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Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is the main genetic modulator of homocysteine. Data suggest a potential association of homozygosity for the TT MTHFR genotype with premature myocardial infarction (MI). We explored whether TT homozygosity is associated with long-term prognosis in patients with premature ST-segment elevation MI (STEMI).A total of 265 consecutive patients who had survived their first STEMI ≤35 years of age were followed for a median of 8 years (5-12). Primary endpoints were cardiac death and secondary endpoints were hospitalizations for acute coronary syndrome, myocardial revascularization, arrhythmic event or ischemic stroke. Serum lipids, homocysteine, folate levels were measured at baseline and all patients were also tested for the MTHFR C677T polymorphism.During follow-up 14 patients died (cardiac death) [5.3%] while 84 (31.7%) met the secondary endpoints. In univariate Cox regression analysis TT homozygosity predicted the occurrence of cardiac death (Hazard ratio (HR): 4.071; 95% confidence interval (CI): 1.404-11.809,Homozygosity for the TT MTHFR is an independent long-term predictor of cardiac death in patients with premature STEMI.

Aim Limited data are available regarding the clinical relevance of platelet function measurements in stable patients with coronary artery disease (CAD). Our aim is to evaluate the agreement between multiple electrode aggregometry (MEA) and light transmittance aggregometry (LTA) in detecting clopidogrel low responders and their prognostic value in CAD patients with type 2 diabetes mellitus (T2DM) on dual platelet inhibition. Methods LTA and MEA were performed in 122 stable cardiovascular patients with T2DM. The upper quartile of patients according to maximum LTA (LTAmax) and MEA measurements were defined as clopidogrel low responders. Agreement between the two methods was evaluated by kappa statistics. We assessed the potential correlation between antiplatelet response and clinical outcome and the optimal cutoff value according to ROC analysis to predict the occurrence of major adverse cardiovascular events (MACE), during 1-year follow-up period. Results Cohen's kappa coefficients (0.214) indicated fair agreement (70.2%) between LTA and MEA. A total of 25 MACE occurred in 108 patients (23.1%). Patients with MACE had higher LTAmax than those without (57.1 ± 16.5 vs 49.3 ± 18.3, respectively, p = 0.023). MEA measurements were similar between patients with and without MACE (30.1 ± 15.4 vs 30.6 ± 20.8, respectively; p = 0.84). Multiple logistic regression showed LTAmax response as an independent predictor of death from cardiovascular causes (Odds Ratio, adjusted:0.2;0.05–0.81). ROC analysis indicated that LTAmax cutoff of 62.5% best predicted death (AUC = 0.67, sensitivity = 78%, specificity = 61.5%). Conclusions The assessment of platelet responsiveness remains highly test-specific. Our results support the prognostic role of LTA, but not MEA testing, for death risk evaluation in stable cardiovascular T2DM patients.

BACKGROUND Our aim was to investigate the role of thromboelastometry (ROTEM) parameters, including maximum clot elasticity (MCE) and platelet component (PLTEM MCE and PLTEM MCF), in early prediction of bleeding events in thrombocytopenic critically ill neonates. MATERIAL AND METHODS This single-center, prospective cohort study included 110 consecutive thrombocytopenic neonates with sepsis, suspected sepsis, or hypoxia. On the first day of disease onset, ROTEM EXTEM and FIBTEM assays were performed and the neonatal bleeding assessment tool was used for the evaluation of bleeding events. RESULTS Most EXTEM and FIBTEM ROTEM parameters significantly differed between neonates with (n = 77) and without bleeding events (n = 33). Neonates with bleeding events had significantly lower PLTEM MCE and PLTEM MCF values compared to those without bleeding events (P

Estrogen-containing medication, prescribed either for contraception in women of reproductive age or for prevention of cardiovascular events and osteoporosis as well as for alleviation of symptoms related to menopause, is associated with changes in the hemostatic balance and contributes to increased risk of development of venous thromboembolic complications. This risk is dose and medication dependent, increases with age, congenital and/or acquired predisposition to thrombosis, and mode of administration. This review attempts to summarize the current knowledge regarding the pathophysiology of oral contraceptive (OC) and hormone replacement therapy (HRT) -induced prothrombotic state in women, the risk of thrombosis associated with administration of various commercially available OCs and HRT, the additional risk in women with hereditary or acquired thrombophilia, and the currently available recommendations regarding massive screening of women for thrombophilia prior to initial prescription or continuation of treatment with OCs and HRT preparations.

Hypercoagulability and thrombosis remain a challenge in severe coronavirus disease 2019 (COVID-19) infections. Our aim is to investigate the hemostatic profile of critically ill COVID-19 patients on therapeutic anticoagulant treatment.Forty one patients were enrolled into the study. We recruited 11 consecutive, COVID-19, patients who received therapeutic anticoagulant treatment on intensive care unit (ICU) admission. Disease severity indexes, biochemical, hematological and haemostatic parameters, endogenous thrombin potential (ETP), plasminogen activator inhibitor-1 (PAI-1) activity and extrinsically activated rotational thromboelastometry assay (EXTEM) were recorded on days 1, 3, 7. We also enrolled 9 ICU non-COVID-19, 21 non-ICU COVID-19 patients and 20 healthy blood donors as control populations.Critically ill COVID-19 patients demonstrated a more hypercoagulable and hypofibrinolytic profile related to those with COVID-19 mild illness, based on EXTEM amplitude at 10 min (A10), maximum clot firmness (MCF) and lysis index at 60 min (LI60) variables (p = 0.020, 0.046 and 0.001, respectively). Similarly, a more hypercoagulable state was detected in COVID-19 ICU patients related to non-COVID-19 ICU patients based on A10 and MCF parameters (p = 0.03 and 0.04, respectively). On the contrary, ETP and EXTEM (clotting time) CT values were similar between patients with severe and mild form of the COVID-19 infection, probably due to anticoagulant treatment given.Critically ill COVID-19 patients showed a hypercoagulable profile despite the therapeutic anticoagulant doses given. Due to the small sample size and the study design, the prognostic role of the hypercoagulability in this clinical setting remains unknown and further research is required in order to be assessed.

