Mj Adams

The effects of alcohol on coagulation and fibrinolytic factors: a controlled trial.

Light-to-moderate alcohol intake is associated with a reduced incidence of ischaemic cardiovascular events, whilst heavy alcohol intake can predispose individuals to stroke. Alcohol-induced changes in coagulation and fibrinolysis may be relevant and are the subject of this controlled trial of varying alcohol intake in 55 predominantly beer-drinking men. Following 4 weeks stabilization maintaining usual drinking habits, participants were randomized to either continue usual alcohol intake or to restrict alcohol by changing to low alcohol beer for 4 weeks. In a final 4 week period, they crossed over to low or usual alcohol intake, respectively. Comparing combined low and usual alcohol periods, an increase in mean weekly alcohol intake from 92 to 410 ml (mean daily intake from 13 to 58 ml) was associated with a decrease in plasma fibrinogen (by 11%, P < 0.001) and platelet count (3%, P < 0.05), but increases in factor VII (7%, P = 0.001), tissue plasminogen activator (tPA; 16%, P = 0.01) and plasminogen activator inhibitor-1 (PAI-1; 21%, P < 0.001). The ratio, tPA/PAI-1, fell from 0.50 to 0.44 (P = 0.02) confirming the relatively greater increase in PAI-1 with alcohol consumption. Two lipid-associated natural anticoagulants, tissue factor pathway inhibitor and beta 2-glycoprotein-I, did not change. The substantial reduction in plasma fibrinogen with alcohol intake may well contribute to the apparent protection alcohol confers against ischaemic coronary and cerebral events. The increase in factor VII and relatively greater increase in PAI-1 than tPA with alcohol intake may attenuate this benefit and indeed may sufficiently predispose individuals to thrombosis to contribute to the increased incidence of ischaemic stroke seen in heavier drinkers. The balance of anticoagulant and procoagulant and fibrinolytic effects in any individual may vary depending on quantity and type of alcoholic beverage ingested, as well as on genetic and other variables, all of which merit further study.

Thrombosis in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a potentially fatal multiorgan inflammatory disease that primarily affects females. Due to the heterogeneity of clinical manifestations and lack of laboratory tests that are both specific and sensitive for the disease, diagnosis of SLE can often be difficult. Although the precise etiology remains to be fully elucidated, it is probable that various environmental, genetic, and hormonal factors contribute to the development of the disease. Patients with SLE have an increased risk for premature thrombosis and/or atherosclerosis, with up to half experiencing a thrombotic event. Furthermore, antiphospholipid antibodies probably play a key role in the development of thrombosis by affecting various hemostatic protein interactions with phospholipids and cell surfaces as well as platelet function. Despite recent advances in knowledge related to the factors that contribute to the pathophysiology of SLE, numerous challenges related to earlier diagnosis as well as the prediction and prevention of thrombotic events remain to be fully addressed.

Hypercoagulability in chronic kidney disease is associated with coagulation activation but not endothelial function

INTRODUCTION Patients with chronic kidney disease exhibit features of a hypercoagulable state and have endothelial dysfunction, which may contribute to their increased cardiovascular risk. We examined the relationship between coagulation activation and vascular function in patients with chronic kidney disease. MATERIALS AND METHODS We measured parameters of the tissue factor pathway of blood coagulation (tissue factor, factor VIIc and factor X); natural inhibitors (tissue factor pathway inhibitor, protein C, free and total protein S, antithrombin III) and markers of coagulation activation (thrombin-antithrombin complexes, prothrombin fragment 1+2) in 66 stage 4&5 chronic kidney disease patients and 36 healthy controls. Their relationship with markers of vascular function (flow mediated dilatation, soluble E-selectin and thrombomodulin) and a mediator of inflammation (interleukin-6) was determined. RESULTS Up-regulation of the tissue factor pathway (increased tissue factor and factor VIIc), increased prothrombin fragment 1+2 and significant reductions in antithrombin III and the ratio of free protein S: total protein S were found in patients compared to healthy controls. Increased tissue factor antigen was significantly and independently correlated with creatinine and interleukin-6 (P<0.001). Factor X and antithrombin III were both reduced in chronic kidney disease and correlated (r=0.58; P<0.001). Changes in coagulation and anti-coagulation were independent of all measures of endothelial function. CONCLUSIONS Significant activation of the TF pathway of coagulation and depletion or reduction of some natural anticoagulants in chronic kidney disease was correlated with the degree of renal dysfunction, but not correlated with the abnormalities of vascular function. These data are consistent with a hypercoagulable state in chronic kidney disease that may be independent of endothelial based regulation but associated with an inflammatory state.

