Haplotypes of the EPCR gene, plasma sEPCR levels and the risk of deep venous thrombosis

BACKGROUND: Binding of protein C (PC) to the endothelial cell PC receptor (EPCR) stimulates PC activation by increasing the affinity of PC for the thrombin-thrombomodulin complex. A soluble form of this receptor (sEPCR) circulates in plasma and inhibits both PC activation and APC anticoagulant activity. OBJECTIVES: The aim of this study was to investigate whether variations in the EPCR gene or plasma sEPCR levels are risk factors for deep venous thrombosis (DVT). PATIENTS/METHODS: In a large case-control study, the Leiden Thrombophilia Study (LETS), sEPCR levels were measured by ELISA. All subjects were genotyped for three haplotype-tagging SNPs, enabling us to detect all four common haplotypes of the EPCR gene. RESULTS: The distribution of sEPCR levels in the control population was trimodal and was genetically controlled by haplotype 3 (H3). This haplotype explained 86.5% of the variation in sEPCR levels. Carriers of two H3 alleles had higher sEPCR levels (439 ng mL(-1)) than carriers of one H3 allele (258 ng mL(-1)), which had higher levels than non-H3 carriers (94 ng mL(-1)). Haplotype 4 was associated with a slightly increased risk (OR = 1.4, 95%CI:1.0-2.2). The risk of subjects with sEPCR levels in the top quartile (>/= 137 ng mL(-1)) was increased compared to that of subjects in the first quartile (< 81 ng mL(-1)), but since there was no dose-response effect, it is most likely that low sEPCR levels reduce the risk of DVT. CONCLUSIONS: Our data do not suggest a strong association between EPCR haplotypes and thrombosis risk, but low sEPCR levels appear to reduce the risk of DVT.     

Genetic modulation of the FVLeiden/normal FV ratio and risk of venous thrombosis in factor V Leiden heterozygotes

Summary. Background and Objectives: The factor (F) V Leiden mutation causes activated protein C (APC) resistance by decreasing the susceptibility of FVa to APC‐mediated inactivation and by impairing the APC‐cofactor activity of FV in FVIIIa inactivation. However, APC resistance and the risk of venous thromboembolism (VTE) vary widely among FV Leiden heterozygotes. Common F5 genetic variation probably contributes to this variability. Patients/methods: APC resistance was determined in 250 FV Leiden heterozygotes and 133 normal relatives using the prothrombinase‐based assay, which specifically measures the susceptibility of plasma FVa to APC. The effects of 12 F5 single‐nucleotide polymorphisms (SNPs) on the normalized APC sensitivity ratio (nAPCsr) and on FV levels were determined by multiple regression analysis. Results: In FV Leiden heterozygotes, VTE risk increased with increasing nAPCsr, reaching an odds ratio (OR) of 9.9 (95% confidence interval [CI] 1.2–80.5) in the highest nAPCsr quartile. The minor alleles of several F5 SNPs, including 327 A/G (Q51Q), 409 G/C (D79H), 2663 A/G (K830R, T2 haplotype), 6533 T/C (M2120T) and 6755 A/G (D2194G, R2 haplotype), increased the nAPCsr in FV Leiden heterozygotes, but not in their normal relatives. Most of these effects could be attributed to a shift in the FV_Leiden/normal FV ratio. Four FV Leiden heterozygotes with extremely high nAPCsr turned out to be pseudo‐homozygotes, i.e. they carried a deleterious mutation on the non‐Leiden allele. Conclusions: In FV Leiden heterozygotes, the prothrombinase‐based nAPCsr is a marker of VTE risk and is modulated by common F5 SNPs that affect the FV_Leiden/normal FV ratio in plasma.     

Factor V Leiden and venous thromboembolism: risk associated with hormone replacement therapy

PURPOSE: To determine the risk-benefit ratio of hormone replacement therapy (HRT) and the cost-effectiveness of screening in perimenopausal and postmenopausal women who are carriers of factor V Leiden, as well as to provide evidence-based clinical recommendations for the primary care provider. DATA SOURCES: Databases searched included EMBASE, BIOSIS, MEDLINE, CINAHL, PubMed, SciSearch, and the Cochrane Database. Two reviewers extracted, reviewed, and concurred upon relevant evidence identified in the data-bases. RESULTS: Results confirmed that all women have a higher risk for the development of venous thrombosis while on HRT. The presence of a genetic mutation, such as factor V Leiden, in combination with HRT dramatically increased an individual's chance for developing venous thrombi. CONCLUSION/IMPLICATIONS: Based on the findings of the studies reviewed, it is recommended that women wishing to initiate HRT be thoroughly screened for known risk factors of thrombosis. If risk factors are identified, genetic testing for factor V Leiden may be warranted.     

