. These results present no evidence for an association of rs1799963 with JME. Further studies including other types of epilepsy are required to investigate the involvement of prothrombin gene in the genetic susceptibility to chronic seizure.

Obesity and thrombin-generation profiles in women with venous thromboembolism

Obesity is a known risk factor for venous and arterial thrombosis but the mechanisms are still unclear. In women, obesity is correlated with low-grade inflammation and recent data show that BMI is positively associated with thrombin generation. We explored the correlations between obesity, inflammation and thrombin generation in women with increased thrombotic risk by looking at a cohort of women with prior venous thrombosis. One hundred and fifty-six women age 18-65 years were enrolled at diagnosis of first venous thromboembolism (VTE). Plasma samples were obtained at least 3 weeks after cessation of anticoagulant treatment. Thrombin generation was determined with the calibrated automated thrombography (CAT) assay and the Innovance ETP assay. Thrombin generation started later but was more pronounced with higher endogenous thrombin generation potential (ETP) determined with CAT in patients with obesity. The Innovance ETP assay showed results consistent with CAT. Furthermore, patients with obesity had significantly higher levels of fibrinogen, C-reactive protein and plasminogen activator inhibitor-I (PAI-I) than patients without obesity. Increased levels of fibrinogen were the main determinant of the prolonged lag-time in patients with obesity whereas higher levels of prothrombin could account for the difference in the ETP between the groups. We found an association between BMI and ETP values using two different methods to measure thrombin generation. Obesity correlated with increased thrombin generation in women with VTE and the main determinants of this hypercoagulable state were increased levels of fibrinogen and prothrombin. This shows a possible link between obesity, low-grade inflammation and increased thrombin generation in women at increased risk for future thrombosis.

Analysis of factor V Leiden and prothrombin mutations in patients with suspected thrombophilia in São Paulo state-Brazil

INTRODUCTION: Prothrombin (factor II) is a thrombin precursor, which induces fibrin formation. A mutation in the prothrombin gene (G20210A) has been described, which is directly associated with high prothrombin levels, hence thrombophilia. G1691A mutation in the factor V Leiden (FVL) gene occurs on exon 10, one of the main cleavage sites for protein C activation, resulting in protein alteration. OBJECTIVE: To identify and estimate the genotype frequency of the three possible genotypes and the frequency of the two existing alleles in the FVL and prothrombin genes in patients with suspected thrombophilia in the state of Sao Paulo. This study may provide more literature and reference data on the incidence of prothrombin genotypes among individuals in Brazil. MATERIAL AND METHODS: Analysis of point mutation by real time polymerase chain reaction (RT-PCR). RESULTS: We obtained a total of 100 individuals, from which 94% had the homozygous G genotype. Only 6% had heterozygous genotype and there was no individual with the homozygous genotype A for FVL gene. As to the prothrombin gene, the frequency was 97% for homozygous G genotype and 3% for the heterozygous genotype. There was no patient with the homozygous A genotype. CONCLUSION: This study demonstrated that the genotype identification of these genes is advisable for patients with suspected thrombophilia in this region.

Étiopathologie du carcinome hépatocellulaire à Bangui, République centrafricaine : caractéristiques cliniques, biologiques et aspects virologiques des patients

Objectives To describe clinical, biological characteristics and virological aspects of patients with hepatocellular carcinoma (HCC). Patients and methods Sera obtained from consent patients with clinical suspicion of HCC. Routine biochemical tests and serological markers of hepatitis B virus (HBV), hepatitis D virus (HDV) and hepatitis C virus (HCV) were searched. A hepatic ultrasound scan was realized and, when a suspected mass lesion was observed, ultrasonographic-guided fine needle aspiration of the hepatic mass lesion was made to ascertain the diagnosis of HCC. Results One hundred and seventy-five sera were collected from 99 men and 76 women. Approximately 96.6% (169/175) of them had previous contact with HBV. HBs surface antigen was positive in 41% (69/169) of whom 53.6% (37/69) were co-infected by the HDV. Three patients (1.71% = 3/175) were positive for anti-HCV antibodies. The other three patients (1.71% = 3/175) did not present any markers for HBV or HCV. At the time of diagnosis, right upper quadrant pain, hepatomegaly, cachexia, were present in all patients. An increase in serum transaminases (70%), high bilirubin concentrations (40%), high AFP levels (40%) and low prothrombin levels (52%) were the most frequent biological abnormalities. Only 17 guided fine needle aspirations of mass lesions were realized. The diagnosis of HCC was confirmed by cytopathological examination for 14 of them. Conclusions HCC is a frequent pathology in the Central African Republic (CAR). According to the ultrasound imaging data, patients consult at terminal stage of the disease. This study confirms also the association between HBV and HCC in CAR. Moreover, in 26.5% of patients HDV is associated with severe forms of the disease.

