Thrombosis Research - Articles in Press

Levels of fibrinogen and thromboelastometry fibrin polymerisation following treatment with desmopressin (DDAVP) - Corrected Proof

Benny Sørensen, Gary Moore, Gerald Hochleitner, Christian Fenger-Eriksen — 2012-02-01

Introduction. Patients who have received platelet inhibitory drugs may constitute a challenge in management of perioperative bleeding . Administration of DDAVP has been proposed to be effective in partially restoring platelet function following aspirin or gpIIb/IIIa antagonists , and reduces transfusion needs after surgery . Thus, in many institutions, DDAVP is part of the established algorithm for management of perioperative bleeding . Furthermore, an increasing number of institutions use thromboelastometry for guidance of transfusion and administration of haemostatic drugs . The importance of correcting fibrinogen and fibrin polymerisation in management of perioperative bleeding has recently been emphasized by a series of experimental and clinical studies . It may be speculated, that DDAVP influences levels of fibrinogen and fibrin polymerisation due to renal water retention. Thus, the aim of the present study was to investigate the effect of DDAVP on levels of fibrinogen and fibrin polymerisation. We tested the null hypotheses that DDVAP do not change levels of fibrinogen nor fibrin polymerisation. Materials and methods. A total of 16 patients (11 Females, 5 males, mean age=36) referred to our Haemophlia Centre for investigation of primary haemostatic defects were enrolled in the study. All patients had received a test dose of DDAVP (Desmopressin, Octostim®, Ferring Pharmaceuticals Ltd, Berkshire, UK) 0.2μg/kg subcutaneous or intranasal. Blood samples (2.8 % citrate) were taken immediately, as well as 6 hours after DDAVP. Levels of fibrinogen [g/L] were measured by a photometric Clauss method using a Sysmex CA-1500 (TOA, Kobe, Japan). Platelet poor plasma was (PPP) diluted with Owrens and activated with bovine thrombin (SysmexFbg, TOA, Kobe, Japan). Furthermore, fibrin polymerisation was evaluated by ROTEM® Thromboelastometry, (TEM Innovations, Munich, Germany). Platelet poor plasma was transferred to a pre-warmed plastic cup containingcytochalasin D, a strong platelet inhibitor (FibTEM®, TEM Innovations), followed by activation with tissue factor (ExTEM®, TEM Innovations). Fibrin polymerisation was evaluated by recording maximum clot firmness (MCF, [mm]). Based on pilot data a sample size of 16 pair would have a 90 % power to detect a difference between FibTEM means of 1.43mm or more with a significance level (alpha) of 0.05 (two-tailed). Recorded data did not follow a Gaussian distribution as evaluated by Kolmogorov-Smirnov test, thus data were compared using a non-parametric Wilcoxon matched-pairs signed-ranks test. A two-tailed P value less than 0.05 was considered statistical significant. Results: Levels of fibrinogen changed significantly from median=2.30g/L (range=1.69-3.95) before DDAVP to median=2.06g/L (range=1.61-3.22) after DDAVP, p=0.0327. The median difference was 0.14g/L (95%CI: -0.08 - 0.43), corresponding a relative change of 6 %. A total of 11 patients had a drop in their level of fibrinogen (changes ranged from −0.01 to −1.18g/L), whereas 2 had an increase (0.08 and 1.12g/L) and 3 showed no change. However, fibrin polymerisation as evaluated by the MCF value of the FibTEMPPP assay showed no significant change. The MCF of the FibTEMPPP before DDAVP was median=25mm (range=14-28) as compared to a MCF of median=22mm (range=16-30) after DDAVP, p=0.1763. The median difference was 1.0mm (95%CI: -0.4 - 3.0), corresponding a relative change of 4 %. A total of 8 patients revealed a reduction in their MCF (changes ranged from −1 to −7mm), whereas 4 patients had an increase (changes ranged from 1 to 3mm) and 3 showed no change (measurements failed in 1 patient due to technical problems). Discussion. Data showed that fibrinogen decreases following administration of DDAVP. However, fibrin polymerisation revealed no statistical relevant changes. Historically, the threshold level of fibrinogen has been 1g/L. However, the critical threshold levels of fibrinogen are currently being challenged, thus depending on the clinical scenario, the suggested new threshold levels may be much higher than 1g/L . Recently, thromboelastometry algorithms have been used to guide haemostatic intervention in the perioperative setting. In these guidelines, intervention with fibrinogen is suggested if the FibTEM is ≤5-7mm . Based on our study it seems unlikely that treatment with DDAVP will have major clinical interference with levels of fibrinogen or fibrin polymerisation. The addition of Cytochalasin D to PPP avoided the bias DDAVP could have caused by increasing the MCF via improvement of platelet function. From a diagnostic point of view, the individual changes in levels of fibrinogen and MCF seems negligible in most cases. However, it should be noted that bleeding patients often develop a low level of fibrinogen and MCF, thus if DDAVP is administered to a patient close to the threshold levels of fibrinogen and/or MCF, this may be clinically important.