Aging of the venous valves as a new risk factor for venous thrombosis in the elderly: the BATAVIA study

Essentials

• Risk of venous thrombosis (VT) related to valve thickness and valvular reflux in unknown.
• Venous valves and reflux were measured by ultrasonography in cases and controls aged 70+.
• Risk of VT was associated with increased valve thickness and valvular reflux >1second.
• Thickening of valves is a generic process: there was no difference between right and left legs.

Summary

Background

Increasing age is the strongest risk factor for venous thrombosis (VT). Increasing age has been related to a thickening of the venous valves and a decreased valvular function. The association between valve thickness and the risk of VT is not known.

Objectives

To assess the association between increased valve thickness and valve closure time (VCT) and the risk of VT.

Methods

Analyses were performed in the BATAVIA study, including 70 cases aged 70 + with a first VT and 96 controls. We performed an ultrasound examination of the valves in the popliteal veins. The valves were imaged with a 9 MHz linear probe using B‐mode ultrasonography. VCT was measured as an indicator for valve function using an automatic inflatable cuff. To estimate the risk of VT, valve thickness was dichotomized at the 90th percentile as measured in controls and VCT was dichotomized at 1 s.

Results

Mean valve thickness of controls was similar in the left (0.36 mm, 95% CI 0.34–0.37) and right (0.36 mm, 95% CI 0.35–0.38) leg. In 45 cases a valve was observed in the contralateral leg with a mean valve thickness of 0.39 mm (95% CI 0.36–0.42). Cases had an increased valve thickness compared with controls: mean difference 0.028 mm (95%CI 0.001–0.055). Valve thickness > 90th percentile increased the risk of VT 2.9‐fold. Mean VCT in controls was 0.38 s, in contralateral leg of cases 0.58 s. VCT > 1 s increased the risk of VT 2.8‐fold (95% CI 0.8–10.4).

Conclusions

Risk of VT was associated with increased valve thickness and valvular reflux of > 1 s.     

C-reactive protein gene polymorphisms and the risk of venous thromboembolism: a haplotype-based analysis.

C-reactive protein (CRP) is a risk predictor for future athero-thrombotic events, and its plasma concentration has a heritable component. CRP has also been suggested to play a role in the pathophysiology of venous thromboembolism. To date, no genetic-epidemiological data are available on the relation of CRP gene variants with the risk of venous thromboembolism. The present study was carried out to investigate the possible association of two previously characterized (an exonic 1059G-->C, and an intronic T-->A) CRP gene polymorphisms in a prospective, matched case-control sample from the Physicians Health Study. Allele, genotype, and haplotype distributions were similar between 130 cases and 130 matched controls. Genotype distributions were in Hardy-Weinberg equilibrium. Further investigation using a haplotype-based matched logistic regression analysis, adjusting for age, smoking, randomized treatment group (likelihood ratio test: X(2)2df = 0.19, P = 0.91) or with further controlling for body mass index, hypertension, and diabetes (likelihood ratio test: X(2)2df = 0.66, P = 0.72) yielded similar null findings. In conclusion, we found no evidence for an association between the CRP polymorphisms/haplotypes tested and the risk of venous thromboembolism.     

Activation of innate immunity in patients with venous thrombosis: the Leiden Thrombophilia Study.

