Thrombosis Research - Articles in Press

The 8-oxoguanine glycosylase I (hOGG1) Ser326Cys variant affects the susceptibility to multi-vessel disease in Taiwan coronary artery disease patients - Corrected Proof

2010-07-28

Abstract: 8-hydroxydeoxyguanosine, the key lesion of oxidative DNA damage, contributes to the development of coronary artery disease (CAD). In humans, 8-hydroxydeoxyguanosine is repaired by the enzyme 8-oxoguanine glycosylase I (hOGG1). We investigated the association between the hOGG1 Ser326Cys polymorphism and the presence and the severity of CAD in a Taiwan population. Genotypes of the hOGG1 Ser326Cys polymorphism were determined from 1397 participants enrolled in this study (378 CAD patients and 1019 controls). CAD severity was indicated both by number of vessels affected (single-vessel disease, SVD vs. multi-vessel disease, MVD), and by individual diffuse score. Real-time polymerase chain reaction was used to determine genotype, using allele-specific TaqMan probes. We found that presence of the hOGG1 Ser326Cys polymorphism was associated with a significantly increased risk of CAD and multi-vessel disease when assuming a dominant model of inheritance (OR: 1.52 [95%:1.082~2.133], p=0.015; OR: 2.26 [95%:1.232~4.156], p=0.007). This result was confirmed by multivariate analysis, after adjustment for age, gender, body-mass index, diabetes hypertension, hypercholesterolemia and smoking (OR: 1.78 [95%:1.127~2.806], p<0.005; OR: 2.44 [95%:1.276~4.651], p<0.001). In the present study, hOGG1 Ser326Cys polymorphism is a novel genetic marker to be independently associated with the development and severity of CAD in Taiwanese population.

The effect of aspirin on endothelial progenitor cell biology: Preliminary investigation of novel properties - Corrected Proof

2010-07-26

Abstract: Atherosclerosis develops in an environment of endothelial injury and inflammation. Circulating endothelial progenitor cells (EPCs) are required for vascular repair and restoration of normal endothelial function. We tested the hypothesis that the nonselective cyclooxygenase (COX) inhibitor aspirin (ASA) exerts an effect on circulating EPCs.Methods: As part of a larger study evaluating the effect of aspirin dose in primary and secondary prevention, subjects (n=32) were assigned randomly to either 81mg or 325mg aspirin daily for two months, and circulating mononuclear cells were enumerated at the beginning of the study and after 2months using fluorescent antibodies against CD34 and CD133 as well as based on aldehyde dehydrogenase (ALDH) activity. Brachial artery endothelial function via flow-mediated dilation (BAFMD) and light transmittance platelet aggregometry in response to physiologic agonists was also determined.Results: Subjects taking aspirin at the time of study entry had a lower numbers of CD133+/34+ cells compared to those not previously exposed (0.01% vs. 0.05% of MNCs, P<0.03). After 2months, subjects randomized to 81 vs. 325mg of ASA had no significant differences in the median numbers of EPCs, although mean numbers trended lower in the high dose group. Patients on chronic ASA therapy continued to have lower numbers of EPCs. Similar effects were observed in CD34 and CD 133 single-positive cells, as well as ALDHbr cells. BAFMD did not differ nor change significantly over time between aspirin dose groups. All patients had decreased ex vivo platelet aggregation in response to arachidonic acid and ADP stimulation.Conclusions: Our preliminary studies suggest that aspirin exerts a time-dependent effect on circulating EPCs. Short-term exposure to differing doses of ASA had indeterminate effects on EPCs levels, suggesting that time of ASA exposure may play a more important role than dose. Determining the responsible mechanism(s) and the overall clinical relevance of these findings will require further investigation.

Monkey business for hemophilia management - Corrected Proof

Gonzalo Hortelano — 2010-07-26

A number of factors make hemophilia an excellent model for gene therapy . It is no surprise then, that virtually every gene therapy strategy conceived to date has been applied to hemophilia, yielding in the process a particularly rich and useful array of research data. Key to the development of successful alternative therapies is the availability of suitable animal models of the human disease. There are colonies of naturally occurring hemophilic dogs and genetically generated KO mice that have proven invaluable in assessing novel therapeutic strategies. Despite their interest and usefulness, current animal models for hemophilia are not without limitations. These shortcomings became particularly evident following preclinical studies in which, following direct administration of adeno-associated virus (AAV) in mice and dogs, circulating FIX levels achieved were found somewhat different in both species. In an illustrative comparative study of this discrepancy, direct injection of AAV2-hFIX in hemophilic mice led to clinically relevant levels of circulating FIX (up to10%) , while injection of the same AAV2-FIX vector in hemophilic dogs did not reached 1% of normal FIX levels . These results made the outcome of AAV2-FIX administration in humans uncertain. In 2002, human trials of hemophilia B based on direct injection of AAV2 were able to transiently elevate FIX activity in selected severe hemophiliac patients . Unfortunately, elevated liver enzymes were also observed in two patients of the highest dose group, whom also had detectable circulating FIX, suggesting some there was some toxicity associated with the administration of AAV2 . Interestingly, no liver complications were found in treated mice or dogs, highlighting the difficulty of predicting the outcome of human trials based on the currently available animal models. Eventually, an immune response directed against a specific portion of the capsid of AAV2 was identified and characterized .

In vitro comparison of dabigatran, unfractionated heparin, and low-molecular-weight heparin in preventing thrombus formation on mechanical heart valves - Corrected Proof

2010-07-26

Abstract: Introduction: Lifelong oral anticoagulation (OAC) therapy is required for the prevention of thromboembolic events after implantation of an artificial heart valve. Thromboembolism and anticoagulant-related bleedings account for ≈75% of all complications experienced by heart valve recipients (2-9% of patients per year). The present study investigated the efficacy of dabigatran, a new direct thrombin inhibitor for oral use, as compared to unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) in preventing thrombus formation on mechanical heart valves in vitro.Material and Methods: Blood (230ml) from healthy young male volunteers was anticoagulated either by dabigatran (1μmol/l), UFH (150IU), or LMWH (100IU). Mechanical heart valve prostheses were placed in an in vitro thrombosis tester and exposed to the anticoagulated blood samples under continuous circulation at a rate of 75 beats per minute.Results: In whole blood with no anticoagulant, the apparatus completely clotted in 15-20minutes. When blood was treated with dabigatran, the mean thrombus weight was 164±55mg, in the UFH group 159±69mg, and in the LMWH group 182±82mg (p-value: 0.704). Electron microscopy showed no significant difference in thrombus formation in any group.Conclusisons: Dabigatran was as effective as UFH and LMWH in preventing thrombus formation on mechanical heart valves in our in vitro investigation. Thus, we hypothesize that dabigatran etexilate might potentially be a useful and competitive orally administered alternative to UFH and LMWH for recipients of alloplastic heart valve prostheses.

Successful treatment of thrombotic thrombocytopenic purpura associated with systemic lupus erythematosus with recombinant human soluble thrombomodulin - Corrected Proof

Mizu Sakai, Takayuki Ikezoe, Kentaro Bandobashi, Akihito Yokoyama — 2010-07-26

Most coagulation abnormalities found in individuals with systemic lupus erythematosus (SLE) are related to the production of anti-phospholipid antibody, which causes thrombotic or thromboembolic events. On the other hand, thrombotic thrombocytopenic purpura (TTP), characterized by microangiopathic hemolytic anemia, thrombocytopenia, renal dysfunction, fever, and neurologic symptoms, is an extremely rare event during the clinical course of SLE. Only four cases of clinical and histopathological TTP were identified among the 257 patients with SLE who underwent renal biopsy . Early recognition and treatment interventions such as plasma exchange are required for this life-threatening disease.

Increased circulating procoagulant activity and thrombin generation in patients with myeloproliferative neoplasms - Corrected Proof

2010-07-26

Abstract: Microparticles (MPs) are membrane fragments ranging in size from 0.1 to 1μm, and are considered as biomarkers reflecting prothrombotic state in many clinical diseases. The clinical course of myeloproliferative neoplasms (MPN) being frequently complicated by thrombotic events, we determined the MPs activity, i.e. circulating procoagulant activity (CPA), in polycythemia vera (PV) and essential thrombocythemia (ET) patients. To evaluate the influence of MPs on the coagulation, a thrombin generation test was realized in the absence and presence of thrombomodulin (TM). Compared with controls, patients had a higher CPA (24.0±9.0 vs 10.6±4.4nM, p<0.001), which was associated with a lower inhibition of the thrombin generation in the presence of TM (20.1±9.5% vs 28.4±11.8%, p<0.001), compatible with a low sensitivity to TM. This sensitivity was influenced by the JAK2V617F mutational status, homozygous patients presenting the lowest inhibition rate of the thrombin generation. Filtration through a 0.22μm membrane increased the sensitivity to TM in plasma, suggesting an influence of MPs in the “TM-resistance” observed in patients. Moreover, MPN patients receiving antiplatelet and/or cytoreductive therapies, our study suggests that CPA might be influenced by cytoreductive therapy. In conclusion, our data evidence in MPN patients the occurrence of an acquired “TM-resistance” partly determined by circulating microparticles. This TM-resistance might contribute to the hypercoagulable state observed in MPN patients, but the predictive value of the “TM-resistance” for thrombosis had not been evaluated.

