Utility of ACMG classification to support interpretation of molecular genetic test results in patients with factor VII deficiency.

Background: The American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) have introduced an internationally shared framework for variant classification in genetic disorders. FVII deficiency is a rare inherited autosomal recessive bleeding disorder with sparse data concerning ACMG classification. Methods: To develop an approach which may improve the utility of molecular genetic test results, 129 patients with FVII deficiency were retrospectively assigned to six subgroups for exploratory analysis: F7 gene wildtype (group 1), ACMG 1 (benign variant) or ACMG 2 (likely benign variant), only (group 2), ACMG 3 (variant of uncertain significance) ± ACMG 1-2 heterozygous or not classified variant (group 3), ACMG 4 (likely pathogenic variant), or ACMG 5 (pathogenic variant) single heterozygous ± ACMG 1-3 single heterozygous (group 4), ACMG 4-5 homozygous or ≥2 ACMG 4-5 heterozygous or ≥1 ACMG 4-5 heterozygous plus either ACMG 1 c.1238G>A modifying variant homozygous or ≥2 ACMG 1-3 (group 5), FVII deficiency and another bleeding disorder (group 6). Results: Eleven of 31 patients (35.5%) in group 5 had abnormal ISTH-BS (n = 7) and/or history of substitution with recombinant factor VIIa (n = 5) versus 4 of 80 patients (5.0%, n = 1 abnormal ISTH-BS, n = 3 substitution) in groups 1 (n = 2/22), 2 (n = 1/29), 3 (n = 0/9), and 4 (n = 1/20). Four of 18 patients (22.2%) with FVII deficiency and another bleeding disorder (group 6) had an abnormal ISTH-BS (n = 2) and/or history of substitution with recombinant factor VIIa (n = 3). Conclusion: Patients with a homozygous ACMG 4-5 variant or with specific combinations of heterozygous ACMG 4-5 ± ACMG 1-3 variants exhibited a high-risk bleeding phenotype in contrast to the remaining patients without another bleeding disorder. This result may serve as a basis to develop a genotype/phenotype prediction model in future studies.

Fondaparinux Pre-, Peri-, and/or Postpartum for the Prophylaxis/Treatment of Venous Thromboembolism (FondaPPP).

We analyzed data for women who received fondaparinux for ≥7 days during pregnancy. The study retrospectively included women who received fondaparinux pre-, peri- and/or postpartum for ≥7 days for prophylaxis/venous thromboembolism (VTE) treatment at German specialist centers (2004-2010). Data on pregnancy, VTE risk factors, anticoagulant treatment, pregnancy outcome and adverse events were extracted from medical records. 120 women (mean age 31.5 years) were included. Among 84 women with prior pregnancies, 41.0% had ≥1 abortion. Anticoagulation was indicated for prophylaxis in 92.5% cases, including 82.5% women with an elevated VTE risk (82.8% thrombophilia, 34.2% VTE history). All women received low-molecular-weight heparin (LMWH) as first-line therapy; 3 also unfractionated heparin. Treatment changed to fondaparinux, due to heparin allergy (41.7%) or heparin-induced thrombocytopenia (10.0%). Fondaparinux was generally well tolerated. Adverse events included bleeding events (n = 5), abortion (n = 2), premature births (n = 2), stillbirth (n = 1), arrested labors (n = 2), injection site erythema (n = 4) and unspecified drug hypersensitivity (n = 6). No VTE events or increased liver enzymes occurred during treatment. In this retrospective study, fondaparinux was effective and well tolerated. Trial registration: ClinicalTrials.gov NCT01004939.

Severe dengue categories as research endpoints-Results from a prospective observational study in hospitalised dengue patients.