BACKGROUND: Previous studies have suggested that levels of inflammatory mediators are risk indicators for venous thrombotic disease. We have sought to confirm and extend these findings by measuring plasma tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8, IL-10 and IL-12p70 levels in a case-control study for venous thrombotic disease. METHODS: The plasma levels of these inflammatory mediators were measured by flow cytometric analysis using a multiplexed bead assay. Patient and control samples came from the Leiden Thrombophilia Study (474 controls and 474 patients). RESULTS: In a subset of patients and controls inflammatory mediators are detectable in plasma. The crude odds ratios (ORs) associated with the presence of detectable markers were 2.1 [TNF-alpha, 95% confidence interval (CI) 1.1, 3.9], 1.7 (IL-1beta, 95% CI 1.1, 2.9), 2.4 (IL-6, 95% CI 1.9, 5.3), 2.8 (IL-8, 95% CI 1.8, 4.4), 0.8 (IL-10, 95% CI 0.3, 1.8), and 1.3 (IL-12p70, 95% CI 0.9, 2.0). Adjustment for putative confounders did not influence the risk estimates. CONCLUSION: TNF-alpha, IL-6, and IL-8 levels are risk determinants for venous thrombosis. Individuals with detectable levels of either of these mediators in plasma have an OR of about 2. In line with these findings, the odds for the anti-inflammatory cytokine Il-10 tend to be < 1. These results add further evidence for the contention that there is an inflammatory component to venous thrombotic disease and may explain why anti-inflammatory agents such as aspirin may be effective for prevention.     

The JAK2 V617F mutation in patients with cerebral venous thrombosis

Summary. Background: It is currently unclear whether or not cerebral venous thrombosis, such as splanchnic venous thrombosis, can be the first manifestation of an underlying myeloproliferative neoplasm. Objective: To determine the prevalence of the JAK2 V617F mutation in patients with a first episode of cerebral venous thrombosis. Patients: In this retrospective cohort study, patients with cerebral venous thrombosis were tested for the JAK2 V617F mutation and were followed until the development of a myeloproliferative neoplasm or censored at the end of follow‐up. Results: Ten of 152 patients (6.6%) carried the JAK2 V617F mutation. Three of them had known acquired risk factors for thrombosis, and five had thrombophilia. Six patients met the diagnostic criteria for myeloproliferative neoplasm at the time of cerebral venous thrombosis, and three additional patients developed the disease during the follow‐up (median duration 7.8 years, range 6 months to 21.3 years), giving an annual incidence of 0.26% patient‐years (95% confidence interval 0.05–0.64). The last patient has no evidence of disease after 3 years of follow‐up. Patients without the JAK2 V617F mutation at the time of cerebral venous thrombosis were retested at the end of the follow‐up and remained negative, with normal blood counts (log‐rank test χ²: 159 [P < 0.0001]). Conclusions: Cerebral venous thrombosis can be the first symptom of a myeloproliferative neoplasm. Patients with cerebral venous thrombosis can carry the JAK2 V617F mutation, irrespective of blood count.     

Expression of the normal factor V allele modulates the APC resistance phenotype in heterozygous carriers of the factor V Leiden mutation

BACKGROUND: Functional defects of the protein C pathway, detectable in plasma as activated protein C (APC) resistance, are a prevalent risk factor for venous thrombosis. The factor V (FV) Leiden mutation causes APC resistance by interfering with the APC-mediated inactivation of both FVa and FVIIIa. Co-inheritance of FV Leiden and quantitative FV deficiency on different alleles, a rare condition known as pseudo-homozygous APC resistance, is associated with pronounced APC resistance and 50% reduced FV levels, because of non-expression of the non-Leiden FV allele. OBJECTIVES: The role of normal FV in modulating the APC resistance phenotype in carriers of FV Leiden was investigated in patients with pseudo-homozygous APC resistance and in model systems. PATIENTS/METHODS: Four functional plasma assays probing both components of APC resistance (susceptibility of FVa to APC and cofactor activity of FV in FVIIIa inactivation) were employed to compare seven clinically and genetically characterized FV Leiden pseudo-homozygotes to 30 relatives with different FV genotypes (including 12 FV Leiden heterozygotes and seven carriers of FV deficiency) and to 32 unrelated FV Leiden homozygotes. RESULTS AND CONCLUSIONS: All assays consistently indicated that FV Leiden pseudo-homozygotes are significantly more APC-resistant than heterozygotes and indistinguishable from homozygotes. Thrombin generation measurements in FV-deficient plasma reconstituted with purified normal FV and FV Leiden confirmed these observations and showed that the expression of the normal FV allele is an important modulator of APC resistance in FV Leiden heterozygotes. These findings provide an explanation for the higher thrombotic risk of FV Leiden pseudo-homozygotes when compared with heterozygotes.     