Anti-tissue factor pathway inhibitor activity in patients with primary antiphospholipid syndrome

The association between antiphospholipid antibodies and an increased risk of thrombosis in antiphospholipid syndrome (aPS) patients is probably caused by numerous mechanisms, including the effects of antibodies to phospholipid‐binding proteins such as β2‐glycoprotein I and prothrombin. In this study, we investigated the inhibition of tissue factor pathway inhibitor (TFPI) in 33 patients with primary antiphospholipid syndrome (PAPS). TFPI was measured in PAPS patients using an amidolytic assay, dependent on the generation of activated factor X (Fxa), and this was compared with 55 healthy subjects. Functional levels of TFPI (mean ± SD) were significantly lower in PAPS patients (0·89 ± 0·37 U/ml) than the control group (1·05 ± 0·15 U/ml) (P = 0·02). The difference was caused by a subset of five patients who had TFPI levels below the lower 99% confidence interval of the normal reference range, representing increased FXa generation in the assay system. IgG fractions were isolated from these five patients and five control subjects, then incorporated into normal plasma to measure FXa generation in the TFPI assay system. FXa generation was increased when polyclonal rabbit anti‐human TFPI IgG (P < 0·0001) or PAPS IgG (P = 0·0001) were added to normal plasma, demonstrating inhibition of TFPI. The apparent anti‐TFPI activity demonstrated in the five subjects with PAPS in this study may represent a significant new mechanism for thrombosis in patients with aPS, as it implies that increased tissue factor FVIIa‐mediated thrombin generation might occur.

Effect of capsaicin and dihydrocapsaicin on in vitro blood coagulation and platelet aggregation

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in developed countries. The growing clinical and financial burden of CVD is, at least in part, being driven by aging populations, and thus represents a significant challenge to health care systemswith regard to diagnosis, prevention and treatment. Novel cost-effective approaches that slow or prevent the onset and/or reduce the incidence of CVD warrant investigation. Capsaicinoids, including capsaicin and dihydrocapsaicin, are the major pungent constituents of ‘hot chilli peppers’ of the Capsicum genus. These spice principles have been documented to increase carbohydrate metabolism [1,2], energy expenditure [3] and lipid metabolism [4–7], in rats and/or humans, and have been successfully used in the treatment of painful conditions such as rheumatoid arthritis, osteoarthritis and peripheral neuropathies [8]. Furthermore, we have recently shown that regular intake of chilli delays copperinduced oxidation of serum lipids in vitro [4] and lowers and improves post-prandial insulin and glucose profiles [9], actions that may help in reducing CVD risk. Their effect(s) however, on haemostasis have not been extensively investigated, and their potential “anti-haemostatic” properties may also reduce CVD risk. Limited reports have demonstrated that capsaicin inhibits platelet aggregation, although these studies investigated platelets from non-human species [10,11]. Furthermore, it was reported that capsaicin had no effect on blood coagulation [12], although the potential effects on individual clotting factors were not determined. The aim of this study was to therefore investigate and compare the effects of capsaicin and dihydrocapsaicin on markers of in vitro haemostasis, viz, blood coagulation and platelet aggregation. This studywas approved by the Human Research Ethics Committee (Tasmanian) Network (Ref No: H0009477). Informed consent was obtained from each subject in accordance with the Declaration of Helsinki of the World Medical Association. For blood coagulation experiments, normal reference plasma (NRP; Helena Laboratories, Beaumont, USA) was spiked with increasing concentrations (3.125100 μmol/L) of capsaicin (Sigma Chemical Company, St Louis, USA) or dihydrocapsaicin (Tocris Bioscience, Ellisville, USA). Each capsaicinoid was initially dissolved in 100% ethanol to make a stock solution of 1 mol/L and then diluted for further experiments in normal buffered saline, pH 7.1. After incubation for 30 minutes at 37 °C, prothrombin time (PT), activated partial thromboplastin time (aPTT) and the activities of clotting factors II, V, VII, VIII:C, IX, X, XI and XII were determined using a STarT4 coagulation analyzer (Diagnostica Stago, Parsippany, USA). For platelet aggregation experiments, venous whole blood was collected using 21 gauge syringes from six healthy subjects