Severe dengue was perceived as one clinical disease entity until the WHO 2009 classification stratified it into severe vascular leakage, severe bleeding, and severe organ dysfunction. The objectives of this study were to investigate the potential use of severe dengue categories as endpoints for intervention research. 271 patients with severe dengue among 1734 confirmed dengue patients were followed prospectively in this hospital-based observational study in Latin America and Asia. We compared the distribution of severe dengue categories according to gender and age (below/above 15y), and determined the relative frequency and the overlap of severe dengue categories in the same patients. In a next step, we extended the analysis to candidate moderate severity categories, based on recently suggested definitions which were adapted for our purposes. Severe vascular leakage occurred in 244 (90%), severe bleeding in 39 (14%), and severe organ dysfunction in 28 (10%) of 271 severe dengue patients. A higher frequency of severe leakage was seen in children or adolescents (<15y) compared to adults. More than 80% of the severe leakage cases, and 30-50% of the cases with severe bleeding or severe organ dysfunction, were defined as severe on the basis of that feature alone. In 136 out of 213 patients with severe leakage alone, neither moderate bleeding manifestation nor hepatic involvement was recorded. On the other hand, moderate leakage manifestations were detected in 4 out of 12 cases that were classified as severe based on bleeding alone. A major proportion of severe dengue patients exhibited clinical manifestations of severe vascular leakage only, which may constitute a useful endpoint for intervention research or pathophysiology studies. Severe bleeding and severe organ manifestation were recorded less frequently and exhibited a higher degree of overlap with severe leakage. Severe bleeding without leakage may be associated with individual predisposition or the presence of comorbidities. More detailed assessments are needed to explore this hypothesis. Candidate moderate disease endpoints were investigated and need to be further validated.

Replacement Therapy in Patients with Von Willebrand Disease-Indications and Monitoring.

In patients with von Willebrand disease (VWD), replacement therapy may be indicated in the case of spontaneous bleeding, surgical interventions and injuries/trauma or as a prophylaxis of spontaneous bleeding episodes. The deficient von Willebrand factor (VWF) is replaced with or without factor VIII (FVIII). Dual VWF/FVIII concentrates can be beneficial in the case of low FVIII level, while repeated dosing may lead to very high FVIII levels, with a potential thrombogenic effect in individual VWD patients. An excessive FVIII:C increase can be limited by using a VWF product with a low level of FVIII, achieving a haemostatic adequate FVIII:C increase after 6 to 12 hours. Replacement therapy in patients with VWD shall be individualised considering VWD type, history and risk of bleeding and risk of thrombosis, as well as indication and the individually variable VWF and FVIII increase. Deviations from the dosages and minimum trough levels mentioned in guidelines or recommendations can be considered in justified cases. The objective of this review is to provide recommendations for specific constellations of replacement therapy based on the VWD-specific guidelines available in Europe, the available evidence, own experiences and the consensus of the interdisciplinary German author group.

Direct oral anticoagulants--pharmacology, drug interactions, and side effects.

The direct thrombin inhibitor, dabigatran, as well as the direct factor Xa inhibtors rivaroxaban, apixaban, and edoxaban, display pharmacodynamic features quite similar to low-molecular-weight heparins, with a time to peak level of 1-4 hours after oral administration, and a half-life between 5 and 14 hours. All drugs display a linear relationship and a high degree of correlation between drug levels in plasma, and the anticoagulant effect. Major differences are the extent of renal elimination (with 80% or more for dabigatran, 66% for rivaroxaban [33% unchanged, active drug, and 33% inactive metabolites], 33% for edoxaban, and finally, 25% for apixaban), and bioavailability, which determines the amount of drug required for attaining the target plasma concentration of the drug. Due to the reliable pharmacokinetics and pharmacodynamics, no routine laboratory monitoring is necessary, although dedicated laboratory assays are available for emergencies and some other specific conditions.

The G534E-polymorphism of the gene encoding the factor VII-activating protease is a risk factor for venous thrombosis and recurrent events.