Our aim is to determine the most appropriate laboratory tests, besides anti-factor Xa (anti-FXa) chromogenic assays, to estimate the degree of anticoagulation with apixaban and compare it with that of rivaroxaban in real-world patients. Twenty patients with nonvalvular atrial fibrillation treated with apixaban 5 mg twice daily and 20 patients on rivaroxaban 20 mg once daily were studied. Conventional coagulation tests, thrombin generation assay (TGA), and thromboelastometry (nonactivated TEM [NATEM] assay) were performed in the 40 patients and 20 controls. The anti-FXa chromogenic assays were used to measure apixaban and rivaroxaban plasma levels. The NATEM measurements showed no significant difference between the 2 groups of patients. Concerning TGA, endogenous thrombin potential (ETP) was significantly decreased in patients on rivaroxaban as compared to those treated with apixaban ( P < .003). A statistically significant, strong inverse correlation between apixaban plasma concentrations and ETP ( P < .001) was observed. Apixaban significantly reduces ETP compared to controls, but to a lesser extent than rivaroxaban. Thrombin generation assay might provide additional information on apixaban exposure, which is required in order to individualize treatment especially for patients with a high bleeding risk. Our findings have to be further investigated in studies with larger sample sizes, in the entire range of apixaban exposure, with other direct oral anticoagulants, and in relation to clinical outcomes.

Background There are scarce data regarding the circadian pattern of symptoms onset in young patients presenting with acute myocardial infarction (AMI). We explored whether young patients with ST-segment elevation AMI exhibit a circadian variation in symptoms onset. Methods We recruited prospectively 256 consecutive patients who had survived their first ST-segment elevation AMI ≤ 35 years of age. Patients were categorized into 4 groups by 6-h intervals over 24 h. Results In 49 patients (19.1%) the clinical presentation of AMI was atypical. The symptoms onset was as follows: 00:01 to 06:00, 19.1%, 06:01 to 12:00, 32.4%; 12:01 to 18:00, 28.1%; and 18:01 to 24:00, 20.3%. There was a significant association between the time of day and the likelihood of symptoms onset (Rayleigh test, p

There is a complex and not fully elucidated association between pulmonary arterial hypertension (PAH) and coagulation disorders. The goal of this study was to evaluate platelet function, coagulation and fibrinolysis in PAH patients at diagnosis, before PAH-specific treatment initiation. We enrolled 20 healthy controls and 30 PAH patients (20 with connective tissue disease (CTD-PAH) and 10 idiopathic (iPAH)). None of the participants was on any antiplatelet or anticoagulation therapy. Blood samples from PAH patients were collected during the initial right heart catheterization. All subjects were assessed with platelet function analyzer-100 (PFA-100), epinephrine (Epi) and ADP-induced light transmission aggregometry (LTA), thromboelastometry (ROTEM) and endogenous thrombin potential (ETP). Our results showed that Epi and ADP-LTA values were significantly lower in newly diagnosed PAH patients compared to controls. Disaggregation was present in 73% of patients, a characteristic not seen in healthy individuals. In ROTEM assay, CT and CFT measurements were significantly higher and a angle lower compared to controls. ETP testing revealed significantly reduced outcomes in AUC, Cmax and Tmax. When CTD-PAH and iPAH patient groups were compared, iPAH ADP-LTA values were significantly decreased compared to CTD-PAH. In conclusion, newly diagnosed PAH patients presented with decreased platelet aggregation, clot propagation and thrombin generation, along with delayed initiation of the coagulation process. These hemostatic deficits could indicate an "exhaustion" of the coagulation process that could be caused by endothelial dysfunction and chronic activation of the procoagulant pathways. Further studies are warranted to confirm these laboratory findings and assess their potential clinical significance.

The contribution of prothrombotic genetic risk factors in the pathogenesis of premature acute myocardial infarction (MI) is controversial. We examined the prevalence of prothrombotic polymorphisms (G1691A of factor V gene [FV Leiden] and G20210A of prothrombin [FII] gene), deficiencies of natural anticoagulants (protein C, protein S and antithrombin III) and antiphospholipid syndrome (APS) in patients with early ST-segment elevation MI (STEMI). We recruited 255 consecutive patients who had survived a STEMI ≤ 35 years of age (224 men). The control group consisted of 400 healthy individuals matched with cases for age and sex. G20210A polymorphism of FII gene was more frequent in young patients than in controls (7.4 vs. 3.5%, p = 0.023). The odds ratio (OR) for STEMI for carriers versus non-carriers was 2.239 (95% CI 1.102–4.250). The adjusted OR for major cardiovascular risk factors was 2.569 (95% CI 1.086–6.074). The risk was increased by 22-fold (95% CI 9.192–66.517) when G20210A polymorphism was present in combination with smoking. There was no difference in the prevalence of FV Leiden between patients and controls (7.8 vs. 6.5%, p = 0.512). There was only one patient (0.4%) with protein C deficiency and one with APS (0.4%). G20210A polymorphism of FII gene may be associated with increased risk of premature STEMI and the risk increases substantially when smoking is present. The contribution of other prothrombotic disorders such as deficiencies of protein C, protein S and antithrombin III and APS was minimal in this cohort.