Pregnancy-associated changes in the hemostatic system in wild-type and factor V Leiden mice.

Pregnancy, oral contraceptive (OC)use and hormone replacement therapy (HRT) are established risk factors for venous thrombosis. Acquired resistance to activated protein C (APC) has been proposed to contribute to the increased thrombosis risk. Mouse models are often used for preclinical testing of newly developed hormone preparations. However, it is not known whether hormone-induced APC resistance is also observed in laboratory animals. To investigate whether hormonal changes modulate APC resistance in mice, we used pregnant mice as a model of hormone-induced APC resistance. The effect of pregnancy on APC resistance was studied in wild-type and factor (F)V Leiden mice. APC resistance was determined in mouse plasma using a thrombin generation-based APC resistance test. APC resistance determinants,i.e. prothrombin, FV, FX, antithrombin and protein S levels,and of tissue factor pathway inhibitor (TFPI) activity were evaluated in plasma from non-pregnant and pregnant mice. In contrast to humans, pregnancy induced a decrease in APC resistance in wild-type and in FV Leiden mice.Pregnant mice had higher levels of prothrombin, FV, FX,protein S and TFPI activity as compared with non-pregnant mice. Pregnancy causes a decrease in APC resistance in mice, which can be explained by the elevation of protein S levels and increased TFPI activity in plasma. Our findings show species specificity in the effects of pregnancy on the major determinants of the protein C system and suggest that protein S and TFPI play an important role in the development of pregnancy-induced APC resistance in humans.

G20210A Prothrombin gene variant in Turkish patients with angiographically documented coronary artery disease

G-->A substitution at position 20210A in the 3'-untranslated region in prothrombin gene is associated with raised plasma prothrombin. G20210A prothrombin variant has been associated with high prothrombin levels and an increased risk of venous thrombosis. To determine the prevalence of the G20210A prothrombin variant among Turkish and to evaluate the potential relevance of this variant to Turkish patients with angiographically documented coronary artery disease (CAD). We conducted a case-control study on 268 unrelated subjects who referred to the cardiology department of the university hospital for coronary angiography. One hundred-thirty seven patients with angiographically documented CAD and 131 subjects without angiographically documented CAD were studied to examine the association of the G20210A prothrombin variant with CAD. Blood samples from the patients and controls were analyzed for the G20210A prothrombin variant by DNA analysis, using polimeraz chain reaction. G20210A prothrombin variant was found in 6 of 137 (4.4%) patients with CAD and 1 of 131 (0.8%) in control subjects (p = 0.064). There were no significant differences in terms of diabetes mellitus, hypertension, dyslipidemia, sex and family history of CAD with and without the G20210A prothrombin variant in the patient group. In agreement with the results of recent meta-analyses, our data from northeast Anatolia show a 5-fold higher prevalence of the G20210A prothrombin variant among the patients with angiographically-documented CAD when compared with those without angiographic signs of significant CAD and its variant cannot be considered as a risk factor for CAD in this region.

Differential effects of high prothrombin levels on thrombin generation depending on the cause of the hyperprothrombinemia.

Hyperprothrombinemia, resulting from the prothrombin G20210A mutation or other causes, is associated with activated protein C (APC) resistance and increased thrombosis risk. When high prothrombin levels are a result of increased hepatic biosynthesis, these effects may be counteracted by concomitantly increased levels of the anticoagulant factors (particularly protein S). Differently, in prothrombin G20210A carriers only prothrombin levels are elevated. To investigate whether prothrombin G20210A carriers have a more severe hypercoagulable state than non-carriers with comparable prothrombin levels. Coagulation factor levels, thrombin generation (Calibrated Automated Thrombogram in the presence and absence of APC) and APC resistance were measured in normal (n = 132), heterozygous (n = 167) and homozygous (n = 3) individuals. Prothrombin levels, thrombin generation and APC resistance were higher in carriers of the prothrombin G20210A mutation (especially those who had experienced venous thrombosis) than in non-carriers, whereas protein S and antithrombin levels were similar among genotype groups. Because individuals with high prothrombin levels in the absence of the prothrombin G20210A mutation tend to have all liver-synthesized factors elevated, carriers of the mutation had lower protein S and antithrombin levels than non-carriers with equally high prothrombin levels. Accordingly, they also generated more thrombin and showed a tendency toward higher APC resistance. Analogous effects, but less pronounced, were observed in homozygotes for the prothrombin A19911G polymorphism, which also upregulates prothrombin levels. Individuals with hyperprothrombinemia as a result of prothrombin gene mutations generate more thrombin and tend to be more APC-resistant than individuals with comparable prothrombin levels because of other causes.