Continuous thrombin infusion leads to a bleeding phenotype in sheep - Corrected Proof

2012-01-30

Abstract: Background: In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We aimed to establish an ovine model to study thrombin effects in vivo.Methods: Thrombin (0.0004-0.42IU/kg/min) was continuously infused in Austrian Mountain Sheep over five hours in the dose escalation study (n=5 animals; 15 experiments). In the dose verification study animals received 0.42IU/kg/min of thrombin vs. saline solution in a cross-over design (n=3 animals; 7 experiments).Results: Thrombin at an infusion rate of 0.42IU/kg/min decreased fibrinogen levels by 75% (p<0.001) and increased degradation products of the fibrinogen beta-chain as shown in a proteomic analysis. Thrombin decreased platelet counts by 36% (p=0.006), prolonged thrombin time by 70% (p=0.012) and activated partial thromboplastin time by 32%. Interestingly, thrombin infusion significantly increased the activity of coagulation factors V and X (p<0.05) and decreased the activity of the coagulation factors VIII and XIII (p<0.05). Accordingly, thrombin displayed predominantly anti-coagulant and anti-platelet effects: 1) thrombin prolonged clotting time/clot formation time 7-fold (p=0.019) and induced a 65% decrease in maximal clot firmness (p<0.001); 2) thrombin reduced collagen- induced platelet aggregation by 85% and prolonged collagen/adenosine diphosphate closure time 3-fold; and 3) thrombin caused lung haemorrhage but not thromboembolism.Conclusion: Protracted intravenous infusion of thrombin over a period of five hours offers a new experimental model to study thrombin effects in a large animal species.

Factors associated with the failure of clopidogrel dose-adjustment according to platelet reactivity monitoring to optimize P2Y12-ADP receptor blockade - Corrected Proof

2012-01-30

Abstract: Introduction: Inter-individual variability in clopidogrel responsiveness is dependent on genetic polymorphisms. We aimed to investigate the impact of 3 genetic polymorphisms involved in clopidogrel metabolism on a strategy of dose-adjustment according to platelet reactivity (PR) monitoring.Materiel and methods: This prospective multicenter study enrolled 498 ACS patients undergoing PCI. PR was measured using the Vasodilator-Stimulated Phosphoprotein index (VASP) and a cut-off value of ≥50% defined high on-treatment platelet reactivity (HTPR). Genetic polymorphisms of cytochrome (CYP) 2C19, Paraxonase-1 (PON1) and ABCB1 were determined by allele specific PCR. Dose-adjustment was performed using up-to 3 additional loading doses (LD) of 600mg clopidogrel in order to obtain a VASP <50% in patients with HTPR following the first LD.Results: CYP 2C19 2*polymorphism (p=0.02), but neither PON1 (p=0.8) nor ABCB1 genotype (p=0.9), was significantly associated with HTPR. The dose-adjustment strategy failed in 11% of patients. ABCB1 polymorphism was significantly associated with a failed dose-adjustment (FDA) (p=0.04). No relation was found between the other genotypes and the efficacy of LD adjustment. In multivariate analysis, BMI and ABCB1 polymorphism were the only factors significantly associated with FDA (p=0.005 and p=0.04 respectively).Conclusion: While CYP 2C19 2* is associated with HTPR after 600mg of clopidogrel, ABCB1 is responsible for the failure of a strategy of loading dose-adjustment according to PR monitoring. These findings may help to define a therapeutic strategy to optimize anti-platelet therapy in ACS patients undergoing PCI.