INTRODUCTION: A single nucleotide polymorphism of the factor VII activating protease (FSAP), FSAP Marburg I (rs7080536) has been identified as a risk factor for venous thrombosis, but its clinical role has so far been controversial in part due to small cohort sizes. The aim of the present case-control study was to elucidate the impact of the FSAP Marburg I polymorphism (FSAP-MI) on the development of venous thromboembolic disease (VTE) with other known sequence variations, including Factor V Leiden (rs6025) and Factor II G20210A (rs1799963). MATERIALS AND METHODS: The study included 891 patients (312 male and 579 female) with a history of deep venous thrombosis (DVT) and/or pulmonary embolism (PE) and 1283 healthy blood donors with no history of venous thromboembolic disease. RESULTS: We found that besides to the well-established aforementioned sequence variations of FV and Prothrombin, the FSAP Marburg I (FSAP-MI) polymorphism was significantly associated with the development of DVTs (1.65 (1.16-2.34) OR (95% CI)) and recurrent thromboembolic events (DVT and PE) (2.13 (1.35-3.36) OR (95% CI)). Comparing patients displaying one or more events FSAP-MI was still associated with the development of recurrent thromboembolic events (1.64 (1- 2.69) OR (95% CI)). CONCLUSIONS: We conclude that FSAP Marburg-I genotyping may be used to determine the risk for thromboembolic disorders in patients with suspected thrombophilia and known DVT or PE.

Platelet and monocyte activation in acute ischemic stroke--is there a correlation with stroke etiology?

An upregulation of platelet CD40 ligand (CD40L) and CD62P has been described in atherosclerotic cardiovascular diseases and among patients with acute cerebral ischemia. Correlation between platelet and monocyte activation and the etiology of ischemic stroke were examined in 41 patients with acute ischemic stroke. Compared to 10 controls, all patients with stroke showed a significantly elevated platelet expression of CD40L (P < .001) and had significantly higher amounts of platelet-monocyte aggregates (P = .002). Plasma levels of interleukin 7 were significantly lower in patients with stroke compared to controls (P = .006). Patients with small artery disease had a significantly higher platelet CD40L expression than patients with cardioembolic stroke (P = .029). Plasma levels of soluble CD40L were significantly higher in patients with large artery disease compared to patients with cardioembolic stroke (P = .047). In conclusion, patients with acute ischemic stroke show an upregulation of platelet CD40L and an activation of cellular coagulation with highest activation in the large artery disease subgroup.

The platelet-rich plasma clot: a standardized in-vitro clot formation protocol for investigations of sonothrombolysis under physiological flows.

No agreement exists about which protocol for in-vitro clot formation is suitable for sonothrombolysis investigations. Lysis rates vary considerably because of different clotting processes and cannot be compared. We aim to establish a new protocol for in-vitro coagulation to permit standardized sonothrombolysis investigations. The proposed procedure is based upon clots prepared from platelet-rich plasma (PRP). This clot material (group A) was compared with the two most commonly used procedures, namely, recalcification of citrate-anticoagulated whole venous blood (group B) and spontaneous clotting of nonanticoagulated venous blood (group C). Histological examinations were performed and clot stability was tested under physiological flow conditions in vitro for all groups (each n = 10). Lysis rates measured by mass loss were compared using buffered plasma and recombinant tissue plasminogen activator (60 kU/ml), or buffered plasma alone. PRP clots displayed a high degree of similarity to emboli specimens in histological examinations and remained stable under pulsatile flow conditions. B and C clots were mechanically unstable and did not resist physiological flow and pressure. Measuring the lysis rate by weighing seems to be inaccurate, with lowest variability in PRP clots. PRP clots appeared more resistant to lysis. PRP clots should be used for standardized sonothrombolysis investigations.

Endogenous plasma activated protein C levels and the effect of enoxaparin and drotrecogin alfa (activated) on markers of coagulation activation and fibrinolysis in pulmonary embolism.