Metabolomics in hypertension.

Hypertension is the most prevalent chronic medical condition and a major risk factor for cardiovascular morbidity and mortality. In the majority of hypertensive cases, the underlying cause of hypertension cannot be easily identified because of the heterogeneous, polygenic and multi-factorial nature of hypertension. Metabolomics is a relatively new field of research that has been used to evaluate metabolic perturbations associated with disease, identify disease biomarkers and to both assess and predict drug safety and efficacy. Metabolomics has been increasingly used to characterize risk factors for cardiovascular disease, including hypertension, and it appears to have significant potential for uncovering mechanisms of this complex disease. This review details the analytical techniques, pre-analytical steps and study designs used in metabolomics studies, as well as the emerging role for metabolomics in gaining mechanistic insights into the development of hypertension. Suggestions as to the future direction for metabolomics research in the field of hypertension are also proposed.

Polymorphisms of the tissue factor pathway inhibitor gene are associated with venous thromboembolism in the antiphospholipid syndrome and carriers of factor V Leiden.

Polymorphisms within the tissue factor pathway inhibitor (TFPI) gene may determine TFPI expression and increase the risk of venous thromboembolism (VTE) in predisposed individuals. We tested this hypothesis by comparing TFPI activity and the frequency of common TFPI polymorphisms, –33T->C, –399C->T and –287T->C, in patients with antiphospholipid syndrome (APS) (n = 24) or factor V Leiden (n = 44) who had a history of VTE (n = 26), compared with those without VTE (n = 42) and also with normal control individuals (n = 56). TFPI activity was measured using a modified amidolytic assay and genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism. We found that only APS patients with a history of venous thrombosis had TFPI activity levels significantly different from control individuals (1.77 ± 0.60 vs 0.77 ± 0.19 U/ml; P = 0.0001), and this was associated with inheritance of the TFPI –33C allele (1.70 ± 0.72 U/ml for TC/CC genotypes vs 0.97 ± 0.56 U/ml for TT; P = 0.01). Multivariate analysis of APS and factor V Leiden patients revealed that the greatest independent contributor to VTE was TFPI activity (adjusted odds ratio = 16.84; 95% confidence interval = 2.47–114.36, P = 0.004), while inheritance of either the TFPI –33C or –399T alleles each increased the odds of VTE by nearly 13 times (95% confidence interval = 2.39–69.91, P = 0.003; and 95% confidence interval = 2.25–71.23, P = 0.004, respectively). These results indicate that the TFPI –33T->C and –399C->T polymorphisms are significantly associated with venous thrombosis in the presence of other risk factors, especially APS, and may be clinically relevant in patients who are prone to hypercoagulability.

D-Dimer levels at different stages of pregnancy in Australian women: a single centre study using two different immunoturbidimetric assays.