Bone morphogenetic protein -7 increases thrombogenicity of lipid-rich atherosclerotic plaques via activation of tissue factor - Corrected Proof

2010-07-26

Abstract: Thrombogenicity of atherosclerotic plaques largely depends on plaque morphology. Tissue factor (TF) expression is higher in lipid-rich than in calcified lesions. Although bone morphogenetic protein (BMP) -7 is a known inhibitor of vascular calcification, the role of BMP-7 in the development of plaque thrombogenicity is uncertain. We hypothesized that increased thrombogenic potential of lipid-rich plaques is attributed to activation of TF by BMP-7. We measured levels of BMP-7 and TF proteins in lipid-rich and calcified carotid plaques, and tested the effects of BMP-7 on TF expression in human monocytes in vitro. Quantitative immunohistochemical analysis of endarterectomy specimens for TF and BMP-7 revealed that lipid-rich plaques contained more TF antigen than calcified ones (158.6±25.3 vs 37.4±8.8 AU, p<0.008). Lipid-rich plaques also expressed higher levels of BMP-7 (60.7±5.2 AU) than calcified lesions (31.8±8.6 AU, p<0.021). In vitro treatment of whole blood with BMP-7 markedly increased the population of TF-positive monocytes from 1.5±0.6 % to 31.0±7.6 % (p<0.001). Stimulation of blood with BMP-7 was accompanied by elevated surface presentation of TF antigen in monocytes as TF-dependent fluorescence intensity increased from 5.0±2.6 AU in unstimulated conditions to 15.8±1.9 AU after incubation with BMP-7 (p<0.002). Our data suggest that BMP-7 contributes to increased thrombogenicity of lipid-rich plaques via enhancement of TF expression.

Venous thromboembolism in acutely ill hospitalized medical patients - Corrected Proof

2010-07-26

Abstract: Background: Acute and chronic illness, immobility, and procedural and pharmacologic interventions may predispose patients in the Internal Medicine Wards to venous thromboembolic disease (VTE). The purpose of this study was to determine the incidence of VTE in these patients.Materials and Methods: A retrospective chart review of cohort of consecutive patients admitted to Internal Medicine wards in Spain between January 1st 2005 and December 31st 2007 was performed. For each patient, demographic data, risk factors for VTE and the diagnosis of VTE during hospitalization was recorded.Results: We analyzed 1,567,659 patients, excluding 28,226 patients who had DVT or PE before admission, and 196,555 who were discharged in the first 48 hours. We identify 12,458 new diagnosed VTE events among 1,344,959 patients (incidence 0.93%) hospitalized more than two days. Hospitalized-acquired VTE risk factors were feminine gender (odds ratio [OR] 1.31; CI95% 1.26-1.35), age >70 (OR 1.08 CI95% 1.04-1.13), acute infectious disease (OR 1.27 CI95% 1.17-1.38), acute respiratory disease (OR 1.23 CI95% 1.17-1.28), dementia (OR 1.22 CI95% 1.14-1.31), neoplasic disease (OR 2.29, CI95% 2.19-2.49), and hemiplegia (OR 1.49, CI95% 1.31-1.69).Conclusions: The number of patients with VTE in an Internal Medicine ward is higher than expected. Several independent risk factors for VTE were identified. Based on the large number of patients who developed a VTE during hospitalization, our data add strength to the argument that VTE prevention should be high on the list of priorities when health care policies are being formed.

Validation of a rapid test (VWF-LIA) for the quantitative determination of von Willebrand factor antigen in type 1 von Willebrand disease diagnosis within the European multicenter study MCMDM-1VWD - Corrected Proof

2010-07-23

Abstract: Background: Accurate measurement of von Willebrand factor (VWF) is a critical requirement for the diagnosis of von Willebrand disease (VWD).Aim of the study: To evaluate the diagnostic efficiency of a rapid quantitative test for the measurement of VWF antigen (VWF:Ag) in type 1 VWD.Patients and methods: VWF:Ag was measured with an ELISA in a robotic instrument, as a reference method, and with a fully automated latex-immunoassay (LIA) on an ACL 9000 analyser in 1,716 subjects enrolled within the Molecular and Clinical Markers for Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD) Study. Among these subjects, 1,049 were healthy controls, 281 healthy family members and 386 affected members from 127 European families with type 1 VWD.Results: The assay linearity range was 10-125IU/dL for LIA (R2=0.99) and 5-133IU/dL for ELISA (R2=0.99). The inter-assay CV for low VWF levels (∼30IU/dL) was 2% for the LIA test and 8.7 % for ELISA. The sensitivity for detection of type 1 VWD affected members was 86% and the specificity 91% for LIA, 87% and 90% for ELISA. A receiver-operator (ROC) analysis disclosed only a marginal difference between the two tests, LIA having a slightly greater area under the curve (0.94 vs. 0.93, p=0.03).Conclusion: VWF:Ag LIA compared well to standard ELISA in this large population of patients and controls, showing better CV. However the lower detection limit for the VWF:Ag LIA compared to the VWF:Ag ELISA means that the LIA assay is less good at discriminating between type 3 VWD and moderate type 1 VWD.

Interferon gamma in the etiology of atherosclerosis and periodontitis - Corrected Proof

Iwona Niedzielska, Szymon Cierpka — 2010-07-23

Abstract: Clinical observations and a few research reports seem to suggest that intraoral infection as well as periodontal teeth could potentially lead to systemic infections including atherosclerosis. The aim of our investigations was to determine whether periodontal disease might aggravate atherosclerosis and whether interferon-gamma (IFNG), widely recognized as a potent multifunctional cytokine, might serve as a marker of the process. This is the first research based on tissue material such as atheromata and periodontal pocket granulation tissue.The study population consisted of 15 patients with periodontitis and atherosclerosis. Control group comprised 15 non-atherosclerotic patients with periodontitis. IFNG, IFNGR1 and IFNGR2 expression was analysed using qRT-PCR profiling in the inflammatory granulation tissue and atheroma. Granulation tissue samples obtained from non-atherosclerotic group showed a significant increase in IFNG and a decrease of IFNGR1, IFNGR2 expression whereas granulation tissue and atheromata of patients with systemic disease demonstrated lower IFNG and higher IFNGR1 and IFNGR2 expression.

Atorvastatin has antithrombotic effects in patients with type 1 diabetes and dyslipidemia - Corrected Proof

2010-07-20

Abstract: Introduction: Diabetes is a prothrombotic state involving a more thrombogenic fibrin network. In the present study we investigated the effects of lipid-lowering therapy with atorvastatin on fibrin network structure and platelet-derived microparticles in patients with type 1 diabetes and dyslipidemia.Materials and Methods: Twenty patients were treated with atorvastatin (80mg daily) or placebo during 2months in a randomized, double-blind, cross-over study. Fibrin network permeability, expression of glycoprotein IIIa, P-selectin and tissue factor on platelet-derived microparticles, plasma endogenous thrombin potential, plasminogen activator inhibitor-1 and tissue plasminogen activator antigen levels were assessed. Additionally, levels of plasma fibrinogen, high-sensitivity C-reactive protein and glycated haemoglobin were measured.Results: During treatment with atorvastatin, fibrin network permeability increased (p=0.01), while endogenous thrombin potential and expression of glycoprotein IIIa, P-selectin and tissue factor decreased (p<0.01). In vitro experiments indicated that platelet-derived microparticles influence the fibrin network formation as fibrin network permeability decreased significantly when platelet-derived microparticles were added to normal plasma. Baseline levels of plasminogen activator inhibitor-1 and tissue plasminogen activator antigen as well as plasma fibrinogen and high-sensitivity C-reactive protein were within reference values and not significantly changed during atorvastatin treatment, while glycated haemoglobin increased 0.3% (p<0.001).Conclusions: Novel treatment effects were found in patients with type 1 diabetes and dyslipidemia during atorvastatin therapy, i.e. a more porous fibrin network, to which reduced expression of glycoprotein IIIa, P-selectin and tissue factor on platelet-derived microparticles may contribute. The observed impairment of glycemic control during long-term statin treatment deserves attention.