INTRODUCTION: There are no published data on the status of endogenous activated protein C (APC) in pulmonary embolism (PE), and no data on the effect of drotrecogin alfa (activated) (DAA) given in addition to therapeutic dose enoxaparin. METHODS: In this double-blind clinical trial, 47 patients with computed tomography (CT)-confirmed acute submassive PE treated with 1 mg/kg body weight of enoxaparin twice daily were randomized to groups receiving a 12-hour intravenous infusion of 6, 12, 18, or 24 µg/kg/hour of DAA or a placebo. Blood samples were drawn before starting DAA infusion, after 4, 8 and 12 hours (at the end of the infusion period), and on treatment days 2, 3, 4, 5 and 6. RESULTS: Initial endogenous plasma activated protein C (APC) levels were 0.36 ± 0.48 ng/ml (<0.10 to 1.72 ng/ml) and remained in the same range in the placebo group. APC levels in patients treated with DAA were 13.67 ± 3.57 ng/ml, 32.71 ± 8.76 ng/ml, 36.13 ± 7.60 ng/ml, and 51.79 ± 15.84 ng/ml in patients treated with 6, 12, 18, and 24 µg/kg/hour DAA, respectively. In patients with a D-dimer level >4 mg/L indicating a high level of acute fibrin formation and dissolution, DAA infusion resulted in a more rapid drop in soluble fibrin, D-dimer, and fibrinogen/fibrin degradation products (FDP) levels, compared to enoxaparin alone. There was a parallel decline of soluble fibrin, D-dimer, FDP, and plasmin-plasmin inhibitor complex (PPIC) in response to treatment with enoxaparin ± DAA, with no evidence of a systemic profibrinolytic effect of the treatment. CONCLUSIONS: In patients with acute submassive PE endogenous APC levels are low. DAA infusion enhances the inhibition of fibrin formation. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00191724.

Sex-based differences in clinical and angiographic outcomes in patients with ST-elevation myocardial infarction treated with concomitant use of glycoprotein IIb/IIIa inhibitors.

BACKGROUND: The widespread use of primary coronary intervention (PCI) has significantly improved the prognosis of men presenting with acute coronary syndromes, but the cardiovascular event rate among women has either levelled off or increased. The purpose of the present prospective study was to compare the clinical outcome of women and men presenting with ST-elevation myocardial infarction (STEMI) undergoing primary PCI with concomitant usage of GP IIb/IIIa inhibitors. METHODS: Between January 2006 and December 2007, 297 consecutive patients presenting with STEMI were prospectively included in this single center investigation. Overall, 82 (27.6%) women and 215 (72.4%) men were treated by PCI with additional bare metal stent implantation and a GP IIb/IIIa inhibitor. RESULTS: Women were significantly older (65 ± 10 vs 60 ± 12 years, p = 0.04), presented with a smaller reference luminal diameter (2.83 ± 0.51 vs 2.94 ± 0.43, p = 0.03) and had a higher prevalence of hypertension (68% vs 53%, p = 0.025) and obesity (30% vs 18%, p = 0.03). The incidence of major adverse cardiac events (MACE, defined as death, re-myocardial infarction, target lesion revascularization and coronary artery bypass graft) during long-term follow-up was similar in women and men (20% vs 26%, p = 0.29). Age, C-reactive protein, platelet count and cardiogenic shock were identified as independent predictors for MACE, whereas gender was not predictive. CONCLUSIONS: In this study, female gender did not emerge as an independent predictor for MACE, but women presenting with STEMI had a higher cardiovascular risk profile; this emphasizes the need for a more extensive therapeutic strategy. Combination therapy with primary PCI and GP IIb/IIIa inhibitors might mitigate gender-related differences in clinical outcomes.

Thrombogenicity of sirolimus-eluting stents and bare metal stents: evaluation in the early phase after stent implantation.

BACKGROUND: Thrombogenicitiy of drug-eluting stents is a matter of controversial debate. The aim of this study was to evaluate the thrombogenicity of sirolimus-eluting stents (SES) compared to bare metal stents (BMS) in a standardised in vitro model. MATERIALS AND METHODS: Nine SES and nine BMS were implanted in tubing loops and nine loops without stent served as controls. Initially and after 90 minutes of blood circulation in a modified chandler loop model, thrombin-antithrombin III complex (TAT), PMN-elastase, factor XIIa, SC5b-9, sP-selectin and platelet count were measured. Expression of CD62P, CD45/41 and PAC-1 on platelets were determined by flow cytometry. RESULTS: After 90 minutes, platelet count decreased significantly in the loops with BMS and SES (p<0.05). Levels of TAT, PMN-elastase and SC5b-9 were significantly elevated after 90 minutes in all loops (p<0.05). sP-selectin significantly increased in the loops with BMS and SES after 90 minutes. No significant changes occurred in any flow cytometric data. Platelet count, sP-selectin, TAT, PMN-elastase, SC5b-9, CD62P, CD41/CD45 and PAC-1 showed no significant difference between BMS and SES. CONCLUSION: These data provide evidence that there is no difference in thrombogenicity of BMS and SES in the in vitro model.