There are scarce data regarding risk factors and prognosis of patients with premature ST segment elevation myocardial infarction (STEMI) and "normal or near normal" coronary arteries (N/NNCAs). We compared the characteristics and long-term prognosis of patients with premature STEMI and N/NNCAs with their counterparts with significant coronary artery disease (CAD). We recruited 330 patients who had STEMI ≤35 years of age and 167 age- and gender-matched controls. All patients underwent coronary angiography. Coronary arteries with no lesions or lesions causing30% reduction in lumen diameter were defined as N/NNCAs, whereas narrowings causing ≥50% diameter reduction formed the significant CAD group. Lipid profile, homocysteine levels, and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism were determined. Sixty patients (18%) had N/NNCAs. Patients with N/NNCAs had lower low-density lipoprotein-cholesterol and higher high-density lipoprotein-cholesterol levels, higher homocysteine levels, and higher prevalence of MTHFR TT genotype (34.6 vs 18%, p = 0.008) compared with patients with significant CAD. After a median follow-up of 8 years, cardiovascular events occurred in 105 (36%) of 291 patients with available follow-up data. Significant CAD was associated with higher risk for recurrent cardiovascular events after adjustment for traditional risk factors (hazard ratio 2.095, 95% confidence interval 1.088 to 3.664, p = 0.022) and additional adjustment for the left ventricular ejection fraction, reperfusion therapy, and persistent smoking (hazard ratio 1.869, 95% confidence interval 1.007 to 3.468, p = 0.041). In conclusion, patients with premature STEMI and N/NNCAs have fewer lipid abnormalities, higher homocysteine levels and prevalence of MTHFR TT genotype, and better long-term prognosis compared with their counterparts with significant CAD.

Background Previous studies suggested a possible negative interference of proton pump inhibitors (PPIs) on clopidogrel’s antiplatelet effect because of the competitive inhibition of the CYP 2C19 isoenzyme. Moreover, carriers of the loss-of-function allele of CYP2C19 polymorphism (CYP2C19*2) display significantly lower responses to clopidogrel. In this study, we investigated the association between CYP2C19*2 genotype, PPI intake and clopidogrel resistance in patients with coronary artery disease (CAD) and their effect on clinical outcome. Methods We recruited 95 patients with CAD receiving chronic clopidogrel therapy in combination with aspirin. Platelet reactivity was simultaneously assessed by INNOVANCE PFA-100 P2Y, ADP-induced light transmission aggregometry (LTA), flow-cytometric vasodilator-stimulated phosphoprotein (VASP)-phosphorylation assay and multiple electrode aggregometry (Multiplate). Cardiovascular outcomes were recorded during 1-year follow-up period. Results Only platelet reactivity assessed by measuring platelet phosphorylated-VASP demonstrated a significant higher platelet reactivity in carriers of CYP2C19*2 (p = 0.023). The other methods displayed higher - but not statistically significant - platelet reactivity in patients carrying the CYP2C19*2 variant as compared with non-carriers. Patients on PPIs demonstrated almost similar suppression of platelet reactivity in comparison with those not treated with PPIs by all platelet function assays. In logistic regression analysis none of the platelet function assays measurements were related with clinical outcomes. Similarly neither CYP2C19*2 genetic variant nor PPI treatment were associated with adverse clinical events. Conclusions PPI co-administration did not influence clopidogrel’s antiplatelet effect on laboratory testing by all platelet function assays used. On the contrary, patients carrying CYP2C19*2 genotype had significantly higher residual platelet reactivity as estimated by VASP-phosphorylation assay.

We examined whether angiotensin-converting enzyme (ACE) insertion (I)/deletion (D) polymorphism is associated with the development of myocardial infarction (MI) ator = 35 years of age. The study sample consisted of 201 patients with premature MI and 140 age- and sex-matched healthy individuals. No difference was found in the distribution of ACE genotypes between the patients and controls. A higher prevalence of the DD genotype among hypertensives was found compared with the non-hypertensive patients (62.5% vs 35.6%, p = 0.01). ACE polymorphism is not associated with the development of premature MI and this might be due to the low prevalence of hypertension in young coronary patients.

Intoduction Increased levels of plasminogen activator inhibitor-1 (PAI-1) have been associated with worse outcome in ALI/ARDS. A single guanosine insertion/deletion (4G/5G) polymorphism in the promoter region of the PAI-1 gene, may play an important role in the regulation of PAI-1 expression. The objective of the study was to evaluate the effect of this polymorphism on the outcome of critically ill patients with ALI/ARDS. Materials and Methods 52 consecutive ventilated patients with ALI/ARDS were studied. Bronchoalveolar lavage was performed within 48 hours from diagnosis. Measurement of plasma and BALF PAI-1 activity and D-dimers levels, and 4G/5G genotyping of PAI-1 were carried out. The primary outcome was 28-day mortality, and secondary outcomes included organ dysfunction and ventilator-free days. Results 17 patients were homozygotes for the 4G allele. Severity scores were not different between subgroups upon study enrollment. 28-day mortality was 70.6% and 42.9% for the 4G-4G and the non-4G-4G patients, respectively (p = 0.06). PAI-1 activity levels and D-dimer in plasma and BALF were not significantly different between the 4G-4G and the non-4G-4G subgroups. In the multivariate analysis, genotype 4G/4G was the only variable independently associated with 28-day mortality (Odds Ratio = 9.95, 95% CI: 1.79-55.28, p = 0.009). Furthermore, genotype 4G/4G and plasma PAI-1 activity levels were independently negatively associated with ventilator free days (p = 0.033 and p = 0.008, respectively). Conclusions ALI/ARDS patients, homozygous for the 4G allele of the PAI-1 gene, experienced higher 28-day mortality. This genotype was associated with a reduction in the number of days of unassisted ventilation and was inversely associated with the number of days without organ failure.