Central thromboembolism is a possible predictor of right heart dysfunction in normotensive patients with acute pulmonary embolism - Corrected Proof

T.M. Berghaus, T. Haeckel, W. Behr, M. Wehler, W. von Scheidt, M. Schwaiblmair — 2010-07-20

Abstract: Background: Right heart dysfunction is a crucial factor in risk stratification of normotensive patients with pulmonary embolism. Apart from biomarkers, determinants of right heart dysfunction in this group of patients are not yet well established.Aim and method: In order to identify such determinants, we analysed data of 252 patients with acute pulmonary embolism admitted to our hospital in 2008.Results: 69 out of 140 patients showed right heart dysfunction by echocardiography within 24 hours after diagnosis, 71 did not. Right ventricular dysfunction was significantly more frequent in patients with central clots on computed tomography (p=0.004), a history of syncope (p<0.001) and among women on oral contraceptives (p=0.003). In multiple regression analysis, only central thromboembolism (p<0.001) was identified as individual predictor of right ventricular dysfunction. Age, gender, body mass index, idiopathic or recurrent thromboembolism, duration of symptoms, preceding surgery, room air oxygen saturation, carcinoma, hypertension, diabetes, renal disease, congestive left heart failure and concomitant lung disease were equally distributed. In comparison with NT-pro brain natriuretic peptide (PPV 67%, NPV 75%, p=0.782) and troponin I (PPV 76%, NPV 62%, p=0.336), central thromboembolism has shown to have a greater statistical power in predicting right heart dysfunction in normotensive patients with pulmonary embolism (PPV 78%, NPV 88%, p<0.001).Conclusion: Among normotensive patients with acute pulmonary embolism, those with central clots seem to be at greater risk for echocardiographically evaluated right ventricular dysfunction.

Purified thromboplastin causes haemostatic abnormalities but not overt DIC in an experimental rabbit model - Corrected Proof

Line Olrik Berthelsen, Annemarie T. Kristensen, Mikael Tranholm — 2010-07-16

Abstract: Validation of animal models of disseminated intravascular coagulation (DIC) to human DIC is crucial in order to translate findings in research models to treatment modalities for DIC in humans. ISTH classifications of overt and non-overt human DIC have proven to have a high diagnostic accuracy, and we have previously established a rabbit model of non-overt DIC based on the ISTH classification of non-overt DIC. In this rabbit model, we used purified rabbit brain thromboplastin to induce DIC and test applicability of ISTH classifications of overt human DIC.Cardiovascular and haematological parameters from rabbits, either saline-injected or administered a 2.5mg thromboplastin/kg bolus and a 15minutes 1.25mg thromboplastin/kg infusion, were determined at four time points over a 90minute period. All groups of rabbits were scored at each time point according to the ISTH classifications of overt DIC.Despite the fact that injection of purified thromboplastin resulted in decreased platelet count, increased prothrombin time, activated partial thromboplastin time, level of thrombin-antithrombin complexes and fibrin degradation products, and pulmonary micro-thrombosis, none of the rabbits were diagnosed as having overt DIC according to ISTH classification.We conclude that purified thromboplastin causes haemostatic abnormalities in the rabbit but this experimental model was not diagnosed as overt DIC.

Evaluation of platelet function and pharmacological platelet inhibition in patients with myeloproliferative disorders using multiple electrode aggregometry - Corrected Proof

2010-07-15

Abstract: Background: The aim of this study was to describe platelet aggregation characteristics by multiple electrode aggregometry (MEA) and to evaluate MEA for its potential to detect platelet dysfunction and response to anti-aggregatory drugs in patients with myeloproliferative disorders (MPD).Methods: We compared the platelet response to arachidonic acid (ASPI test), adenosine diphosphate (ADP test) and thrombin receptor activating peptide (TRAP test) in hirudin-anticoagulated blood of 55 patients with polycythaemia vera and essential thrombocythaemia and 75 controls.Results: Comparing MPD patients and controls no statistically significant difference indicative of platelet dysfunction was found in MPD patients. Analysis of covariance revealed platelet- and leukocyte count as a significant influencing factor on MEA function. Furthermore we could demonstrate that ASA and clopidogrel treatment results in a statistically significant lower ASPI (Controls: p<0.0001, MPD: p<0.0001) and ADPtest value (MPD: p=0.00125) compared to untreated patients thereby validating the method for monitoring of anti-aggregatory therapy.Conclusion: In this study MEA was confirmed as a valid method for monitoring of ASA and clopidogrel treatment in patients with MPD and normal control subjects. The platelet and leukocyte count were identified as major influencing factors on MEA aggregation tests both in MPD patients and controls. No functional platelet abnormalities were detected in MPD patients.

French clinical practice guidelines on the management of patients on vitamin K antagonists in at-risk situations (overdose, risk of bleeding, and active bleeding) - Corrected Proof

2010-07-15

Abstract: The present report from several French medical societies in the field and the French National Authority for Health provides an expert consensus for the management of patients on vitamin K antagonists in at-risk situations (overdose, risk of bleeding, and active bleeding). Asymptomatic VKA overdose is defined as an International Normalized Ratio (INR) value above the upper limit of the therapeutic target. In this case, the guidelines describe the rapid reduction of the INR down to the therapeutic range, either by omitting a dose or using vitamin K. Regarding the haemorrhagic complications, the guidelines address the management of these patients according to the severity of bleeding, and especially focus on the use of prothrombin complex concentrate. Finally, the consensus addresses the management of patients in cases of elective or emergency surgery or other invasive procedures, and discusses whether treatment should be continued or not, and whether VKA substitution by heparin – “bridging anticoagulation” - is needed.

Thrombolysis for massive pulmonary embolism in pregnancy - A report of three cases and follow up over a two year period - Corrected Proof

2010-07-14

We read with interest the recent literature review submitted by te Raa et al regarding treatment options in massive pulmonary embolism during pregnancy . As highlighted venous thromboembolism is a leading cause of maternal mortality in the UK . Treatment options for pregnant patients that develop pulmonary embolism are often considered high risk with possible effects to both mother and foetus. We describe our total experience of thrombolysis for the treatment of massive PE in pregnancy over the last 5years.

Thrombosis in children and neonates - Corrected Proof

Gili Kenet, Ulrike Nowak Gottl — 2010-07-14

In this series of six reviews to be published in thrombosis research over the next three issues, we explore the many facets of thrombosis and anticoagulant therapy in children, in order to provide readers with insight into current trends in research striving to explain pediatric thrombosis epidemiology, pathophysiology and therapy. Ultimately, improved prevention and treatment paradigms, grounded in robust and cumulative scientific evidence, will emerge.

Sources of variation in factor VIII, von Willebrand factor and fibrinogen measurements: Implications for detecting deficiencies and increased plasma levels - Corrected Proof

Juergen Bach, Hanne Haubelt, Peter Hellstern — 2010-07-14

Abstract: Objectives: Differences in pre-analytical and assay conditions, inappropriate reference ranges, or inflammation may have the potential to impair clinical decisions based on measurements of factor VIII (FVIII), von Willebrand factor (VWF) and fibrinogen (Fg). This study examined the impact on FVIII, VWF and Fg in plasma of freezing and thawing, different citrate anticoagulant concentrations, and inflammation, as determined by high-sensitivity C-reactive protein (hsCRP).Materials and Methods: FVIII was determined prior to freezing and after thawing using a one-stage clotting assay (FVIII:C), an amidolytic assay (FVIII:AM) and an enzyme immunoassay (FVIII:Ag). Samples were anticoagulated with 106 or 129mmol/L of citrate. FVIII, VWF and Fg were quantified in 300 individuals to establish reference ranges and to investigate associations with hsCRP.Results: Freezing and thawing reduced FVIII:C and FVIII:AM markedly. FVIII coagulant activities were not significantly different between samples anticoagulated with 106 or 129mmol/L of citrate, respectively. FVIII, VWF and Fg were significantly associated with hsCRP. FVIII:C was greater than FVIII:AM and FVIII:Ag in all experiments, indicating that the presence of activated FVIII may lead to overestimation of FVIII:C.Conclusions: Standardized freezing and thawing of plasma samples appears to be indispensable if reliable FVIII results are to be obtained. Because inflammation can potentially mask deficiency states or mimic an increased risk of thrombosis, FVIII, VWF and Fg determinations should be supplemented by measurements of hsCRP.

Clinical characteristics of patients with factor V Leiden or prothrombin G20210A and a first episode of venous thromboembolism. Findings from the RIETE Registry - Corrected Proof

2010-07-14

Abstract: Background: The clinical characteristics of patients with factor V Leiden or prothrombin G20210A presenting with a first episode of venous thromboembolism (VTE) have not been thoroughly studied.Methods: RIETE is an ongoing registry of consecutive patients with acute VTE. We compared the clinical characteristics of patients with factor V Leiden, prothrombin G20210A, or no thrombophilia, at presentation with a first episode of VTE.Results: As of May 2009, 22428 patients had been enrolled with a first episode of VTE. Of these, 345 had factor V Leiden, 261 had prothrombin G20210A, and 2399 tested negative. Sixty-two percent of the VTE episodes in women with factor V Leiden or prothrombin G20210A (40% in men) were associated with an acquired risk factor. Among women, pregnancy or contraceptive use accounted for 63% and 67% of such risk factors. Patients with factor V Leiden presented with pulmonary embolism (PE) less likely than those with prothrombin G20210A (31% vs. 51%; p<0.001) or with negative testing (31% vs. 45%, p<0.001). In addition, PE patients with Factor V Leiden presented with hypoxaemia (Sat O2 levels<90%) less likely than those with prothrombin G20210A (4.5% vs. 17%; p<0.001) or with no thrombophilia (4.5% vs. 20%; p<0.001).Conclusions: Most VTE episodes in women (not men) with factor V Leiden or prothrombin G20210A were associated with an acquired risk factor (mostly pregnancy or contraceptive use). Only 4.5% of patients with factor V Leiden presenting with acute PE had hypoxaemia.