Incomplete resolution of deep vein thrombosis under reduced flow conditions - Corrected Proof

Brian C. Cooley, Chao-Ying Chen, Ryan Hess, Gregory Schmeling — 2012-01-30

Abstract: Introduction: Post-thrombotic syndrome is a major complication of deep vein thrombosis (DVT), occurring in up to 1/3 of first-episode DVT patients. Non-ambulatory patients have increased risk for both DVT and post-thrombotic syndrome. Experimental models are lacking that can serve as reasonable in vivo clinical analogues for poor resolution of DVT that can lead to post-thrombotic syndrome.Materials and methods: A murine model of combined DVT and reduced flow was developed that results in persistent vein wall remodeling of poorly resolved thrombus. An electrolytic-injury model of venous thrombosis was created in the femoral veins of adult CD-1 mice, either with or without upstream flow reduction (10% of normal flow), with subsequent histomorphometric and immunohistochemical evaluation out to 28days.Results: Most venous thrombi with normal flow resolved, with little evidence of thrombus or vein wall changes 4 or more days after thrombus induction. In contrast, reduced flow had a prolonging effect on thrombus presence, resulting in long-term remodeling of the thrombus and vein wall, persistent out to 28days. There was little evidence of monocyte or neutrophil infiltration in remodeled tissue, with only partial smooth muscle cell phenotypic presence, suggesting a fibrotic nature of the residual thrombus.Conclusions: Flow reduction inhibits thrombotic resolution in veins with resultant long-term thrombus presence and subsequent vein wall remodeling. This model may offer clinical analogy to unresolved DVT that leads to post-thrombotic syndrome.

Prospective Study of the Frequency and Outcomes of Patients with Suspected Pulmonary Embolism Administered Heparin Prior to Confirmatory Imaging - Corrected Proof

Jeffrey A. Kline, Michael R. Marchick, Christopher Kabrhel, D. Mark Courtney — 2012-01-30

Abstract: Objectives: The administration of empiric systemic anticoagulation (ESA) before confirmatory radiographic testing in patients with suspected pulmonary embolism (PE) may improve outcomes, but no data have been published regarding current practice. We describe the use of ESA in a large prospective cohort of emergency department (ED) patients and report the outcomes of those treated with ESA compared with patients not receiving ESA.Methods: 12-center, noninterventional study of ED patients who presented with symptoms concerning for PE. Clinical data including pretest probability and decision to start ESA were recorded at point of care by attending physicians. Patients were followed for adverse in-hospital outcomes and recurrence of venous thromboembolism.Results: ESA was initiated 342/7932 (4.3%) of enrolled patients, including 142/618 (23%) patients with high pretest probability. Patients receiving ESA had more abnormal vital signs and were more likely to have a history of venous thromboembolism than those who did not receive ESA. Overall, 481/7,932 (6.1%) had PE diagnosed, 72/481 (15.0%) with PE had ESA, and 72/342 (21%) of ESA patients had PE. Three patients (0.9%, 95%CI: 0.2-2.5%) who received ESA suffered hemorrhagic complications compared with 38 patients (0.5%, 95%CI: 0.4-0.7%) who did not receive ESA.Conclusions: In this multicenter sample, ED physicians administered ESA to a small, generally more acutely ill subset of patients with high pretest probability of PE, and very few had hemorrhagic complications. ESA was not associated with any clear difference in outcomes. More study is needed to clarify the risk versus benefit of ESA.