The pathogenetic mechanism of premature myocardial infarction (MI) remains unknown. We explored the association of homocysteine and its main genetic modulator methylenetetrahydrofolate reductase (MTHFR) 677C-T polymorphism with the development of MIor=35 years of age.We performed a case-control study of 147 patients with a first MIor=35 years and 103 age and sex-matched controls. We assessed plasma lipids, homocysteine, folate, vitamin B(12) levels and MTHFR 677C-T polymorphism.Patients with premature MI had higher homocysteine levels (13.9+/-8.6 vs. 11.8+/-4.9 mmol/l, p=0.02) and higher prevalence of TT homozygocity compared to controls (27.1% vs. 14.6%, p=0.02). Thirty-four patients (23.6%) had angiographically "normal" coronary arteries. Subgroup analysis according to angiographic findings ("normal" coronary arteries versus significant coronary heart disease) showed that only patients with MI and "normal" coronary arteries (MINCA) had higher homocysteine levels compared to controls (17.6+/-12.2 vs. 11.8+/-4.9 mmol/l, p0.001). The prevalence of TT genotype was higher only in patients with MINCA compared to controls (44.1% vs. 14.6%, p=0.001) (odds ratio 4.6, 95% confidence interval (CI), 1.9-11, p=0.001). This association remained after adjusting for conventional risk factors (odds ratio 3.4, 95% CI, 1.1-10.4, p=0.03). The adjusted odds ratio for MINCA of young individuals with MTHFR TT genotype and folate levels in the lowest quartile (or=5 ng/ml) was 6.1 (95% CI, 1.1-31, p=0.04).Homozygocity for the 677C-T mutation of MTHFR is independently associated with the development of premature MINCA.

Tissue factor (TF), plays a key role in pathophysiology of disseminated intravascular coagulation (DIC) and multiple organ dysfunction syndrome. Tissue factor pathway inhibitor (TFPI) regulates tissue factor activity. Several studies in septic patients have shown that TF production exceeds TFPI production,promoting a prothrombotic state.

Endothelial protein C receptor (EPCR) is a candidate mediator in the pathogenesis of thrombosis, as several data in the literature indicate that polymorphisms such as EPCR 4678G/C and 4600A/G are associated with either protective effect or increased risk of thrombosis, respectively. We investigated the prevalence of these polymorphisms in patients with thrombotic disorders as well as their impact on the risk of thrombosis, the age of first thrombotic episode, and recurrence. The prevalence of the rare EPCR alleles 4600G and 4678C was comparable in patients and controls. However, in a subset analysis, we observed that 4600G allele was more prevalent among patients who developed thrombosis at younger age (

TMA = transcription-mediated amplification.From the Department of Hygiene and Epidemiology, Athens University Medical School, Athens; the Second Regional Blood Transfusion and Comprehensive Haemophilia Care Center, Laiko General Hospital, Athens; the Red Cross General Hospital, Athens; the First Department of Internal Medicine, Western Attica General Hospital, Athens; and the Department of Nephrology, Laiko General Hospital, Athens, Greece.

Background Angiotensin-converting enzyme (ACE) gene polymorphism has been associated with an increased incidence of myocardial infarction. Recent studies have investigated a potential influence of ACE gene polymorphism on fibrinolysis or endothelial function. It has been previously established that essential hypertension is accompanied by endothelial dysfunction and fibrinolytic balance disorders. The aim of our study was to study the relation between ACE gene polymorphism and fibrinolytic/hemostatic factors as well as endothelial cell damage markers in patients with hypertension. Methods The following parameters were evaluated in 104 patients with previously untreated hypertension: plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) antigen, fibrinogen, D -dimer, and von Willebrand factor (vWF). The genotype of the ACE gene was also determined (by the polymerase chain reaction method), and patients were characterized according to the observed alleles as deletion/deletion (DD), insertion/insertion (II), or insertion/deletion (ID). Results Those with DD genotype (n = 42) had significantly higher plasma levels of PAI-1 antigen ( P =.012), tPA antigen ( P =.0001), fibrinogen ( P =.0002), D -dimer ( P =.0001) and vWF ( P =.0004) compared with ID (n = 30) or II (n = 32) genotypes. The ACE gene genotypes appeared to be significant predictors for plasma PAI-1 antigen, tPA antigen, fibrinogen, D -dimer, and vWF even after adjustment for age, sex, body mass index, triglyceride and cholesterol levels, and blood pressure. Conclusions Our findings suggest that the ACE/DD genotype is associated with hemostasis balance disturbances reflecting hypercoagulability and endothelial damage in patients with untreated hypertension. (Am Heart J 2000;140:760-5.)