von Willebrand factor and its propeptide: the influence of secretion and clearance on protein levels and the risk of venous thrombosis

BACKGROUND AND OBJECTIVES: Elevated levels of factor (F)VIII are associated with an increased risk of thrombosis. FVIII levels are determined mainly by von Willebrand factor (VWF). We have investigated the contribution of secretion and clearance rates to the elevated VWF antigen (VWF:Ag) and to the risk of thrombosis. VWF is secreted in equimolar amounts with its propeptide, which has a shorter half-life. VWF propeptide can be used as a measure of VWF secretion and allows estimation of the VWF half-life. METHODS AND RESULTS: We have measured VWF propeptide, VWF:Ag, FVIII:Ag and FVIII activity (FVIII:C) in the Leiden Thrombophilia Study. In controls, high VWF propeptide was associated with high VWF:Ag, FVIII:Ag and FVIII:C. In contrast to mature VWF:Ag, VWF propeptide was not influenced by blood groups. Using an ELISA-based assay we have shown that VWF propeptide lacks ABO antigens. Levels were higher in men and increased with age. A long VWF half-life was also associated with high VWF:Ag, FVIII:Ag and FVIII:C. The VWF half-life was influenced by blood group (10 h in O vs. 12 h in non-O individuals), but not by sex, and only slightly by age. VWF propeptide was higher in thrombosis patients than in controls. The VWF half-life was similar in patients and controls (11.4 and 11.1 h, respectively). CONCLUSIONS: Both secretion and clearance rates are important determinants of VWF and FVIII levels. However, mainly high VWF and FVIII levels caused by increased secretion seem to be associated with thrombosis. ABO blood group influences the clearance rates of VWF rather than VWF secretion rates.     

The JAK2 V617F mutation frequently occurs in patients with portal and mesenteric venous thrombosis

BACKGROUND: Myeloproliferative disorders (MPDs) represent a risk factor for thrombosis in the portal, mesenteric, and hepatic districts. OBJECTIVE: We aimed to assess whether the Janus kinase 2 (JAK2) V617F mutation, an acquired mutation that occurs in MPD patients, is a risk factor for portal and mesenteric venous thrombosis (PMVT) independently of the presence of overt MPDs. PATIENTS AND METHODS: The medical histories of 99 patients presenting with PMVT were obtained. The presence of the JAK2 V617F and VHL 598C > T mutations was determined by polymerase chain reaction followed by restriction enzyme analysis and direct cycle sequence analysis. RESULTS: Over a 10-year period of observation, of the 99 patients presenting with PMVT, the JAK2 V617F mutation was detected in heterozygous state in 17 individuals [17.2%; 95% confidence interval (95% CI) 10.9-25.9]. None of the patients presenting with the JAK2 V617F mutation carried an inherited thrombophilic risk factor. Seven patients with (43.8%; 95% CI 19.8-70.1) and two without (2.4%; 95% CI 0.3-8.4) the JAK2 V617F mutation had a diagnosis of MPD at the occurrence of the venous thrombotic event. After a median follow-up of 41 months (range 3-114 months), three out of the 10 patients carrying the JAK2 V617F mutation were then diagnosed as having idiopathic myelofibrosis (n = 2) or polycythemia vera (n = 1), whereas in seven patients a MPD was not detected. Two of the 83 patients without the JAK2 V617F mutation went on to develop MPDs. CONCLUSIONS: Determination of the JAK2 V617F mutation may contribute to the search for genetic determinants of PMVT and may be useful to recognize patients who should be carefully observed for the subsequent development of overt MPDs.     

Familial thrombophilia and lifetime risk of venous thrombosis.