Prolonged travel and venous thromboembolism findings from the RIETE registry - Corrected Proof

2010-07-13

Abstract: There is a lack of information on clinical risk factors for venous thromboembolism (VTE) development following prolonged traveling. Clinical characteristics and additional risk factors for VTE in travelers were analyzed in RIETE, an ongoing registry of patients with symptomatic, confirmed acute VTE. Of 26,172 patients enrolled in RIETE as of May 2009, 2% developed VTE in association with recent traveling. Travelers were ten years younger, had significantly more previous VTE events (20% vs. 16%; OR: 1.4; 95%CI: 1.1-1.7) and their body mass index (BMI) was 28.4±5.1 vs. 27.7±5.2 in other patients from the registry (P=0.004). 115 (20%) of recent travelers had previous VTE compared to 16% among others patients (OR: 1.4; 95%CI: 1.1-1.7). Recent travelers used hormones significantly more frequently (8.7% vs. 3.7%; OR: 2.5; 95% CI: 1.8-3.3) and more often had a positive thrombophilia test (16% vs. 8.7%; OR: 2; 95%CI: 1.6-2.6). Travelers used LMWH prophylaxis significantly less frequently than other patients in the registry (2.4% vs. 13%; OR 0.2; 95%CI: 0.1-0.3). There were differences in VTE risk in professional drivers compared to passengers. The current study demonstrates four risk factors for VTE development after long traveling: high BMI, previous VTE, hormone use and thrombophilia. Studies of prophylactic antithrombotic therapy in high risk travelers are warranted.

Antithrombotic efficacy of an oral low molecular weight heparin conjugated with deoxycholic asset on microsurgical anastomosis in rats - Corrected Proof

2010-07-12

Abstract: Introduction: Thrombogenic occlusion, at the anastomotic microvessels, contributed impaired blood flow to flap failure. The effect of an orally active low molecular weight heparin (LMWH) derivative conjugated with deoxycholic acid (DOCA) on the patency of anastomosis of the crushed rat artery was investigated, expecting its antithrombogenic effect.Materials and Methods: 60 Femoral arteries of 30 rats, divided into three groups of 20 each, were reanastomosed. LMWH-DOCA conjugate was orally administered prior to and after operation for 5 consecutive days. On the sixth day of operation, the patency of the anastomosed artery was evaluated.Results: The patency of oral LMWH-DOCA (10mg/kg) group was significantly enhanced from 15% to 45%, compared to non-treated control group. On the other hand, when the dosage of LMWH-DOCA was reduced to 1mg/kg, its efficacy on anastomosis was not as efficacious in terms of patency. The intima of crushed artery was impaired and thrombus formation was examined in the control group. In the drug treated group, the patency was only compromised by a thin layer of thrombus that covered the inner layer of the vessel without causing any damage to the internal elastic lamina.Conclusion: The medication of oral LMWH-DOCA conjugate has been vetted in microvascular anastomosis of the crushed artery. LMWH-DOCA was potentially useful for improving the patency in compromised vessels after microsurgery.

VKORC1 V66M mutation in African Brazilian patients resistant to oral anticoagulant therapy - Corrected Proof

2010-07-09

Abstract: Warfarin-based anticoagulant therapy is associated with large variability in dose response. Genetic variability in the VKORC1 and CYP2C9 genes is associated with increased warfarin sensitivity. In addition, rare coding region mutations in VKORC1 have been associated with resistance to warfarin. VKORC1 and CYP2C9 variability associated with altered warfarin response is less well characterized in African and mixed-raced populations such as Brazilians. To determine genetic variability associated with altered warfarin response among Brazilian patients, sixty-two adult patients with extreme resistance or sensitivity to warfarin were genotyped for variants in CYP2C9 and VKORC1. Of the 51 patients on low doses of warfarin, the VKORC1 – 1639 (3673) G>A polymorphism associated with warfarin sensitivity was present in 48 (94.1%), including 97% of Caucasians, 82% of African-descent patients, and all 7 (100%) patients of Indian descent. Additionally, 52.9% of warfarin sensitive patients had at least one CYP2C9*2 or CYP2C9*3 decreased metabolism allele, 63.6% of Caucasians and 54% of African-descent patients. Of the 11 patients on high doses of warfarin, sequencing of VKORC1 revealed a nonsynonymous V66M mutation in two warfarin resistant patients, both of African-descent. Brazilian patients requiring low doses of warfarin have a high frequency of VKORC1 and CYP2C9 variants associated with warfarin sensitivity. The presence of the rare VKORC1 V66M in two warfarin high dose outlier patients implies that this variant may be more frequent among African Brazilians and has implications for future warfarin studies in other populations of African descent.

Cloning, expression, and hemostatic activities of a disintegrin, r-mojastin 1, from the mohave rattlesnake (Crotalus scutulatus scutulatus) - Corrected Proof

2010-07-05

Abstract: Interactions with exposed subendothelial extracellular proteins and cellular integrins (endothelial cells, platelets and lymphocytes) can cause alterations in the hemostatic system associated with atherothrombotic processes. Many molecules found in snake venoms induce pathophysiological changes in humans, cause edema, hemorrhage, and necrosis. Disintegrins are low molecular weight, non-enzymatic proteins found in snake venom that mediate changes by binding to integrins of platelets or other cells and prevent binding of the natural ligands such as fibrinogen, fibronectin or vitronectin. Disintegrins are of great biomedical importance due to their binding affinities resulting in the inhibition of platelet aggregation, adhesion of cancer cells, and induction of signal transduction pathways. RT-PCR was used to obtain a 216bp disintegrin cDNA from a C. s. scutulatus snake venom gland. The cloned recombinant disintegrin called r-mojastin 1 codes for 71 amino acids, including 12 cysteines, and an RGD binding motif. r-Mojastin 1 inhibited platelet adhesion to fibronectin with an IC50 of 58.3nM and ADP-induced platelet aggregation in whole blood with an IC50 of 46nM. r-Mojastin 1 was also tested for its ability to inhibit platelet ATP release using PRP resulting with an IC50 of 95.6nM. MALDI-TOF mass spectrum analysis showed that r-mojastin has a mass of 7.95676kDa.

Prospective external validation of the new scoring system for disseminated intravascular coagulation by Japanese Association for Acute Medicine (JAAM) - Corrected Proof

2010-07-01

Abstract: Introduction: A new disseminated intravascular coagulation (DIC) scoring system was recently announced by Japanese Association for Acute Medicine (JAAM). We have conducted a prospective external validation study to assess the accuracy of this scoring system.Materials and methods: All patients admitted to the ICU in a tertiary academic hospital in 2007 were prospectively observed. All patients younger than 15years of age, those who stayed in the ICU for less than 24hours, had cardiac surgery, hematological diseases, recent chemotherapy or radiotherapy or liver cirrhosis were excluded. The remaining patients were then screened using the JAAM DIC scoring system.Results: DIC was diagnosed by the JAAM DIC scoring system in 45 of the 242 patients screened (18.6%). The DIC patients were older, had a higher Acute Physiology and Chronic Health Evaluation (APACHE) II score and stayed in the ICU longer in comparison to the non-DIC patients. However, hospital mortality was similar between the two groups (p=0.98). There was no difference in the JAAM DIC score between the surviving and non-surviving DIC patients (p=0.40). A multivariate logistic regression analysis revealed the DIC diagnosed by JAAM to have a non-significant low odds ratio for hospital mortality (OR 0.29, 95%CI 0.08-1.08, p=0.066).Conclusion: We have reported an external validation study of the JAAM DIC scoring system, which was conducted outside of the centers where data for developing the score were collected. DIC diagnosed by this scoring system was not related to hospital mortality.

Identification, Evaluation and Treatment of Prasugrel Low-Response after Coronary Stent Implantation – A Preliminary Study - Corrected Proof

Horst Neubauer, Andreas Kaiser, Birgit Busse, A. Mügge — 2010-06-29

Prasugrel was approved in 2009 and is a third generation thienopyridine with the advantage of a faster, less variable and more effective antiplatelet response than clopidogrel . Like the other thienopyridines ticlopidine and clopidogrel, prasugrel inhibits ADP-induced platelet activation by irreversibly blocking the P2Y12 platelet receptor and is an inactive prodrug needing cytochrome P450 (CYP) isoenzyme dependent platelet activation to obtain the active metabolite . In contrast to clopidogrel (metabolized by the hepatic CYP system through a two step pathway), prasugrel is transformed first through hydrolization of esterases followed by a single CYP-dependent oxidative step into its active metabolite . The CYPs involved in bioactivation of prasugrel are mainly CYP3A4 and CYP2B6 and to a lesser degree CYP2C9 and CYP2C19 . The active metabolites of clopidogrel and prasugrel are equipotent platelet inhibitors . Clopidogrel has several limitations, especially hypo-responsiveness in up to 30 % of the patients treated (dependent on dose, method and time of measurement), causing an increased rate of major adverse cardiac events . We investigated if prasugrel treatment contains the risk of a low response as measured by ADP-induced platelet aggregation, and if so, how this can be overcome. Furthermore we assessed if this phenomenon is possibly linked to ADP P2Y12 receptor defect or to polymorphism of selected cytochrome enzymes.