We report a case of a 35-year-old previously healthy man treated with clomiphene for infertility, who presented with blurred vision in his left eye due to ocular vein occlusion as documented by fluorescein angiography. The patient was heterozygous for the factor V Leiden (FV Leiden) mutation and for the 1298 A-C polymorphism of the methylene-tetrahydrofolate reductase (MTHFR) gene. He was treated with clopidogrel and is now free of symptoms. Because congenital thrombophilia is a moderate risk factor for central retinal vein occlusion and the administration of clomiphene may trigger this process, we recommend screening of young patients for FV Leiden before clomiphene treatment.

A 29-year-old primigravida at 27 weeks of twin gestation was admitted to the hospital because of vomiting, nausea and persisting abdominal pain. This was the third consecutive admission within a mo...

Our objectives are to describe the progression of HIV disease and to assess the influence of hemophilia-related variables, age at infection, and antibodies to cytomegalovirus infection (anti-CMV) in a Greek cohort of 158 HIV-1-positive hemophilic men, who received prospective follow-up for up to 16 years after infection. A total of 79 patients had died, representing a cumulative progression rate of 72.4% (95% confidence interval [CI], 56.6-83.3). A significant proportion of the mortality (30%) resulted from conditions not formally related to AIDS, with liver failure and cerebral hemorrhage predominant. At 16 years after seroconversion, 66 patients had developed clinical AIDS, a cumulative progression rate of 58.2% (95% CI, 47.1%-86.3%) whereas 15 years after infection 81.5% (95% CI, 74.2%-87.9%) of the patients had AIDS or a CD4 cell count200 cells/mm3. Hemophilia-related variables or presence of anti-CMV were not significantly associated with disease progression. Age at infection was a strong prognostic factor for all three endpoints. Appropriate modeling showed a nonlinear age effect, with a steeper increase of relative hazard for patients40 years of age at seroconversion. The age effect remained significant even after controlling for current CD4 cell count. Further investigation is required to elucidate the mechanisms of the age effect and the contribution of HCV coinfection on the disease progression.The Greek Hemophilia Cohort Study encompasses 158 HIV-positive men with documented seroconversion dates. The present study estimated the rate of progression to all-cause mortality, clinical AIDS, or advanced immunodeficiency 16 years after seroconversion and evaluated the independent effects of hemophilia-related factors, age at seroconversion, and cytomegalovirus status early in the course of infection. Seroconversion dates extended from September 1980 to December 1985. By 1996, after a median follow-up of 11.6 years, 117 of the 158 men had developed AIDS or had a CD4 cell count of 20 cells/cu. mm, and 79 had died. The estimated cumulative incidence rates of clinical AIDS and death over 16 years since infection were 58.1% and 72%, respectively. 30% of the mortality was due to diseases not formally associated with AIDS (e.g., liver failure and cerebral hemorrhage). A significant association existed between older age at seroconversion and more rapid progression to both AIDS and death, with a particularly steep gradient for patients over 40 years old at seroconversion. The age effect remained significant even after controlling for current CD4 cell count. Increases of 1.32-fold and 1.33-fold in the risks of dying and developing AIDS, respectively, were obtained with every one unit decrease in CD4 cell count on the square root scale. Severity of hemophilia, dosage of clotting factor concentrates, and antibodies to cytomegalovirus were not associated with either AIDS risk or mortality. Further investigations are urged to clarify the mechanisms underlying the age effect observed in this study.

The role of systemic risk factors (age, smoking, diabetes, arterial hypertension) in the development of retinal vein occlusion (RVO) is well established. However, the association of RVO with genetic predisposition to thrombosis remains poorly understood. The aim of the study was to assess any possible additional effect of genetic predisposition to the already well known 'classical' risk factors of RVO in a cohort of elderly Greek patients. Fifty-one elderly patients with RVO and 51 healthy individuals matched for age and sex were evaluated for systemic risk factors (smoking, diabetes, dyslipidemia, arterial hypertension) and coagulation defects (lupus anticoagulant, natural inhibitors of coagulation). Additionally, genotyping was performed for mutations/polymorphisms involved in haemostasis such as: FV G1691A, FV G4070A, FIIG 20210A, MTHFR C677T and A1298C, PAI-1-675 4G/5G, F XIII exon 2G/T, EPCR A4600G and G4678C. We identified systemic risk factors in the majority of the patients Hypertension (P=0.001), dyslipidemia (P=0.029) and diabetes (P=0.01) are associated with RVO in the majority of the patients. The prevalence of prothrombotic risk factors was not significantly different in the patients with RVO compared to controls. Apart from systemic risk factors, genetic predisposition to thrombosis does not seem to have an important association with RVO in this group of elderly patients.

Summary. We illustrate the usefulness of direct mutation detection for genetic counselling by showing its application to an extremely large mild haemophilia A pedigree (91 haemophiliacs) originating from the village of Aiani in Macedonia, northern Greece. The causative mutation has already been shown to be an A to T transversion in codon 280 of the FVIII gene which replaces Asn 280 (AAC) by He (ATC) and which creates a new Bam HI restriction site in exon 7. The latter permitted direct, rapid and reliable detection of the mutation in relevant family members. All major branches of the family were shown to share the mutation, and carrier status was diagnosed or excluded for 23 possible carriers. Other interesting characteristics of the Aiani haemophilic population are a slightly higher longevity and fecundity than that observed in the general population and a wide range of FVIILC levels (5–25%) associated with the mutation.