BACKGROUND: We started a large multicenter prospective follow-up study to provide reliable risk estimates of venous thrombosis in families with various thrombophilic defects. OBJECTIVES: This paper describes data collected at study entry on venous events experienced before study inclusion, i.e. the baseline data. PATIENTS/METHODS: All individuals (probands, relatives) registered in nine European thrombosis centers with the factor (F)V Leiden mutation, a deficiency of antithrombin, protein C or protein S, or a combination of these defects, were enrolled between March 1994 and September 1997. As control individuals, partners, friends or acquaintances of the thrombophilic participants were included. Incidence and relative risk of objectively confirmed venous thrombotic events (VTEs) prior to entry were calculated for the relatives with thrombophilia and the controls. RESULTS: Of the 846 relatives with thrombophilia (excluding probands), 139 (16%) had experienced a VTE with an incidence of 4.4 per 1000 person years. Of the controls, 15 of the 1212 (1%) controls had experienced a VTE with an incidence of 0.3 per 1000 person years. The risk of venous thrombosis associated with familial thrombophilia was 15.7 (95% CI 9.2-26.8) and remained similar after adjustment for regional and sex-effects (16.4; 95% CI 9.6-28.0). The highest incidence per 1000 person years was found in relatives with combined defects (8.4; 95% CI 5.6-12.2), and the lowest incidence was found in those with the FV Leiden mutation (1.5; 95% CI 0.8-2.6). CONCLUSIONS: Considerable differences in the lifetime risk of VTE were observed among individuals with different thrombophilia defects.     

Risk factors for venous thrombosis in medical inpatients: validation of a thrombosis risk score.

BACKGROUND/OBJECTIVES: The occurrence of and risk factors for venous thrombosis (VT) complicating hospital admission in unselected medical inpatients have not been widely studied. PATIENTS and METHODS: In a 400-bed teaching hospital we identified all cases of VT complicating hospital admission between September 2000 and September 2002 using discharge codes and chart review. Controls were randomly selected adult inpatients frequency matched to cases for medical service. RESULTS: The incidence of VT complicating hospital admission was 7.6 per 1000 admissions. On average, VT was diagnosed on the fifth hospital day. The median age of the 65 cases and 123 controls was 68 years and 45% were men. Cases had a 4-fold higher death rate than controls [95% confidence interval (CI) 1.9, 8.8]. At admission, trauma within 3 months, leg edema, pneumonia, platelet count > 350 x 10(3) mm(-3) and certain cancers were associated with risk of VT. Age, body mass index, and acute myocardial infarction were not associated with VT risk. One of three published VT risk models was able to risk stratify patients and was associated with a 2.6-fold increased risk of VT (95% CI 1.3, 5.5). Use of VT prophylaxis did not differ in cases and controls; prophylaxis was used < 1/3 of hospital days in 52% of patients. CONCLUSIONS: VT was common among medical inpatients. Of the risk factors identified, elevated platelet count has not been previously reported. Only one of three published risk scores was associated with risk of inpatient VT. Future study should improve upon risk prediction models for in-hospital VT among medical patients.     

Homocysteine, MTHFR and risk of venous thrombosis: a meta-analysis of published epidemiological studies

CONTEXT: It has been suggested that elevated total plasma homocysteine levels are associated with the risk of venous thrombosis. OBJECTIVE: To assess the relationship of homocysteine and the MTHFR 677TT genotype and the risk of venous thrombosis by conducting a meta-analysis of all relevant studies. DATA SOURCES AND SELECTION: Studies (case-control or nested case-control) were identified by searches of electronic literature for relevant reports published before July 2003 on homocysteine and the MTHFR 677TT genotype and venous thrombosis as an end-point, by hand-searching reference lists of original articles (including meta-analyses) on this topic and by contact with investigators in the field. DATA EXTRACTION: A meta-analysis of 24 retrospective (n = 3289 cases) and three prospective studies (n = 476 cases) was carried out to examine the association of homocysteine with venous thrombosis. A meta-analysis of 53 studies (n = 8364 cases) of the MTHFR 677TT genotype (that increases homocysteine) was carried out to assess if this association is causal. DATA SYNTHESIS: A 5 micromol L(-1) higher measured homocysteine level was associated with a 27% (95% CI: 1-59) higher risk of venous thrombosis in prospective studies and a 60% (95% CI: 10-134) higher risk in retrospective studies. The 677TT genotype was associated with a 20% (95% CI: 8-32) higher risk of venous thrombosis compared with the 677CC genotype. In contrast with non-American studies, the 677TT genotype had no effect on venous thrombosis in North America, due probably to the higher intake of folate and riboflavin in North America. CONCLUSION: This meta-analysis of prospective and retrospective studies demonstrates a modest association of homocysteine with venous thrombosis. The elevated risk associated with the MTHFR 677TT genotype provides some support for causality.     