A Cost-effectiveness Analysis of Diagnostic Algorithms of Deep Vein Thrombosis at the Emergency Department - Corrected Proof

Jenny M. Norlin, Johan L. Elf, Peter J. Svensson, Katarina Steen Carlsson — 2010-06-28

Abstract: Introduction: Suspected cases of deep vein thrombosis are common at emergency departments and they often require extensive and costly diagnostic testing. The objective of this study was to evaluate whether a diagnostic algorithm based upon pre-test probability and D-dimer in diagnosing deep vein thrombosis may be cost-effective from a societal perspective in a Swedish setting.Material and Methods: The cost-effectiveness of two alternative diagnostic algorithms were calculated using decision analysis. An algorithm which out ruled deep vein thrombosis among low probability patients with negative D-dimer was compared to a traditional algorithm including compression ultrasonography and/or contrast venography for all patients. For sensitivity analysis, a third reversed algorithm, where D-dimer was followed by pre-test probability, was analyzed. Estimates of probabilities were obtained from a prospective management study, including 357 outpatients with clinical suspicion of deep vein thrombosis. Direct costs were estimated using prices from Scania, Sweden. Indirect costs were estimated using time spent at the local emergency department and gross average wages in Sweden.Results: The total cost of the pre-test probability and D-dimer algorithm was estimated to €406 per patient and the traditional algorithm was estimated to €581 per patient. Reversing the order of the score and test resulted in an estimate of €421 per patient.Conclusion: At no significant difference in diagnostic efficacy the algorithm based upon pre-test probability and D-dimer was cost-effective, while the reversed algorithm and diagnostic imaging for all patients were not.

Selectins in human amniotic fluid and cord blood plasma. A preliminary report - Corrected Proof

2010-06-28

We investigated soluble forms of E-, P- and L-selectins in three fluid compartments of a pregnant woman, i.e. in the amniotic compartment, fetal blood compartment and maternal blood compartment , and we would like to share our observations made during a preliminary study. To the best of our knowledge, the selectins have not been identified in the amniotic fluid or cord blood (fetal blood) yet, but only in maternal blood.

Serum Lipoprotein (a) Levels in Patients with First Unprovoked Venous Thromboembolism is not Associated with Subsequent Risk of Recurrent VTE - Corrected Proof

2010-06-28

Abstract: Introduction: Case-control studies suggest that elevated lipoprotein (a) (Lp(a)) is a risk factor for first venous thromboembolism (VTE). Lp(a) has not been prospectively investigated as a possible risk factor for recurrent VTE in first unprovoked VTE patients. We sought to determine if serum Lp(a) levels in patients with unprovoked VTE who discontinue anticoagulants after 5 to 7months of therapy predict VTE recurrence in a prospective cohort study.Materials and Methods: Serum Lp(a) measurements were obtained from 510 first unprovoked VTE patients treated for 5 -7months with anticoagulants in a 12 center study. Patients were subsequently followed for a mean of 16.9months (SD±11.2) for symptomatic VTE recurrence which was independently adjudicated with reference to baseline imaging.Results: There was no significant association between Lp(a) as a continuous variable and recurrent VTE nor in gender stratified subgroups. No statistically significant differences were observed in the median Lp(a) concentrations between patients who recurred and those who did not recur (median (interquartile range): 0.09g/L (0.17) versus 0.06g/L (0.11) respectively; p=0.15). The Lp(a) cut-off point of 0.3g/L was not significantly associated with recurrent VTE for the overall population nor in gender stratified subgroups.Conclusions: Elevated serum Lp(a) does not appear to be associated with recurrent VTE in patients with history of first unprovoked VTE and may not play a role in identifying patients with unprovoked VTE at high risk of recurrence. There was no optimal predictive threshold for the overall population or for sex sub-groups and Lp(a)≥0.3g/L was not a significant predictor of recurrent VTE.

Aspirin resistance following pediatric cardiac surgery - Corrected Proof

2010-06-15

Abstract: Introduction: Aspirin is often used to prevent thrombosis in pediatric cardiac surgery. The primary study aim was to assess aspirin resistance in this context. Secondary aims were to evaluate (1) the relationship between elevated inflammatory markers and thrombosis and (2) aspirin's effect on these levels.Materials and Methods: This was a prospective observational study of children undergoing cardiac surgery managed with and without aspirin. Aspirin response was assessed using the VerifyNow™ system and urinary 11-dehydrothromboxane B2 (uTxB2) measurements. Laboratory studies of inflammation were also obtained.Results: 101 subjects were studied; 50 received aspirin. Six subjects (5.9%), 5 aspirin-treated, experienced symptomatic thrombosis. When measured by VerifyNow™ resistance was 43% after aspirin suppositories and 14% after additional days of oral aspirin. There was no correlation with thrombosis. Upper quartile post-operative day (POD) #5 uTxB2 was correlated with thrombosis in aspirin treated subjects (p<0.01). High risk aspirin-treated subjects who experienced thrombosis had higher POD#5 uTxB2. This finding did not reach statistical significance (p=0.07). Elevated pre-operative C-reactive protein (CRP) was independently associated with thrombosis (p<0.02) in all subjects and in high risk subjects (p=0.01). Inflammatory markers were not affected by aspirin.Conclusions: Aspirin inhibited ex-vivo platelet function with a low incidence of resistance. Elevated POD#5 uTxB2 and pre-operative CRP were correlated with thrombosis in aspirin treated subjects. Further studies are needed to determine whether children with high levels of uTxB2 despite aspirin therapy and/or those with elevated preoperative CRP are at increased risk for thrombosis.

Measurement of procoagulant platelet subpopulations in whole blood: Development of an assay for population-based studies - Corrected Proof

Beth A. Bouchard, Aimee K. Paradis, Kathleen E. Brummel-Ziedins — 2010-06-14

In the hemostatic and thrombotic processes, thrombin is generated at the activated platelet surface through the concerted assembly and function of the macromolecular, enzymatic complexes, intrinsic tenase and prothrombinase, to effect the generation of factor Xa and thrombin, respectively . Platelet activation can be accomplished by nanomolar concentrations of thrombin and is central to appropriate coagulation complex assembly and function . Assembly of a functional intrinsic tenase [for review see ] or prothrombinase [for review see ] complex is dependent upon several protein/protein and protein/membrane interactions. However, platelet activation and complex formation may not be sufficient. Several laboratories have identified discrete subpopulations of platelets that differentially regulate procoagulant enzyme complex and function . When all platelets are thrombin-activated to a level consistent with maximal procoagulant activity, only a subpopulation of the activated platelets are capable of prothrombinase or intrinsic tenase complex assembly . In these studies, factor Va and factor Xa binding colocalize to the same subset of activated platelets , as do components of the intrinsic tenase complex (factor VIIIa, factor IXa, and factor X) . Likewise, platelet activation with low levels of thrombin plus collagen, or thrombin plus a glycoprotein VI agonist, convulxin, generates a subpopulation of platelets, referred to as COATED-platelets, that express high levels of membrane bound α-granule proteins, such as factor Va , which are thought to be covalently bound to the platelet surface via serotonin . COATED-platelets can also bind high levels of factor Xa .

Evaluation of a Post-operative Thrombin Inhibitor Replacement Protocol to reduce Haemorrhagic and Thrombotic Complications after Paediatric Liver Transplantation - Corrected Proof

2010-06-14

Abstract: Introduction: Bleeding and thrombotic complications contribute to morbidity and mortality following paediatric orthotopic liver transplantation (OLT). However, the pathophysiology of haemostasis during paediatric OLT is not well understood. This report consists of two complimentary studies examining the frequency of haemostatic complications before and after the introduction of a post-operative thrombin inhibitor replacement therapy protocol at a single institution.Materials and Methods: A retrospective study of 40 patients who underwent 43 liver transplants between July 1992 and July 2002, identified bleeding to be the most frequent complication associated with OLT (30%), however thrombotic complications were also common (12.5%). In 2003, following a detailed analysis of haemostatic profiles of children undergoing OLT, a thrombin inhibitor replacement protocol was introduced. A prospective clinical outcome audit was undertaken from April 2003 to September 2008 to determine the effect of the new protocol on haemostasis.Results: Commencement of the thrombin inhibitor replacement protocol significantly reduced the incidence of thrombosis (from 5 to 1, p<0.05), graft loss (from 4 to none, p<0.05), mortality due to thrombosis or bleeding (from 3 to none, p<0.05) and was associated with a 50% reduction in frequency of major bleeding.Conclusion: In conclusion, the introduction of a post-operative thrombin inhibitor replacement therapy protocol following paediatric OLT significantly improved haemostasis-related morbidity and mortality outcomes in children.