Thrombomodulin is a cell surface-expressed glycoprotein that serves as a cofactor for thrombin-mediated activation of protein C (PC), an event further amplified by the endothelial cell PC receptor. The PC pathway is a major anticoagulant mechanism that downregulates thrombin formation and hedges thrombus formation. The objectives of this review were to review recent findings regarding thrombomodulin structure, its involvement in the regulation of hemostasis and further discuss the implication, if any, of the genetic polymorphisms in the thrombomodulin gene in the risk of development of thrombosis. We performed a literature search by using electronic bibliographic databases. Although the direct evaluation of risk situations associated with thrombomodulin mutations/polymorphisms could be of clinical significance, it appears that mutations that affect the function of thrombomodulin are rarely associated with venous thromboembolism. However, several polymorphisms are reported to be associated with increased risk for arterial thrombosis. Additionally studies on knock out mice as well studies on humans bearing rare mutations suggest that thrombomodulin dysfunction may be implicated in the pathogenesis of myocardial infraction.

There are limited and controversial data regarding the impact of 4G/5G polymorphism of the plasminogen activator inhibitor-1 (PAI-1) gene in the pathogenesis of premature myocardial infarction (MI). We explored whether 4G/5G polymorphism of the PAI-1 gene is associated with the development of MIor= 35 years of age. We recruited 201 consecutive patients who had survived their first acute MIor= 35 years of age (mean age = 32.2 +/- 3.4 years). The control group consisted of 140 healthy individuals matched with cases for age and sex, without a family history of premature coronary heart disease. 4G/5G polymorphism of PAI-1 was tested with polymerase chain reaction and reverse hybridization. 4G allele carriers (4G/4G and 4G/5G genotypes) of PAI-1 were less frequent in patients than in controls (69.6 vs. 83.6%, P = 0.007). 4G carriership of the polymorphism of PAI-1 was associated with lower risk for acute MI (odds ratio 0.45, 95% confidence interval 0.23-0.88, P = 0.02) after adjusting for major cardiovascular risk factors. Patients possessing the 4G allele had higher PAI-1 plasma levels (32.2 +/- 25 vs. 22.2 +/- 11.3 ng/ml, P = 0.006) but lower lipoprotein(a) levels (10.1 [2.1-29.9] vs. 15.3 [8.2-57.1] mg/dl, P = 0.03) compared to 5G/5G homozygotes. Our data indicate that the 4G allele of the PAI-1 4G/5G polymorphism is less frequent among survivors of MI at very young age compared with matched controls.

We herein report a 56-year-old man who presented with abdominal pain, diarrhea and a 22-kg-weight loss over 4 months. He was on acenocoumarol treatment because of portal, splenic and mesenteric vein thrombosis (PSMVT) 3 months before, with admission international normalized ratio (INR):1.6. Doppler ultrasonography and helical computerized tomographic scan of the abdomen showed complete thrombosis of the extrahepatic portal vein extending into the superior mesenteric vein and splenic vein. The manifestation of thrombosis was in the absence of provocative stimuli or local cause. The patient had a negative history of venous thromboembolism. Thrombophilia workup revealed double heterozygosity for factor V Leiden and prothrombin G20210A mutation. He was immediately started with intravenous unfractionated heparin, followed by oral anticoagulation with target INR 2-3. Five days after a Doppler examination showed significant improvement in the flow within the portal vein, and a computerized tomographic scan of the abdomen 1 month later showed extensive recanalization of the portal venous system. The patient is now 36 months out from the second PSMVT episode and is doing well although maintaining oral lifelong anticoagulation. The case is of particular interest in that PSMVT was the first manifestation of this combined disorder. We conclude that all patients presenting with unexplained PSMVT should be investigated for the presence of a hypercoagulable state. Anticoagulation should be considered in all patients with this diagnosis and should be a lifelong therapy in those with an underlying thrombophilia.

Introduction There are limited and controversial data regarding the impact of G-455A polymorphism of β-fibrinogen gene in the pathogenesis of premature myocardial infarction (MI). We examined whether the G-455A polymorphism of β-fibrinogen gene is associated with the development of MI ≤ 35 years of age. Methods We recruited 181 consecutive patients who had survived their first acute MI ≤ 35 years of age (mean age = 32.2 ± 3.4 years). The control group consisted of 129 healthy individuals matched with cases for age and sex, without a family history of premature coronary heart disease. G-455A polymorphism of β-fibrinogen was tested with polymerase chain reaction and reverse hybridization. Results There was a higher prevalence of carriers of the A allele (GA+AA genotype) in controls than in patients (odds ratio [OR] 0.57, 95% confidence interval [CI] 0.36 to 0.91, p = 0.02). G-455A polymorphism of β-fibrinogen gene was associated with lower risk for acute MI (OR 0.46, 95% CI 0.25 to 0.83, p = 0.01) after adjusting for major cardiovascular risk factors. Fibrinogen levels were higher in patients compared to controls [332 (292-385) vs. 311 (262-373) mg/dL, p = 0.01], but the adjusted for classical risk factors fibrinogen levels did not differ (OR 1.003, 95% CI 0.99 to 1.01, p = 0.37). Patients possessing the A allele did not differ in their fibrinogen and lipid levels compared to patients with the -455GG genotype. Conclusions Our data indicate that the presence of the G-455A polymorphism of β-fibrinogen gene has a “protective effect” against the development of non-fatal acute MI ≤ 35 years of age in Greece.