The role of thrombin receptors PAR1 and PAR4 for PAI-1 storage, synthesis and secretion by human platelets - Corrected Proof

2012-01-27

Abstract: Introduction: Arterial thrombi contain more platelets than venous thrombi and are more resistant to fibrinolysis. This resistance could partly be due to plasminogen activator inhibitor 1 (PAI-1) secreted by platelets. The aim of this study was to elucidate differences between thrombin receptors protease-activated receptor (PAR) 1 and 4 and platelet storage, secretion and synthesis of platelet PAI-1, as compared to other platelet α-granule proteins such as VEGF and endostatin.Materials and methods: Human isolated platelets were incubated with thrombin (0.5U/ml), PAR1-activating peptide (AP) (0.4-30μM) or PAR4-AP (1.5-300μM) for up to 24hours. ELISA, western blot and fluorescence microscopy were used to measure secretion, contents and localization of PAI-1, VEGF and endostatin.Results: Our results show that PAI-1 and VEGF might be co-localized and that endostatin does not co-localize with either PAI-1 or VEGF. PAI-1 and VEGF show a similar secretion pattern, being more sensitive to low grade PAR1 activation, but secretion was also observed with higher concentrations of PAR4-APs. PAI-1 is secreted in an active form. PAI-1 mRNA was found in platelets, and elevated levels of PAI-1 were detected after 24hours incubation of platelets.Conclusions: PAI-1 and VEGF, but not endostatin, might be stored in the same α-granule in human platelets. PAI-1 and VEGF also show a similar secretion pattern, being more sensitive to PAR1 than to PAR4 activation, but the secretion is not exclusively selective. Our results also show that platelet PAI-1 is increased if incubated for 24hours, both with addition of PAR1-activating peptide and without activation, which could indicate de novo synthesis.

Thrombosis and Acute coronary syndrome - Corrected Proof

Rosanna Abbate, Gabriele Cioni, Ilaria Ricci, Marco Miranda, Anna Maria Gori — 2012-01-27

Abstract: Acute coronary syndromes (ACS) represent the main clinical manifestation of atherosclerotic progression in the coronary district. Thrombosis plays a critical role in the patho-anatomical of ACS, as disruption of an atherosclerotic plaque exposes flowing blood to subendothelial collagen, tissue factor, and other procoagulant molecules that trigger activation of platelets and formation of fibrin within the vessel lumen.Endothelial damage/dysfunction, inflammation and coagulation are closely related to the pathophysiology of ACS and may be inter-related.Platelets play key roles in both the formation of the atheromatous plaque and clinical presentation of acute atherothrombotic events following plaque rupture. In the pathogenesis of the ACS, blood clotting activation has a crucial role and thrombin generation and TF may represent useful markers for the identification of patients at high risk of vascular events. Lipoprotein-associated phospholipase A2 (Lp-PLA2) represents the crossroads between lipid metabolism and inflammatory response.

Evaluation of multiple electrode aggregometry in whole blood using Multiplate® Mini Test cells - Corrected Proof

Kurtis R. Lee, Veerle J.E. Verheyden, Andrew D. Mumford — 2012-01-27

Abstract: Multiple electrode aggregometry (MEA) enables rapid platelet function testing in whole blood using 600 μL disposable standard test cells (STC). However, newly available 350μL mini test cells (MTC) could potentially be advantageous in some clinical settings where sample volume is limiting. In order to evaluate the diagnostic performance of MTC, we have estimated assay imprecision, correlation and agreement between area under curve (AUC) determined using MTC and STC in whole blood from healthy donors and from 119 cardiac surgery patients. Imprecision was similar with ADP, AA and TRAP test reagents using STC and MTC, but was markedly higher with the unvalidated ADR reagent. AUC determined using MTC and STC and the ADP, AA and TRAP reagents correlated strongly although MTC yield consistently lower AUC values reflecting fewer platelets in the smaller test cell. Agreement between AUC from STC and MTC was less strong, probably reflecting a composite effect of imprecision from both assay formats. MTC and STC are equally valid for MEA but AUC values obtained using one test format cannot be directly transformed to the other. Therefore, STC and MTC cannot be used interchangeably and AUC results must be compared to separately determined reference intervals.