The plasma levels of activated thrombin activatable fibrinolysis inhibitor and thrombomodulin in Behçet Disease and their association with thrombosis - Corrected Proof

2010-06-10

Abstract: Objective: To investigate the plasma levels of activated thrombin activatable fibrinolysis inhibitor (aTAFI) and thrombomodulin (TM) in Behçet disease (BD) and their relationship with thrombosis.Methods: Plasma aTAFI and TM levels were measured by ELISA in 89 patients with BD (18 having venous thrombosis) and in 86 healthy controls.Results: Compared with healthy controls, the BD group had significantly lower levels of aTAFI (13.49±8.88µg/ml vs. 26.76±11.57µg/ml, p<0.0001) and significantly higher levels of TM (3.26±1.85ng/ml vs. 2.6±0.69ng/ml, p=0.0003). Neither aTAFI, nor TM levels differed significantly between BD patients with and without thrombosis (p>0.05). Despite a tendency to positive correlation (r=0.37, p=0.0004) between plasma levels of aTAFI and TM in healthy controls, there was a tendency for negative correlation (r=-0.51, p<0.0001) between these two parameters in BD patients.Conclusion: The plasma aTAFI and TM levels do not seem to be related with the presence of thrombosis observed in BD. Increased plasma TM levels in BD may simply reflect endothelial cell activation and dysfunction.

Pooled analysis of trials may, in the presence of heterogeneity inadvertently lead to fragile conclusions due to the importance of clinically relevant variables being either hidden or lost when the findings are pooled - Corrected Proof

R.D. Hull, J. Liang, R. Brant — 2010-06-09

Evidence-based Medicine Guidelines based on venographic end-points recommend low-molecular-weight-heparin (LMWH prophylaxis) for patients undergoing elective hip and knee surgery. These level 1A recommendations highlight the benefit of such therapy. The recommendations are based on randomized clinical trials that do not have important limitations and can apply to most patients in most circumstances without reservation . Surveys indicate that more than 90% of patients who have undergone either elective hip or knee surgery have received thromboprophylaxis . Clearly an unmet need is the availability of oral antithrombotic inhibitors that do not require anticoagulant monitoring and which simplify the administration of thromboprophylaxis by avoiding parenteral administration both in hospital and out-of-hospital settings.

Application of Akaike information criterion to evaluate warfarin dosing algorithm - Corrected Proof

2010-06-09

Abstract: Introduction: Several factors responsible for inter-individual differences in response to warfarin have been confirmed; however, unidentified factors appear to remain. The purpose of this study was to examine a simple method to evaluate whether optional variables are appropriate as factors to improve dosing algorithms.Materials and Methods: All patients were Japanese. Genotyping of selected genes was conducted, and other information was obtained from medical record. Dosing algorithms were constructed by multivariate linear regression analyses and were evaluated by the Akaike Information Criterion (AIC).Results and Conclusions: Multivariate analysis showed that white blood-cell count (WBC), concomitant use of allopurinol, and CYP4F2 genotype are apparently involved in warfarin dose variation, in addition to well-known factors, such as age and VKORC1 genotype. We evaluated the adequacy of these variables as factors to improve the dosing algorithm using the AIC. Addition of WBC, allopurinol administration and CYP4F2 genotype to the basal algorithm resulted in decreased AIC, suggesting that these factor candidates may contribute to improving the prediction of warfarin maintenance dose. This study is the first to evaluate the warfarin dosing algorithm by AIC. To further improve the dosing algorithm, AIC may be a simple and useful tool to evaluate both the model itself and factors to be incorporated into the algorithm.

Use of recombinant human soluble thrombomodulin in the management of HELLP syndrome complicated by DIC - Corrected Proof

T. Ikezoe, N. Ikenoue, N. Uchikawa, S. Kojima, T. Fukaya, A. Yokoyama — 2010-06-07

Hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome is a severe complication in pregnancy characterized by microangiopathic hemolytic anemia, liver dysfunction, and thrombocytopenia, occurring in 0.1-0.2% of all pregnancies and 10-20% of cases with severe pre-eclampsia/eclampsia. Approximately 70% of cases develop prior to delivery with a peak frequency between gestational weeks 27 and 38, with 10% developing before week 27 and the remaining cases occurring postpartum, usually within 48h of delivery . Interestingly, more than 50% of cases show excessive body weight gain and unexplained generalized edema preceding the syndrome, suggesting a possible role of endothelial cell damage in the pathogenesis of HELLP syndrome . In addition, 21-55% of cases with HELLP syndrome are complicated by disseminated intravascular coagulation (DIC), triggered by endothelial cell damage . Moreover, previous studies have found that plasma levels of von Willebrand factor (vWF), a marker of endothelial cell damage, are significantly increased in patients with HELLP syndrome compared to healthy pregnant volunteers .

Drop of PT Quick percent value is associated with both symptomatic and asymptomatic intracranial hemorrhage in patients treated with rt-PA for acute ischemic stroke - Corrected Proof

A. Alonso, C.E. Dempfle, K. Szabo, K. Zohsel, M.G. Hennerici — 2010-06-01

Clinical trials have demonstrated the value of thrombolytic therapy (TT) in the early phase of acute ischemic stroke . Alteplase was approved for treatment of acute ischemic stroke by the European Medicines Evaluation Agency (EMEA) in 2002, excluding patients with severe stroke, altered coagulation parameters like low platelet count or oral anticoagulation therapy amongst others for safety and efficacy reasons. Treatment with alteplase and similar plasminogen activators is, however, associated with an increased risk of intracranial hemorrhage (IH), or hemorrhagic transformation of ischemia after reperfusion. This is also true for daily practice as evidenced by the SITS-MOST trial: among 6483 patients the proportion of patients with symptomatic IH at 7days was 7.3% .

Corrigendum to “Alternatively spliced isoforms of tissue factor pathway inhibitor” [Thrombosis Research 125 (2010) S52–S56] - Corrected Proof

Susan A. Maroney, Paul E. Ellery, Alan E. Mast — 2010-06-01

The authors regret that Fig. 1A was repeated as A in the published manuscript. The correct A appears below. The authors would like to apologise for any inconvenience caused.

Antithrombin and heparin cofactor II-mediated inactivation of α-thrombin by a synthetic, sulfated mannogalactan - Corrected Proof

2010-05-31

Abstract: Introduction: A mannogalactan from Pleurotus ostreatoroseus (MgPr) was chemically sulfated to give MgPr-S1, which was evaluated for its anticoagulant and antithrombotic activities, bleeding tendency, and platelet aggregation.Materials and methods: MgPr-S1 was partially characterized by HPSEC-MALLS, methylation analysis, and 13C NMR spectroscopy. Its anticoagulant activity was determined by assays of aPTT, TT, α-thrombin and factor Xa residual activity, heparin cofactor II (HCII)-, or antithrombin (AT)-mediated inhibition. The antithrombotic effect was evaluated in rats using a venous thrombosis model and the bleeding tendency was also tested in vivo. Platelet aggregation was investigated by an adaptation of the method of Born [1].Results: The hydroxyl groups of β-D-Manp units and OH-2 and OH-4 of the (1→6)-linked α-D-Galp units were preferentially substituted. The anticoagulant activity of MgPr-S1 was mainly by thrombin inhibition with antithrombin and HCII, and had an effect on platelet aggregation induced by ADP and α-thrombin. It almost completely inhibited thrombus formation in vivo at a dose of 6mg/kg and heparin inhibited thrombus formation at a dose of 0.200mg/kg.Conclusions: These results suggested that the chemically sulfated mannogalactan could act as an alternative to heparin as anticoagulant.

Allele-allele interaction within the F13A1 gene: A risk factor for Ischaemic Heart Disease in Spanish population - Corrected Proof

2010-05-31

Atherothrombotic diseases are a group of diseases that cause the majority of population morbidity in industrialized countries. Genetic variation in causative or susceptible genes constitutes the basis of molecular mechanisms, that together with environmental factors, lead to disease development .