Activation of platelets and cellular coagulation in cerebral small-vessel disease.

Platelets and monocytes play a pivotal role in the initiation and progression of large-vessel atherosclerosis. An up-regulation of various platelet and coagulation activation markers has been described in cardiovascular diseases and in patients with acute cerebral ischemia. In the present study the role of platelets and cellular coagulation activation in cerebral small-vessel disease (cSVD) was assessed. In 24 patients with cSVD but without established large-vessel disease, whole blood samples were obtained. Patients were divided into three subgroups (Fazekas 1, 2 and 3) according to extent of cSVD based on morphological magnetic resonance imaging criteria. Surface expression of CD40L and CD62P on platelets, tissue-factor exposition on monocytes and platelet-monocyte aggregates were measured with flow cytometry. Plasma levels of soluble CD40L, interleukin (IL)-6 and IL-7 were assessed by ELISA. Patients with cSVD show a significantly elevated expression of platelet CD40L (P < 0.001) and CD62P (P < 0.023), significantly elevated amounts of platelet-monocyte aggregates (P < 0.004), a significantly enhanced tissue-factor exposition on monocytes (P < 0.019) and significantly lower plasma levels of IL-7 compared to 10 healthy controls. However, this platelet and monocyte activation did not correlate with the severity of cSVD. Patients with cSVD show an up-regulation of the platelet CD40L and CD62P system and an activation of cellular coagulation which might contribute to the initiation and progression of cSVD.

Effect of atorvastatin on haemostasis, fibrinolysis and inflammation in normocholesterolaemic patients with coronary artery disease: a post hoc analysis of data from a prospective, randomized, double-blind study.

BACKGROUND: Treatment with HMG-CoA reductase inhibitors (statins) reduces the morbidity and mortality of coronary artery disease (CAD). In addition to their lipid-lowering actions, pleiotropic effects of statins have been demonstrated. OBJECTIVE: The aim of the present study was to assess if atorvastatin therapy has an impact on haemostasis, fibrinolysis and inflammation in normocholesterolaemic patients with CAD. METHODS: Fifty-four patients with CAD who had received atorvastatin treatment for at least 8 weeks (mean dosage 30 mg/day) and 54 patients with CAD who had not received atorvastatin therapy were selected from a larger prospective, randomized, double-blind study for inclusion in this post hoc analysis. Patients were matched by their total cholesterol levels. All patients were normocholesterolaemic. RESULTS: In the atorvastatin-treated patients significantly lower plasma levels of thrombin-antithrombin complexes (p < 0.05), plasminogen activator inhibitor-1 activity (PAI-1) [p < 0.05], soluble vascular cell adhesion molecule-1 (p < 0.05), soluble platelet selectin (p < 0.05) and high-sensitivity C-reactive protein (p < 0.05) were measured compared with patients not on atorvastatin therapy. Additionally, a strong trend towards lower soluble intercellular adhesion molecule-1 plasma levels was detected in patients treated with atorvastatin. No differences were found in tissue-type plasminogen activator antigen, plasmin-plasmin inhibitor complexes, fibrinogen, D-dimer and activated factor XII values. CONCLUSION: Atorvastatin appears to have an effect on coagulation activation, fibrinolysis and inflammation in patients with CAD. Reduction in PAI-1 and reduced thrombin formation may have an impact on cardiovascular morbidity and mortality in patients with CAD.

Multicentre analytical evaluation of a new point-of-care system for the determination of cardiac and thromboembolic markers.