Objectives : To determine the prevalence of hepatitis viruses B (HBV) and C (HCV) co-infections in HIV-infected patients and the overall impact of these co-infections on deceased AIDS patients survival. Methods : One hundred and eighty-one patients (159 males, 22 females) infected with HIV, attending an academic AIDS unit in Athens, Greece, constituted the study population. The study population consisted of 124 homo/bisexual men, 34 heterosexuals, 12 intravenous drug users (IDU) and 11 blood transfusion recipients. Virological markers tested for HBV infection included HBsAg, anti-HBs and total anti-HBc by enzyme-linked immunoassays. Detection of HCV antibodies was carried out by third generation enzyme-linked immunoassay, and repeatedly positive samples were further tested by a supplemental enzyme-linked immunoassay; only sera reactive by both methods were considered to be HCV-positive. Results : The prevalence of HBV markers was 67.4%: 71.8% in homo/bisexuals, 35.3% in heterosexuals, 91.7% in IDUs and 90.9% in blood transfusion recipients ( P =0.00004). The prevalence of HCV antibodies was 13.8%: 8.1% in homo/bisexuals, 8.8% in heterosexuals, 58.3% in IDU and 45.5% in blood transfusion recipients ( P =0.000001). The prevalence of HCV antibodies was not significantly higher in homo/bisexuals than in heterosexuals ( P =0.8). Co-infection with HBV or HCV, or both, did not influence the survival of deceased AIDS patients ( n =73). Conclusions : HBV infection was equally prevalent among homo/bisexuals and IDU with HIV infection, whereas HCV infection was more prevalent in IDU than in homo/bisexuals with HIV infection. The prevalence of HCV infection was equal among heterosexuals and homo/bisexuals, indicating that if sexual transmission of HCV occurs, homo/bisexuals are not at greater risk than heterosexuals. Finally, the survival of deceased AIDS patients was not affected by the presence of HBV and HCV co-infections.

This study was designed to investigate both resistance to activated protein C (APC-R) and the factor FV Q506 mutation incidence in patients with a history of acute myocardial infarction (AMI) and patients with primary hypertension (PH), a high-risk group for arterial thrombosis. Eighty patients with a history of AMI (group A), 160 patients with a history of PH (group B), and 124 age-matched controls without arterial disease (group C) were studied. APC-R was determined using the Coatest APC Resistance Kit of Chromagenix, Sweden. The prevalence of the FV Q506 mutation was estimated by DNA analysis (Bertina method). The prevalence of the FV Q506 mutation was 20%, 13.75%, and 8% in groups A, B, and C, respectively (A v C P = .0466). The prevalence of APC-R was 47.5% in group A v 13% in group C (P < .0001) and 36.25% in group B v 13% in group C (P < .0001). The response to activated protein C expressed as mean value +/- SD was 2.05 +/- 0.33 in group A v 2.56 +/- 0.46 in group C (P < .05) and 2 +/- 0.22 in group B v 2.56 +/- 0.46 in group C (P < .05). These findings suggest that patients with a history of AMI or PH have a significantly increased incidence of both APC-R and FV Q506 mutation compared with the control group. These findings support the hypothesis that these anticoagulant defects may be risk factors for arterial thrombosis.

This study was aimed at investigating haemostasis parameters in patients (pts) with arterial hypertension (AH) before any medical treatment and to correlate these findings with those in healthy normal Greek population 83 pts (48 m, 35 w) mean age 49.8 +/- 10.1 yrs, body mass index 23.4 +/- 1.5 with mild to moderate AH and 42 healthy volunteers matched for sex (24 m, 18 w), age 51.2 +/- 10.5 yrs and body mass index 22.8 +/- 1.46 were studied. Fibrinogen, vWF, plasminogen, ECLT, a2 antiplasmin, tPA-Ag, PAI-1 in all pts and in the control group were measured. Mean age and BMI did not significantly differ between the two groups. The hypertensive patients had significantly higher levels of fibrinogen (327.75 +/- 51.36 vs. 272.84 +/- 46.8 mg/dl), tPA-Ag (8.81 +/- 3.32 vs. 5.76 +/- 2.54 ng/ml) and PAI-1 (11.8 +/- 10.9 vs. 7.91 +/- 5.5 IU/ml), whereas a2 antiplasmin level was significantly lower (98.71 +/- 15.40 vs. 107.84 +/- 17.52%). No differences were found between hypertensives and normal subjects in vWF, plasminogen and ECLT. These preliminary data suggest that in pts with mild to moderate AH, before any medical treatment, there are significantly higher levels of fibrinogen, tPA-Ag and PAI-1 compared with normal volunteers, whereas there are significantly lower a antiplasmin levels. These findings indicate a disturbance in the haemostasis balance with hypercoagulability and fibrinolytic deficiency.