Algorithms using clinical and genetic data (CYP2C9, VKORC1) are relevant to predict warfarin dose in patients with different INR targets - Corrected Proof

2010-05-31

Oral anticoagulant treatment (OAT) often induces severe adverse drug events due to a large individual susceptibility. The use of warfarin is specifically connected with dose variation, which could be influenced by clinical factors, such as age, weight or height, and genetic variation . Many studies have demonstrated an important role of the cytochrome P450 CYP2C9 enzyme polymorphisms . Gage et al. have shown that genetic variation may influence the warfarin maintenance dose: a transition c.430C>T in exon 3 [rs1799853] is denoted as CYP2C9*2 while a transition c.1075A>C in exon 7 [rs1057910] is denoted as CYP2C9*3. The wild type of this genotype is called CYP2C9*1. The presence of CYP2C9*2 or CYP2C9*3 induced a reduction of warfarin dose of 14% to 20% and 21% to 49%, respectively. Moreover an influence of the c.-1639G>A polymorphism [rs9923231], located in the promoter region of the gene of the vitamin K epoxide reductase complex subunit 1 (VKORC1) has been demonstrated. This polymorphism is in stong linkage disequilibrium with another VKORC1 polymorphism, i.e., c.1173C>T [rs9934438] in intron 1 , and is associated with a higher sensitivity to warfarin. All these polymorphisms could decrease the daily warfarin maintenance dose, alone or in combination . Algorithms, using both clinical and pharmacogenetic data, have been proposed to estimate the warfarin maintenance dose in patients with a target INR range of 2 to 3 . The aim of our study was to evaluate the accuracy of two of these algorithms in two patient groups with different INR target ranges. We chose to test the algorithm published by Sconce et al. and another algorithm published by Gage et al. , also proposed "on line" on a website: www.WarfarinDosing.org. These algorithms were mostly tested within the INR target 2 to 3 for patients on warfarin treatment. Between February 1996 and July 2002, 80 patients were included in a research program (PHRC N° 94-093HP), supported by the French Ministry of Health, to compare efficacy and safety of low (INR 1.5 – 2) and conventional regimens (INR 2.3 – 3) of warfarin in patients having an indication for long-term secondary prophylaxis with OAT. All of them were below 70years old, carried at least one hereditary thrombophilia (8 antithrombin-, 11 protein C-, and 11 protein S deficiencies, 38 heterozygous factor V Leiden mutations, 4 heterozygous factor II 20210A mutations, and 8 combined abnormalities or homozygous factor V Leiden mutations), and had a past history of two or more thrombotic events. After a period of at least three months of conventional OAT (INR between 2 and 3), the patients were randomized to treatment with warfarin, in an open label design, with two target INR targets; group A – INR between 2.3 and 3, and group B – INR between 1.5 and 2. Twenty eight male and 52 female patients were included, 40 in each group. The mean age of the population at the time of inclusion was 43.3±15.9years (range=19-68). During the follow-up, which was 30months after the randomization, INR was tested every 3weeks in local laboratories and results of INR and actual warfarin dose were noted. Every 6months after inclusion, noted survey of INR, warfarin dose and clinical data (thrombotic or hemorrhagic events) were recorded during a visit in the hospital. The weight and height and other treatments were registered at inclusion. The daily maintenance warfarin dose was determined by calculation of the mean of all noted warfarin doses between the 6th and the 30th month. This maintenance dose in our patients was stable with a low mean coefficient of variation of 2.61%±2.80 (standard deviation).

Corrigendum to “A family having type 2B von Willebrand disease with an R1306W mutation: Severe thrombocytopenia leads to the normalization of high molecular weight multimers” [Thrombosis Research 125 (2010) e17–e22] - Corrected Proof

2010-05-31

The authors regret that an incorrect version of appeared in the above published article. The correct version appears below. The authors would like to apologize for any inconvenience caused.

Prophylaxis against Prosthetic Mitral Valve Thrombosis with Unfractionated Heparin Administered by an Elastometric Infusion Pump - Corrected Proof

Per Morten Sandset, Harald Arnesen, Anne Flem Jacobsen, Håkon Wergeland — 2010-05-31

In pregnant women with mechanical prosthetic heart valves, oral anticoagulants offer the best protection against thromboembolic complications including heart valve thrombosis, but their use is teratogenic and is associated with pregnancy loss . Substitution of oral anticoagulants with therapeutic doses of either unfractionated or low molecular weight heparins reduces the risk of fetal damage, but major concerns remain regarding their efficacy to prevent thrombotic complications, especially heart valve thrombosis .

African-Americans have a higher rate of proximal deep vein thrombosis at presentation - Corrected Proof

Tyler W. Buckner, Chirag Amin, Denise A. Esserman, Nigel S. Key — 2010-05-24

Abstract: There is growing evidence that venous thromboembolism (VTE) incidence and mortality rates differ among ethnic/racial groups. Specifically, it has been demonstrated previously that blacks with VTE have a higher proportional rate of pulmonary embolism (PE), as well as a higher case fatality rate from PE compared to whites. We compared the location of DVT (proximal vs. distal) in blacks and whites at the time of diagnosis. We reviewed all lower extremity Doppler ultrasound studies that were positive for acute DVT at our Institution over a 3-year period. Subjects were classified by self-reported race (black or white), disposition at diagnosis (inpatient or outpatient), and clot location (proximal or distal). Outpatients were also examined to determine the presence of potential provoking factors for DVT as well as dates of symptom onset and presentation. We found that the rate of any proximal DVT compared with distal only was significantly higher in black patients compared to white patients (OR=1.4; 95%CI: 1.0, 1.8; P=0.032). This difference remained significant in the outpatient group (OR=2.0; 95% CI: 1.0, 3.8; P=0.048), but not in inpatients (OR=1.3; 95% CI: 0.9, 1.9; P=0.11). The average number of days from symptom onset to presentation was increased for black patients (5.6days) compared to white patients (4.5days). We conclude that blacks with acute lower extremity DVT are more likely to present with proximal DVT than whites, which might account in part for the higher rate of PE seen in black patients.

The antithrombotic effect of a novel hirudin derivative after reconstruction of carotid artery in rabbits - Corrected Proof

2010-05-18

Abstract: Introduction: Hirudin is a direct thrombin inhibitor that has potential mechanistic advantages over indirect inhibitors. Peptides containing the RGD motif competitively inhibit binding of fibrinogen to glycoprotein IIb/IIIa on platelets, thus inhibiting platelet aggregation. A novel hirudin derivative, recombinant RGD-hirudin (r-RGD-hirudin), was engineered by fusing the tripeptide RGD sequence to the native hirudin. We tested the antithrombotic effect of r-RGD-hirudin using a carotid artery reconstruction model in rabbits.Materials and methods: A fusion gene encoding r-RGD-hirudin was constructed and expressed at high levels in Pichia pastoris. Following traumatic injury and anastomosis, 42 New Zealand White rabbits were randomized to receive normal saline, abciximab, wild-type hirudin, or r-RGD-hirudin. Fibrinogen concentration, aPTT, TT, PT, and PAGm were measured prior to and following the operation. Carotid angiography and pathological examination of the anastomotic site were performed to compare patency rates among the groups.Results: The r-RGD-hirudin significantly prolonged aPTT, TT, PT and inhibited PAGm following carotid anastomosis in rabbits. The median dose of r-RGD-hirudin (0.5mg/kg) had a therapeutic effect equal to that of wild-type hirudin (1.0mg/kg) and higher than that of abciximab (0.2mg/kg) with regard to patency rates.Conclusions: Compared to wild-type hirudin or antiplatelet agent, the novel anticoagulant, r-RGD-hirudin was capable of inhibiting both thrombin activity and platelet aggregation, and was demonstrated to be effective in the prevention of thrombosis.

Enhanced platelet reactivity and thrombosis in Apoe-/- mice exposed to cigarette smoke is attenuated by P2Y12 antagonism - Corrected Proof

2010-05-10

Abstract: Introduction: Smoking increases the risk of acute arterial thrombosis, including myocardial infarction, likely due to multi-factorial effects on the vasculature. Heightened platelet reactivity may be among the adverse effects of smoke exposure.Methods: To examine the effects of smoke exposure on platelet function in an atherosclerotic environment, Apoe-deficient female mice, maintained on a Western diet, were exposed (4hrs/d, 5d/wk) to sidestream cigarette smoke in a whole-body exposure chamber for12weeks. A separate group of wild type C57BL/6J mice were also exposed to smoke in an identical fashion.Results: In comparison to control Apoe-/- mice exposed to filtered ambient air, smoke-exposed Apoe-/- mice displayed a 1.8±0.3 fold enhanced ADP-induced fibrinogen binding ex vivo (P<0.001) and had a shorter time to thrombotic occlusion following ferric chloride injury of the carotid artery (median time to thrombosis of 8 vs. 13min; P=0.015). Administration of the direct-acting P2Y12 antagonist cangrelor blunted ex vivo fibrinogen binding and attenuated thrombosis (median time 20min) in Apoe-/- mice exposed to sidestream smoke. The effects of smoke exposure required a proatherosclerotic background, as wild-type C57Bl/6J mice exposed to smoke displayed similar fibrinogen binding and thrombotic occlusion times as did control mice.Conclusions: Our results demonstrate that exposure to smoke heightens platelet reactivity and thrombosis in Apoe-/- mice and implicate signaling through platelet P2Y12 receptor as a mediator of the adverse consequence of smoke exposure. These results may partially explain the recent observations that smokers derive greater clinical benefit from the P2Y12 antagonist clopidogrel than do non-smokers.