Cerebral venous thrombosis: a case report

The incidence of cerebral venous thrombosis (CVT) is three to four cases per one million in adults and seven cases per one million among children. We discuss a case of CVT in a 19-year-old woman with a history of migraine headaches. A CVT most commonly presents acutely, with a wide variety of signs and symptoms in young adults and children. The most common presenting complaint in adults is severe headache. The most frequently thrombosed sinuses are the lateral, cavernous, and superior sagittal sinuses. Risk factors include any genetic or acquired prothrombotic conditions, including pregnancy and the peripartum period. Computed tomography may show the classic "delta sign," although magnetic resonance imaging with magnetic resonance venography is more sensitive. Treatment is controversial at this time. Options include fibrinolysis, anticoagulation, mechanical thrombectomy, or surgery. Despite the lack of agreement on management of CVT, the prognosis of CVT has improved, due to an increasing frequency of diagnosis.     

The risk of venous thromboembolism associated with the factor V Leiden mutation and low B-vitamin status.

Venous thromboembolism (VTE) is a multi-factorial disease involving numerous genetic and environmental risk factors. In this study we investigated the occurrence and the risk associated with factor V Leiden, hyperhomocysteinemia and low folate and vitamin B12 levels in young patients with thrombosis. We studied 78 patients (33 females/45 males, mean age 33 years) with a history of thrombosis in a lower limb. Additionally, 98 healthy subjects (45 females/54 males, mean age 44 years) were included. Serum levels of homocysteine (Hcy), folate and vitamin B12 were assayed. Factor V Leiden and methylenetetrahydrofolate reductase (MTHFR) C677T mutations were investigated in all subjects. Factor V Leiden was highly prevalent in the patients (39% heterozygous, 10% homozygous vs. 6.3% heterozygous in controls). An increase in the risk of idiopathic VTE was associated with Hcy levels > 15.2 micromol/l (odds ratio, OR = 2.83), folate < 15.1 nmol/l (OR = 7.49) and vitamin B12 < 182 pmol/l (OR = 11.97). Low levels of folate or vitamin B12 were independently and strongly associated with the risk of VTE in a multivariate model (OR for idiopathic thrombosis = 16.44 and 10.76, respectively). Twenty patients (53%), carriers of factor V Leiden, had low levels of vitamin B12, compared to 28% of patients who were non-carriers of the mutation (p = 0.03). In contrast, none of the control carriers of the mutation had a low level of vitamin B12. The risk of VTE associated with lower levels of vitamin B12 and folate was stronger than that introduced by elevated Hcy levels. The increased risk of VTE, accompanied by factor V Leiden, may be related to confounding environmental factors.     

Frequency and characteristics of the JAK2 V617F mutation in 23 cerebral venous sinus thrombosis patients with thrombocytosis

ObjectiveTo analyse the frequency and characteristics of the Janus kinase 2 (JAK2) V617F mutation in patients with cerebral venous sinus thrombosis (CVST) with thrombocytosis.MethodsThe study enrolled CVST patients with thrombocytosis that had undergone JAK2 V617F mutation detection to determine the frequency of the JAK2 V617F mutation in this cohort. Correlations between patient demographics, whole blood cell counts, targeted sequencing results and JAK2 V617F mutation status were determined.ResultsA total of 23 patients were enrolled in the study: 11 (47.8%) with the JAK2 V617F mutation and 12 (52.2%) without the JAK2 V617F mutation. The mean platelet count was significantly higher in patients with the JAK2 V617F mutation than in patients without the mutation (478.1 ± 107.4 × 10⁹/l versus 374.4 ± 54.1 × 10⁹/l, respectively). There were no significant differences in age, sex, white blood cell count or haemoglobin level between the two groups. Other than single nucleotide polymorphisms, no hot-spot mutations associated with myeloid tumours other than the JAK2 V617F mutation were detected in four CVST patients that underwent targeted sequencing.ConclusionThe JAK2 V617F mutation was frequently detected in CVST patients with thrombocytosis and it was associated with higher platelet counts.