Background Myeloproliferative neoplasms (MPNs) have been associated with a high incidence of thrombosis and bleeding episodes, which significantly contribute to disease-related morbidity and mortality. Clinical data indicate an association of the JAK2V617F mutation, seen in nearly all polycythemia vera (PV) cases and almost 60% of those with essential thrombocythemia (ET) and myelofibrosis (MF). The mutation is also seen in 37% of patients with in splachnic vein thrombosis (SVT) and its presence was associated with an increased risk for SVT. However, the prevalence of JAK2V617 seems to be low in patients with other thromboembolic events in unusual sites such as cerebral sinus, upper limb deep venous thrombosis (DVT). Additionally, activating mutations of MPL gene, seen in 3% of ET and 5% of MF patients, are considered as a significant risk factor for microvessel disturbances and have been associated with an increased risk of arterial thrombosis. Retinal vein occlusion (RVO) is a thrombotic complication in an uncommon site that may result in sight threatening disease. In this study we investigated the prevalence of JAK2V617F and MPLW515L/K mutations in a prospectively assembled cohort of patients with RVO, hypothesizing that some cases may be associated with an underlying undiagnosed MPN. Patients and Methods We studied 52 (23 males and 29 females) consecutive patients with no evidence of an underlying MPN who had been diagnosed with RVO confirmed with fluorangiography from January 2007 to September 2011. The mean age was 70 years (range: 49-85) Twenty eight patients (53.8%) presented with central RVO and 24 patients with branched RVO (46.5%). DNA was extracted from peripheral blood samples by standard procedures. The JAK2V617F mutation was detected using a tetra-primer amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) assay with a sensitivity of 1% and the allele burden was estimated with a semi-quantitative method. MPLW515L/K were detected using allele-specific PCR (AS-PCR) assays with a sensitivity of 1%. Results Overall, MPN associated mutations were detected in 5/52 cases. JAK2V617F was detected in 2/52 cases (3.8%; 95%CI-1.4%-9%), while MPL exon 10 mutations were detected in 3/52 (5.7%; 95%CI-0.6%-12%). The JAK2V617F allele burden in the two positive patients was 45% and 52% respectively. Both patients who carried the JAK2V617F mutation were female. The first patient had been already diagnosed with ET according to the WHO criteria at the time of RVO screening. She was receiving hydroxyurea and aspirin and her platelet count was normal. The second patient who also carried the JAK2V617F mutation had a PLT count of 850.000/μl at the time of screening and was diagnosed with ET within the 3 following months. The patients with MPL mutations presented with normal blood counts. Conclusions Our findings indicate that a latent MPN could underlie RVO even in the absence of conventional diagnostic criteria. Our results represent the first report that MPL mutations could underlie RVO cases and suggest that routine screening of RVO cases for MPN mutations may be useful, especially in older patients. Disclosures: No relevant conflicts of interest to declare.

SummaryDemographic data of the Greek haemophilia A and B population for the period 1972-1993 were analyzed. Prevalence at birth including known not-registered patients was calculated at 23.1 per 100,000 male births. However, the observed prevalence in 1993 was only 61% of the expected. Since 1975 the proportion of mild cases had significantly increased. Adjusted by age, severity and HIV status reproductive fitness of haemophiliacs was 0.62. Overall mortality was 2.6 times higher than in the general population, but 7.9 times among patients with severe haemophilia and 16.4 among HIV(+) haemophiliacs. Fifty out of 78 deaths occurred among HIV(+) patients and 28 of these were caused by AIDS. Inhibitor patients did not show excess mortality due to bleeding. Cancer mortality was equal to normal, but the number of deaths from ischaemic heart disease was 0.25 of the expected. Risk of death due to cerebral haemorrhage was 3.8 times higher in HIV(+) haemophiliacs than in HIV(-).

The antibody responses and the prevalence patterns of antibodies to hepatitis C virus (anti-HCV) in a cohort of patients (n = 210) with bleeding disorders were studied using a first-generation and a second-generation enzyme immunoassays (EIA-1, EIA-2) as well as a second-generation recombinant immunoblot assay (RIBA-2). The anti-HCV prevalence as determined by EIA-1 and EIA-2 was 183/210 (87.1%) and 197/210 (93.8%), respectively (p = 0.0026). None of the 17 EIA-2(+)/EIA-1(-) samples was scored nonreactive by RIBA-2. At follow-up, samples of 123 patients were tested. Twenty-nine out of 111 patients reactive by EIA-1 seroreverted according to EIA-1 while the seroreversion rate with EIA-2 was 0 out of the 121 (p10(-8)). The anti-HCV prevalence by EIA-2 was 150/154 (97.4%) in anti-HIV-1-positive individuals and 47/56 (83.9%) in the anti-HIV-1-negative ones (p = 0.001). However, high assay signals (OD 492 nm2.0) were observed in 94/150 (62.7%) and 45/47 (95.7%) of the anti-HIV-1-positive and -negative patients, respectively (p = 10(-5)). The decreasing anti-HCV reactivity among anti-HIV-1-positive individuals was mainly due to diminishing c33c reactivity. Seroconversion to anti-HCV was observed in 3/7 (42.9%) cases with acute icteric non-A, non-B hepatitis by both EIA-1 and EIA-2, while the remaining 4 cases had detectable levels of anti-HCV 1-18 months before the acute episode.

Peripheral blood lymphocyte subsets and the incidence of LAV/HTLV-III antibodies were studied in 63 patients with hemophilia A who had been transfused with a low dose regimen of commercial (U.S.A.) factor VIII concentrates. Five patients with hemophilia B were also included in this study. In hemophilia A patients a significant reduction in the percentage and absolute numbers of T4+ cells and of the T4/T8 ratio and a significant increase in the percentage of T8+ and Leu-7+ cells were observed. These abnormalities were independent of the presence of anti-LAV/HTLV-III. In hemophilia B patients a significant increase of T8+ cells and a decrease of T4/T8 ratio was noted while the percentage of Leu-7+ cells was normal. A significant negative correlation of F VIII units transfused and T4/T8 ratio was seen only in LAV/HTLV (-) patients, suggesting that F VIII per se could cause immunodysregulation. Seropositive patients were found to have consumed a larger amount of F VIII units than seronegative patients during the period 1980-1984 (p less than 0.005).

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).