Proteomics: Bases for protein complexity understanding - Corrected Proof

2012-01-27

Abstract: In the post genomic era we became aware that the genomic sequence and protein functions cannot be correlated. One gene can encode multiple protein functions mainly because of mRNA splice variants, post translational modifications (PTM) and moonlighting functions. To study the whole population of proteins present in a cell to a specific time point and under defined conditions it is necessary to investigate the proteome. Comprehensive analysis of the proteome requires the use of emerging high technologies because of the complexity and wide dynamic range of protein concentrations. Proteomics provides the tools to study protein identification and quantitation, protein-protein interactions, protein modifications and localization. The most widespread strategy for studying global protein expression employs two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) allowing thousands of proteins to be resolved and their expression quantified. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has emerged as a high throughput technique for protein identification and characterization because of its high sensitivity, precision and accuracy. LC-MS/MS is well suited for accurate quantitation of protein expression levels, post-translational modifications and comparative and absolute quantitative analysis of peptides. Bioinformatic tools are required to elaborate the growing number of proteomic data. Here, we give an overview of the current status of the wide range of technologies that define and characterize the modern proteomics.

Foreword - Corrected Proof

Giovanni de Gaetano, Chiara Cerletti — 2012-01-27

Bridging with the Low molecular weight heparin certoparin in patients requiring temporary discontinuation of oral anticoagulation – the non-interventional, retrospective REMEMBER study - Corrected Proof

Ralf Eisele, Nima Melzer, Cornelia Englert, Peter Bramlage, Michael Spannagl — 2012-01-27

Abstract: Background: Bridging of a temporal discontinuation of oral anticoagulation with low molecular weight heparins (LMWH) is common practice. Specific data on the LMWH certoparin are however limited despite its wide spread use. We aimed to assess effectiveness and safety of certoparin in this indication under real world conditions.Methods: Retrospective, non-controlled, non-interventional study at hospital- and office based physicians in Germany documenting 259 patients undergoing surgery (trauma, orthopaedic or general), endoscopy or a coronary intervention and who were receiving certoparin to bridge the temporal discontinuation of oral anticoagulation.Results: Patients had a mean age of 70.4±11.5years, a body weight of 81.9±16.2kg and 40.9% were female. Most received oral anticoagulation because of atrial fibrillation (57.5%) and most underwent surgery (55.6%). Oral anticoagulant treatment was discontinued a mean of 6.4±2.3days prior to the intervention and resumed 3.6±3.8days after the intervention. Certoparin, used in 74.9% of patients at a full therapeutic dose of 1x8,000IE, was started about 3days after VKA discontinuation when the mean INR was 1.7±0.4 and patients received the last dose of certoparin a mean of 16.2±7.1h before the intervention. It was resumed 12.2±7.7h after the intervention and stopped at day 7 when the INR had reached 2.2±0.4. No case of clinically overt deep vein thrombosis and no case of pulmonary embolism was reported. Seven patients (2.7%) had any minor and 4 (1.5%) any major bleeding complication.Conclusions: The analysis suggests that certoparin is safe and effective to bridge the temporal discontinuation of oral anticoagulation during surgery, endoscopy or a coronary intervention. It further confirms that current use patterns are appropriate in daily practice in Germany to ensure patient safety.

Effect of the Factor Xa Inhibitor Rivaroxaban on Arterial Thrombosis in Wild-Type and Apolipoprotein E-Deficient Mice - Corrected Proof

Nana-Maria Wagner, Tobias Dressel, Katrin Schäfer, Stavros Konstantinides — 2012-01-27

Abstract: Rivaroxaban is a potent and specific direct inhibitor of coagulation factor Xa. Recent studies have highlighted its effectiveness in the prevention of venous thrombosis and embolic stroke due to atrial fibrillation. To evaluate the antithrombotic effects of rivaroxaban in an in vivo model of arterial thrombosis, photochemical vascular injury was induced in wild-type mice by intravenous rose bengal (50mg/kg body weight [BW]) followed by illumination of the left common carotid artery using a 543nm helium-neon laser beam. Rivaroxaban, injected concomitantly with rose bengal at doses of 1.0, 1.5, 2.0, or 3.0mg/kg BW, dose-dependently prolonged the times to first thrombotic occlusion and stable thrombosis. Quantitative analysis of carotid flow curves revealed higher blood volumes passing through the injured artery with increasing rivaroxaban doses (P<0.01 and P<0.001 vs. vehicle for 2.0 and 3.0mg/kg , respectively), suggesting a dose-dependent effect on vascular patency. Consistently, a significantly higher proportion of mice that received 2.0 and 3.0mg/kg rivaroxaban exhibited patent carotid arteries at the end of the flow monitoring period compared to vehicle alone (P<0.05 and P<0.001, respectively). Histological analysis showed complete thrombotic arterial occlusion in vehicle-treated mice compared to less thrombotic material in mice injected with 3.0mg/kg rivaroxaban (P<0.05). Rivaroxaban also prolonged the time to cessation of tail bleeding in a dose-dependent manner, starting at 1.5mg/kg. Similar findings were obtained in apolipoprotein E-knockout mice. Rivaroxaban may exert beneficial effects by preventing arterial thrombosis and vascular occlusion after endothelial injury.