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Bleeding tendency as a result of clotting factor deficiency is common knowledge. The counterpart, i.e. thrombophilia due to high clotting factor levels, is not surprising, but an association between factor level and thrombosis risk has just recently been described. The role of high factor VIII (FVIII) levels is well documented. The risk of high FVIII levels for the first event is similarly high as that of APC resistance. There is a familiar background of high FVIII levels. Alterations within the FVIII or the von Willebrand factor genes seem to be not causal since polymorphisms of these genes are not associated with venous thromboembolism. An increased FVIII/VWF ratio indicates a reduced FVIII clearance. Probably, the low-density lipoprotein receptor-related protein, i.e. the receptor mediating the hepatic clearance of the FVIII-VWF-complex, is involved. The well known prothrombin G20210A polymorphism is associated with high prothrombin levels perhaps contributing to thrombosis risk via clot resistance against fibrinolysis.

Venous thromboembolism, oral contraceptives and high prothrombin levels

The G20210A prothrombin mutation, associated with elevated prothrombin levels, is a risk factor for venous thromboembolism (VTE) and displays a strong interaction with oral contraceptives (OC). No data are available on VTE risk of OC use in women with high prothrombin levels, either associated or not with the mutation. The aim of this study was to evaluate the risk of VTE in OC users with high prothrombin levels, either including or excluding carriers of the prothrombin mutation. Prothrombin levels were measured by a chromogenic assay in 152 women who suffered from VTE in reproductive age and in 296 healthy women. Subjects carrying thrombophilic alterations other than the G20210A prothrombin mutation were excluded. Prothrombin levels were stratified into quartiles. The OR of subjects in the upper quartile were 3.10 [95% confidence interval (CI) 1.73-5.55] and 2.07 (95% CI 1.11-3.85) in all women and in those not carrying the prothrombin mutation, respectively. Among the 152 patients, 88 had experienced VTE during OC; in the control group we considered as OC users the women who had used OC for at least 6 months in the 2 years before presentation but had stopped the treatment at least 3 months before the time of blood sampling (n = 127). For the interaction between OC and prothrombin levels only the two extreme strata of prothrombin were considered. Women with the lowest prothrombin levels and who did not use OC were used as reference category. The VTE risk of using OC in subjects with prothrombin levels in the upper quartile was increased 5.4-fold (95% CI 2.38-12.3) and 3.5-fold (95% CI 1.48-8.22) in all women and in those not carrying the prothrombin mutation, respectively. We conclude that elevated prothrombin levels, even in women without the G20210A prothrombin mutation, are associated with an increased risk for venous thromboembolism and that oral contraceptive use potentiates such association.

A family history can display a synergistic effect of atherogenic and prothrombotic risk in pregnancy

A family history can display a synergistic effect of atherogenic and prothrombotic risk in pregnancy

G20210A prothrombin gene polymorphism and prothrombin activity in subjects with or without angiographically documented coronary artery disease.

G20210A prothrombin mutation has been associated with high prothrombin levels and an increased risk of venous thrombosis. The role of this common polymorphism, as well as that of prothrombin levels, in determining the risk of arterial disease is still somewhat controversial. We determined the prevalence of the G20210A mutation and prothrombin activity in 660 individuals, of whom 436 had angiographically documented severe coronary artery disease (CAD patients) and 224 had normal coronary angiography (CAD-free control subjects). Heterozygosity for the 20210A allele was found in 5.3% of the CAD patients versus 3.1% of the CAD-free subjects (P=0.21). Similarly, no statistically significant difference was found between CAD patients with or without previous myocardial infarction (4.5% versus 5.3%, respectively; P=0.73). The genotype-phenotype correlation study showed a significant influence of the 20210A allele on prothrombin activity, with higher levels in carriers compared with noncarriers (153.2% versus 122.2%, respectively; P<0.001). Prothrombin activity was significantly higher in CAD patients than in CAD-free subjects (132.8% versus 123.3%, respectively; P<0.005). By multiple logistic regression, prothrombin activity in the upper tertile of the control distribution was significantly associated with CAD compared with prothrombin activity in the lower tertile (adjusted odds ratio 1.86, 95% CI 1.01 to 3.4). In a population with a clear-cut definition of the phenotype, the G20210A prothrombin mutation was not significantly associated, per se, with either angiographically documented CAD or myocardial infarction, whereas it significantly influenced prothrombin activity. In our population, high prothrombin activity itself was independently associated with CAD but not with the presence or absence of previous myocardial infarction.