BACKGROUND To date there is minimal data available on D-Dimer levels at different stages of pregnancy. PATIENTS AND METHODS We prospectively measured D-Dimer levels in 632 consecutive pregnant women from March 2007 to January 2009. The median age of the participants was 31 years (range; 18-42) with a median weight of 78 kilograms (range; 46-137). All subjects were investigated during each trimester with two different immunoturbidimetric assays; D-Dimer PLUS and INNOVANCE D-Dimer. D-Dimer levels were determined using a Sysmex® CA 1500 analyser. RESULTS Our data demonstrate that D-Dimer levels in pregnancy show different patterns of rise within the first trimester, depending on the assay used; D-Dimer PLUS=0.88 (SD: mean ratio), INNOVANCE D-Dimer=0.72 (SD: mean ratio). Furthermore, the rise in mean results was greater for the INNOVANCE D-Dimer assay compared to the D-Dimer PLUS assay as shown by the ratio of third to first trimester results of 3.68 and 1.96 respectively. Both D-Dimer assays demonstrated moderate levels of intra-subject variability, with overall mean CVs of 16.5% (D-Dimer PLUS) and 16.9% (INNOVANCE D-Dimer). Furthermore, we studied the association between D-Dimer levels and occurrence of diseases of pregnancy. For both assays, there was no consistently interpretable evidence of an association between raised mean D-Dimer levels or rising D-Dimer levels and any of the diseases or conditions associated with pregnancy. CONCLUSION Our data suggest that the INNOVANCE D-Dimer assay increases significantly with the advancement of pregnancy, and is more sensitive than D-Dimer PLUS assay in the pregnant population.

Acute effect of a high-carbohydrate low-fat meal on platelet aggregation.

Conflicting information is available regarding patient preparation with respect to the fasting and feeding states prior to blood collection in order to conduct platelet aggregation tests. Some literature suggests avoidance of only high-fat foods and allowance of non-fat foods and clear liquids; others suggest a fast of 8–10 hours. We conducted a study in 16 healthy subjects aged 44.0 ± 12.7 (mean ± SD) years to investigate and compare the effects of fasting and a high-carbohydrate low-fat meal on measures of platelet aggregation. Blood samples collected after an overnight fast of 10–12 hours and those collected at 40 and 120 minute postprandially (post-high-carbohydrate low-fat meal; 1900 kJ energy; 69, 16 and 15% of energy from carbohydrate, protein and fat, respectively), were tested for platelet aggregation in response to adenosine diphosphate. There was a significant reduction in maximum aggregation and area under the aggregation curve from fasting to 120 minute post meal (overall p < 0.001). Serum triglyceride concentrations did not change significantly from fasting to postprandial state (p = 0.53). Although there was a significant association between serum insulin, plasma glucose and measures of platelet aggregation, correcting for the effects of these metabolic parameters did not alter the results, providing evidence that other, currently unknown, factors associated with food consumption affect postprandial platelet aggregation. We propose that protocols for control of pre-analytical variables in platelet aggregation studies should make a fasting sample mandatory rather than “preferable” unless the objective of the study is to measure acute effects in response to a medication or food.

A rapid flow cytometric technique for the detection of platelet-monocyte complexes, activated platelets and platelet-derived microparticles

Platelet activation occurs in a variety of clinical situations in which it directly contributes to the pathology. This study reports a simple flow cytometric assay for platelet activation which measures platelet‐derived microparticles, activated platelets and platelet–monocyte complexes. Pre‐ and post analytical conditions were investigated and optimized and a normal range established on 20 healthy controls. Twenty patients pre‐ and post percutaneous coronary intervention (PCI) were tested with the technique. Soluble activation markers sCD40 ligand and sP‐selectin and plasma phospholipid levels were measured in both groups. There was a significant increase in activated platelets and platelet–monocyte complexes between normal and pre‐PCI (P = 0.005 and 0.0275, respectively) suggesting an activated state. There was a significant fall in activated platelets post‐PCI (P = 0.0027) which was mirrored by a fall in soluble CD40 ligand, soluble P‐selectin and plasma phospholipid levels (P = 0.0066, <0.0001 and 0.0032, respectively) consistent with antiplatelet therapy administered during the process. This is a reliable and rapid method for the assessment of ex vivo platelet activation which may be an aid in diagnosis and help guide therapy for patients with thrombotic disease.