Reduced platelet response to aspirin in patients with coronary artery disease and type 2 diabetes mellitus - Corrected Proof

S.B. Mortensen, S.B. Larsen, E.L. Grove, S.D. Kristensen, A.-M. Hvas — 2010-05-10

Abstract: Introduction: Diabetes mellitus is complicated by accelerated atherosclerosis, resulting in an increased risk of coronary artery disease (CAD) and thrombosis. Despite the proven benefits of aspirin, previous studies indicate a reduced cardiovascular protection from aspirin in diabetic patients. We aimed to investigate whether diabetes mellitus influenced the platelet response to aspirin in patients with CAD.Materials and Methods: Platelet aggregation and activation were evaluated during aspirin treatment in 85 diabetic and 92 non-diabetic patients with CAD. Adherence to aspirin was carefully controlled. All patients had CAD verified by coronary angiography and were taking 75mg non-enteric coated aspirin daily.Results: Diabetic patients showed significantly higher levels of platelet aggregation compared to non-diabetic patients evaluated by VerifyNow® Aspirin (p=0.03) and Multiplate® aggregometry using arachidonic acid (AA) 0.5mM (p=0.005) and 1.0mM (p=0.009). In addition, platelet activation determined by soluble P-selectin was significantly higher in diabetics compared to non-diabetics (p=0.005). The higher AA-induced aggregation was associated with higher levels of HbA1c. Compliance was confirmed by low levels of serum thromboxane B2 (below 7.2ng/mL). Diabetics had significantly higher levels of serum thromboxane B2 (p<0.0001).Conclusions: Diabetic patients with CAD had significantly higher levels of both platelet aggregation and activation compared to non-diabetic patients with CAD despite treatment with the same dosage of aspirin. These findings may partly explain the reduced cardiovascular protection from aspirin in diabetic patients.

Incidence and time course of asymptomatic deep vein thrombosis with fondaparinux in patients undergoing total joint arthroplasty - Corrected Proof

Toshio Yamaguchi, Masahiro Hasegawa, Rui Niimi, Akihiro Sudo — 2010-05-10

Abstract: Introduction: There are many reports concerning fondaparinux prophylaxis of asymptomatic deep vein thrombosis (DVT) after total hip arthroplasty (THA) or total knee arthroplasty (TKA), but little is known about the time course of aymptomatic DVT development during the administration of fondaparinux. The aim of the present study was to define the incidence and time course of aymptomatic DVT development during administration of fondaparinux, and to assess the efficacy of fondaparinux in resolving DVT.Materials and Methods: We studied consecutive71 patients who underwent THA surgery, and 30 patients who underwent TKA surgery with fondaparinux prophylaxis. Patients received once-daily subcutaneous injections of 2.5mg of fondaparinux for 14days after surgery. DVT was diagnosed by ultrasonography, and it was scheduled on the day of surgery on day 1, day 4, and day 14 after surgery.Results: In patients who received fondaparinux for 14days after THA surgery, the incidence of DVT was 0% on the day of the surgery, 13.6% at day 1, 27.1% at day 4, and 11.9% at day 14. In patients who received fondaparinux for 14days after TKA surgery, the incidence of DVT was 4.2% on the day after surgery, 50.0% at day 1, 58.3% at day 4, and 20.8% at day 14. The incidence of DVT after THA or TKA surgery at day 14 was significantly reduced compared to that at day 4.Conclusion: The incidence of asymptomatic DVT up to day 4 was high, but with 14days continued treatment of fondaparinux, the incidence of asymptomatic DVT occurring at postoperative day 4 was significantly reduced at day 14.

The Influence of Direct Thrombin Inhibitors on the Formation of Platelet-leukocyte Aggregates and Tissue Factor Expression - Corrected Proof

Christina Christersson, Matilda Johnell, Agneta Siegbahn — 2010-05-10

Abstract: Introduction: High concentrations of platelet-monocyte aggregates (PMAs) have been found in patients with myocardial infarction (MI). Oral direct thrombin inhibitors (DTIs) are under evaluation as long-term antithrombotic treatment. The aim was to evaluate whether DTIs affect the formation of platelet-leukocyte aggregates, TF expression and procoagulant microparticles (MPs).Material and Methods: DTIs were added to an experimental whole blood model before platelet activation with thrombin or ADP. The concentrations of PMAs, platelet-granulocyte aggregates (PGAs), the amount of platelets bound per leukocyte and MPs were investigated by flow cytometry. TF mRNA and activity were recorded in all settings. TF activity was evaluated in a MI population treated with or without an oral DTI.Results: In vitro, thrombin and ADP increased the formation of PMAs and PGAs as well as TF mRNA expression. DTIs reduced the amount platelets bound to monocytes (p=0.02) and to granulocytes (p=0.001) upon thrombin stimulation together with a reduction of TF mRNA. In contrast, the ADP-induced formation of PMAs, PGAs and TF mRNA was not affected by the DTIs. Both thrombin and ADP stimulation increased the amount of TF-expressing MPs, which was effectively inhibited by the DTIs (p=0.02-0.002). In the MI population, the DTI reduced the TF activity (p<0.001).Conclusion: DTIs modulate the formation of PMAs, PGAs and the TF production therein. Together with a reduction of procoagulant MPs, these results may contribute to the clinical benefit found of oral DTIs. Targeting different mechanisms in platelet and coagulation activation may be of importance due to the lack of effect of DTIs on ADP-induced platelet-leukocyte aggregates and TF production.

Smoking at least 10 cigarettes per day increases platelet inhibition by clopidogrel in patients with ST-segment-elevation myocardial infarction - Corrected Proof

2010-05-10

Abstract: Background: Recent data suggest that cigarette smoking (CS) might decrease the risk of cardiovascular events in patients with ST-segment-elevation myocardial infarction (STEMI) or established cardiovascular disease. Although it may be related to the effect of CS on the metabolism of clopidogrel, the association between the extent of CS and clopidogrel-induced platelet inhibition has not been well defined.Patients and methods: We tested the association between smoking status and inhibition of platelet aggregation (IPA) in response to a clopidogrel loading of 600-mg in 20 healthy subjects. We then enrolled 138 consecutive STEMI patients treated with primary coronary stenting. On-clopidogrel platelet reactivity (PR) was assessed with conventional aggregometry and the VerifyNow P2Y12 assay, according to smoking status.Results: After 6hours post-loading in healthy subjects, CS patients on ≥10 cigarettes/day showed a significantly higher value of 5μmol/L ADP-stimulated IPA (P=0.006), and had a trend toward a greater value of 20μmol/L ADP-stimulated IPA (P=0.093) compared with non-smokers. In STEMI patients, there was no difference in PR between non-smokers (n=66) and CS patients<10 cigarettes/day (n=16). CS patients on ≥10 cigarettes/day (n=56) demonstrated lower PR with 5 and 20μmol/L ADP (40.9±16.1% versus 46.6±11.7%, P=0.028, and 53.8±16.6% versus 59.2±12.2%, P=0.040, respectively) and lower P2Y12 reaction units (204±85 versus 270±69, P<0.001) than non-smokers. On multivariate analyses, CS≥10 cigarettes/day was the only predictor of low on-clopidogrel PR (≤33%; the lowest quartile of 5μmol/L ADP-induced PR; odds ratio 4.651, 95% confidence interval 1.181–18.519, P=0.028).Conclusion: CS seems to increase antiplatelet response to clopidogrel in healthy volunteers and STEMI patients. Smoking 10 or more cigarettes/day can significantly decrease on-clopidogrel platelet reactivity in these populations.

Dabigatran versus enoxaparin for prevention of venous thromboembolism after hip or knee arthroplasty: A pooled analysis of three trials - Corrected Proof

2010-05-03

Abstract: Background: Three randomized, double-blind trials compared dabigatran, an oral direct thrombin inhibitor, with enoxaparin for the primary prevention of venous thromboembolism (VTE) in patients undergoing elective total hip and knee arthroplasty.Objectives and Methods: We conducted a pre-specified pooled analysis of these trials. 8,210 patients were randomized, of whom 8,135 were treated (evaluable for safety) with dabigatran 220mg or 150mg once-daily, or subcutaneous enoxaparin (40mg once-daily or 30mg twice-daily). Efficacy analyses were based on the modified intention-to-treat population of 6,200 patients with an evaluable outcome. The common risk difference (RD) of treatment effect between each dabigatran dose and enoxaparin was estimated using fixed-effects models, and statistical heterogeneity was estimated using the I2 statistic.Results: The composite outcome of major VTE (proximal deep vein thrombosis and/or pulmonary embolism) and VTE-related mortality occurred in 3.3% of the enoxaparin group versus 3.0% of the dabigatran 220mg group (RD vs. enoxaparin -0.2%, 95% CI -1.3% to 0.9%, I2=37%) and 3.8% of the 150mg group (RD vs. enoxaparin 0.5%, -0.6% to 1.6%, I2=0%). Major bleeding occurred in 1.4% of the enoxaparin group versus 1.4% of the dabigatran 220mg group (RD vs. enoxaparin -0.2%, -0.8% to 0.5%, I2=40%) and 1.1% of the 150mg group (RD vs. enoxaparin -0.4%, -1.0% to 0.2%, I2=0%).Conclusions: Oral dabigatran was as effective as subcutaneous enoxaparin in reducing the risk of major VTE and VTE-related mortality after hip or knee arthroplasty and has a similar bleeding profile.