Polymorphisms in the Prothrombin Gene and their Association with Plasma Prothrombin Levels

To find genetic causes of high plasma prothrombin levels, an established prothrombotic risk factor. we searched for sequence variations in the prothrombin gene. We selected subjects with the 20210-GG genotype (since the 20210-A allele is already known to be associated with high levels) and elevated prothrombin levels (> or = 130 U/dl) from the Leiden Thrombophilia Study (LETS). No mutations were found in the 1 kb promoter region of the prothrombin gene in seven individuals with an isolated high prothrombin level. Comparison of the allelic frequencies of four different polymorphisms in the prothrombin gene in healthy volunteers and in the control subjects among the selected LETS individuals indicated a higher frequency of the 19911-G allele in the latter group (allele frequency 52 vs. 78%, respectively). Homozygous carriers of the 19911-G allele had 8 U/dl higher plasma prothrombin levels than 19911-AA carriers. This difference in prothrombin levels did not affect the thrombotic risk in 20210-GG carriers. In heterozygous 20210-A carriers the odds ratio increased from 1.6 (95% CI: 0.6-4.3) in subjects with 19911-A to 4.7 (1.6-14.0) in subjects with 19911-G on the other prothrombin allele.

The impact of molecular medicine upon early cardiovascular drug development.

Heart disease remains the leading cause of death and morbidity in the western world despite significant advances in cardiovascular (CVS) therapeutics. Since the 1960s clinical pharmacologists have been very much involved in bringing forward new classes of CVS drugs. This started with the introduction of β-adrenoceptor blockers in the early 1960s and has continued with calcium antagonists, ACE inhibitors and more recently endothelin antagonists. The principles for classical drug design have been based upon interaction with: receptor sites (β-adreno-ceptor blockers), enzymes (thrombolytics and anticoagulants) and ion channels (calcium antagonists, antiarrhythmics). The advent of molecular biology has added a fourth principle for CVS drug development – that of molecular therapeutics. While classical approaches will still provide new CVS drugs (e.g. platelet IIb/IIIa receptor antagonists and directly acting thrombin inhibitors), there is now anticipation that molecular therapeutics will provide a very specific means of intervention thus potentially avoiding some of the problems associated with the non specificity of conventional approaches, e.g. side-effects associated with lack of cardioselectivity with β-adrenoceptor blockers, risk of bleeding with anticoagulants, cough with ACE inhibitors, controversy following retrospective analysis of the use of calcium antagonists. In the broader sense molecular therapeutics includes both gene therapy and oligonucleotide therapy. However the advances in genetic analysis (identification of polymorphisms associated with diseases) mean that there may be a further impact upon drug development – namely the use of known gene polymorphisms to select patient populations at risk. We will review the status of each of these and prospects for the future.

The G20210A Mutation of the Prothrombin Gene in Patients with Previous First Episodes of Deep-Vein Thrombosis: Prevalence and Association with Factor V G1691A, Methylenetetrahydrofolate Reductase C677T and Plasma Prothrombin Levels

A common G to A transition at nucleotide 20210 of the prothrombin gene is associated with an increased risk for deep-vein thrombosis (DVT) and high plasma levels of prothrombin. We calculated the prevalences of prothrombin G20210A, factor V G1691A (also associated with high risk for DVT) and homozygous methylenetetrahydrofolate reductase (MTHFR) C677T (associated with increased susceptibility to develop hyperhomocysteinemia) in 118 patients with a first episode of DVT and in 416 healthy controls. 15.9% of the patients and 2.3% of the controls had prothrombin G20210A (odds ratio [OR]: 8.7, 95% C.I.: 3.8–21.4); 21.1% of the patients and 3.2% of the controls had factor V G1691A (OR 7.8, 3.9–17.1); 20.5% of the patients and 21% of the controls had homozygous MTHFR C677T (OR: 1.0, 0.7–1.2). Exclusion of patients with other hereditary risk factors for DVT did not substantially modify the results. Mutant factor V and prothrombin coexisted in three patients but in no control. The concomitant presence of the MTHFR mutation did not increase the thrombotic risk associated with prothrombin G20210A. 63.2% of individuals with prothrombin G20210A had plasma levels of prothrombin in the upper quartile of distribution. After adjustment for age and sex, subjects with prothrombin levels in the upper quartile carried a slightly higher risk for thrombosis than those with lower prothrombin concentrations (OR: 1.9, 1.1–3.2). In conclusion, we found that prothrombin G20210A is relatively common in Italy and is associated with high prothrombin levels and an 8.7-fold increase in the risk for DVT. Such risk is independent of the coexistence of other known inherited risk factors for thrombosis and increases in patients with associated mutant factor V. Whether it is due to the associated increase in plasma prothrombin levels remains to be established.