BACKGROUND: The cobas h 232 point-of-care analyzer by Roche is the instrument successor of the Cardiac reader allowing the quantitative determination of troponin T, creatine kinase MB, myoglobin, NT-proBNP and D-dimer. METHODS: In this study 1329 patients with acute coronary syndromes, heart failure, thromboembolic or other diseases and 945 healthy donors were assessed. Comparisons versus central laboratory methods were carried out with 2379 samples from these individuals; out of these, 1591 samples gave quantitative results within the measuring range and were included in the evaluation. RESULTS: The point-of-care assays for creatine kinase MB, myoglobin, NT-proBNP and D-dimer were within a relative bias range of -5.9 to +6.9% compared to the laboratory assay. The troponin T assay showed a bias of -11.0% and after change of the calibration procedure of +1.9%. None of the five point-of-care assays had a relative difference between the new system and the precursor device that was higher than +5.0%. Within-series coefficients of variation of patient samples were found in a range from 4.8 to 14.8%. No significant interference was observed with lipemic, hemolytic and icteric blood or at different hematocrit values. CONCLUSIONS: Due to its good analytical agreement with the laboratory methods and with its precursor device, the cobas h 232 system can be reliably used to support on-site decision making for cardiovascular patients in acute and non-acute settings.

Factor V Leiden mutation enhances fibrin formation and dissolution in vivo in a human endotoxemia model.

Disseminated intravascular coagulation in sepsis is associated with microvascular thrombosis and organ dysfunction. It was expected that prothrombotic disposition such as factor V Leiden (FVL) mutation would worsen clinical outcome. Astonishingly, clinical trial and animal experimental data indicate that FVL can be associated with improved survival. This study investigated the effect of FVL on the response to endotoxin of the coagulation and fibrinolytic system in humans. Fourteen healthy male subjects without FVL and 15 healthy males with heterozygous FVL received an intravenous bolus dose of endotoxin, 2 ng/kg of body weight. Blood samples were drawn before and 1, 2, 4, 6, and 24 hours after administration of the endotoxin. Injection of endotoxin led to a more pronounced increase in soluble fibrin in patients with FVL than in controls. Patients with FVL displayed a more sustained increase in plasmin-plasmin inhibitor complex after 4, 6, and 24 hours. Patients with FVL mutation also displayed higher levels of D-dimer and fibrinogen-fibrin degradation products in plasma after 24 hours. Patients with FVL generate higher levels of soluble fibrin, which may serve as cofactor in tissue plasminogen activator-induced plasminogen activation, leading to a more sustained activation of fibrinolysis with production of more fibrinogen- and fibrin-degradation products.

Effect of atorvastatin on cellular adhesion molecules on leukocytes in patients with normocholesterolemic coronary artery disease.

BACKGROUND: Expression of cellular adhesion molecules on leukocytes plays a key role in coronary artery disease (CAD). The aim of the present study was to assess whether atorvastatin therapy has an impact on the expression of cellular adhesion molecules on leukocytes in patients with normocholesterolemic CAD. PATIENTS AND METHODS: In 54 patients with CAD and atorvastatin treatment and 54 CAD patients without atorvastatin therapy, expression of CD40L, CD11a, CD11b, CD54, CD62L and CD41 on leukocytes was measured using flow cytometry. All patients were normocholesterolemic. RESULTS: Atorvastatin treatment led to a significantly lower expression of CD40L, CD11b and CD54 on monocytes (p<0.05) and neutrophils (p<0.05). Expression of CD11a was significantly lower on monocytes (p<0.05) in atorvastatin-treated patients. CONCLUSION: The present results indicate that atorvastatin apparently improves chronic inflammation and may have a beneficial effect on hemostasis by reducing the expression of cellular adhesion molecules on leukocytes.

Sudden death: do cytokines and prothrombotic peptides contribute to the occurrence of ventricular fibrillation during acute myocardial infarction?

AIMS: Sudden cardiac death (SCD) is frequently caused by ventricular fibrillation (VF) occurring in the course of acute myocardial infarction (AMI). It has not been investigated yet, to what extent markers of coagulation activation and inflammation differ between patients with and without VF in the acute phase of AMI.