Increased Activity of Vitamin K-Dependent Clotting Factors in Human Hyperlipoproteinaemia – Association with Cholesterol and Triglyceride Levels

Levels of blood coagulation factors, cholesterol and triglyceride were measured in human plasma. Prothrombin was significantly elevated in type IIa hyperlipidaemia; prothrombin and factors VII, IX and X in type IIb; and prothrombin and factors VII and IX in type V. Multiple regression analysis showed significant correlation between the levels of these plasma lipids and the vitamin K-dependent clotting factors (prothrombin, factors VII, IX and X). Higher cholesterol levels were associated with higher levels of prothrombin and factor X while higher triglyceride levels were associated with higher levels of these as well as factors VII and IX. Prothrombin showed a significant cholesterol-triglyceride interaction in that higher cholesterol levels were associated with higher prothrombin levels at all levels of triglyceride, with the most marked effects in subjects with higher triglyceride levels. Higher prothrombin levels were noted in subjects with high or moderately elevated (but not low) cholesterol levels. Ultracentrifugation of plasma in a density of 1.21 showed activity for prothrombin and factors VII and X only in the lipoprotein-free subnatant fraction. Thus, a true increase in clotting factor protein was probably present. The significance of the correlation between levels of vitamin K-dependent clotting factors and plasma lipids remains to be determined.

An evaluation of the utility of benzoyl-DL-arginine-β-naphthylamide for the assay of thrombin activity in blood

Thrombin continuously hydrolyzes the model substrate, benzoyl-DL-arginine-β-naphthylamide (BANA) over a 24-hour period, the extent of hydrolysis being proportional to the added thrombin. The apparent inhibition of the hydrolysis of BANA by thrombin in the presence of albumin may be explained by the capacity of albumin to bind the substrate molecule as well as the product, β-naphthylamine. The clotting activity of thrombin is essentially unaffected by the presence of 4% albumin. In view of the binding of BANA to albumin and binding of β-naphthylamine to albumin and other plasma proteins affecting the apparent prothrombin activity in plasma, only relative values for prothrombin can be obtained with BANA as a substrate. These values confirm, however, that plasma from normal subjects have considerably higher prothrombin levels than that of coronary patients on dicumarol therapy. The adsorptivity of benzoyl-DL-arginine-β-naphthylamide and β-naphthylamine by blood proteins would restrict the assay of thrombin with the model substrate by the Bratton and Marshall reaction to such circumstances when determination of relative thrombin concentrations in blood are sufficient, and that other means should be employed for determinations of the absolute levels of the clotting factors.

A Study of Coagulation Factor Levels in Women during Labour and in Their Newborn Infants

Summary1. Coagulation factors levels were measured in 10 normal mothers and in their infants within 15 min of birth and at 48-96 hrs of age.2. In the mothers the levels of fibrinogen (532 mg/%), factors VIII (196%), IX (130%) and X (122%) were elevated; the levels of prothrombin (107%) and factor V (108%) were normal ; and the level of factor XI (69%) was reduced.3. The infants blood examined within 15 min of birth had a slightly elevated factor VIII level (138%), slightly reduced fibrinogen (195 mg/%) and factor V levels (79%), low levels of prothrombin (55%) and factors IX (27%), X (35%) and XI (32%).4. Blood from the infants at 48-96 hrs of age showed little change from the birth levels of factors V (89%), and VIII (116%) and a slight increase in factor XI level (39%). Four of the infants had received vitamin K1 and had higher levels of prothrombin and factors IX and X than the 7 who had not received vitamin K1.5. These results are compared with those of previous studies and the possible mechanisms underlying the changes is discussed.