Podoplanin expression in advanced atherosclerotic lesions of human aortas - Corrected Proof

2012-01-27

Abstract: Thrombus formation on disrupted atherosclerotic lesion is a key mechanism of cardiovascular events. Podoplanin (Aggrus), expressed on the surface of several tumor cells, is an endogenous ligand for C-type lectin-like receptor 2 (CLEC-2), and is involved in tumor cell-induced platelet aggregation and its malignant potency. Podoplanin, which is also expressed in lymphatic endothelial cells, facilitates blood/lymphatic vessel separation. However, podoplanin expression in atherosclerotic lesion has not been investigated. To clarify podoplanin expression in atherosclerotic lesion and to assess its importance for the onset of cardiovascular events, we examined podoplanin expression in abdominal aortas obtained from 31 autopsy cases. Immunohistochemical analysis indicated that podoplanin was localized to smooth muscle cells and macrophages. Moreover, podoplanin immunoreactivity was increased in advanced atherosclerotic lesions containing necrotic core, many macrophages and smooth muscle cells, compared with early lesions composed of smooth muscle cells and small numbers of macrophages. Furthermore, Western-blot and real time-PCR analyses showed that podoplanin expression was significantly enhanced in advanced atherosclerotic lesions, compared with early lesions. These results suggest that podoplanin contributes to thrombotic property of advanced stages of atherosclerosis and that it might be a novel molecular target for an anti-thrombus drug.

Old and new oral anticoagulants for venous thromboembolism and atrial fibrillation: A review of the literature - Corrected Proof

Cecilia Becattini, Maria Cristina Vedovati, Giancarlo Agnelli — 2012-01-24

Abstract: Heparin, fondaparinux and vitamin K antagonists (VKAs) are effective for the prevention and treatment of venous thromboembolism. VKAs reduce by almost 60% the rate of cardioembolic complications in patients with atrial fibrillation. The risk for bleeding and the inconvenience for laboratory monitoring, dose adjustment and drug or food interactions are the main limits for VKAs while parenteral administration is the main limit for heparin and fondaparinux. New oral anticoagulants with more predictable anticoagulant response and no need for laboratory monitoring have been shown to be effective for the prevention and treatment of venous thromboembolism and for the prevention of stroke and systemic embolism in patients with atrial fibrillation. The pharmacokinetic and pharmacodynamic profile of the new agents differ for mechanisms of action – mainly anti Xa and one antithrombin agent- bioavailability, half life, renal or live clearance. Drug interactions have been described with the new agents and inhibitors or inducers of P-gp or CYP3A4. Overall, in the prevention of venous thromboembolism after major elective orthopaedic surgery dabigatran was shown to be non-inferior, rivaroxaban and apixaban to be superior to enoxaparin. Both, rivaroxaban and dabigatran were shown to be non-inferior to low-molecular weight heparin and VKAs for the treatment of venous thromboembolism. Dabigatran 150mg twice daily reduced the incidence of both ischemic and hemorrhagic stroke in patients with atrial fibrillation respect to warfarin. In these patients rivaroxaban and apixaban reduced the incidence of hemorrhagic stroke with a similar incidence of ischemic stroke. No bleeding concern emerged with the new anticoagulant agents in this indication.

Assessment of a Cohort of Primarily Pediatric Patients with a Presumptive Diagnosis of Type 1 von Willebrand Disease with a Novel High Shear Rate, Non-citrated Blood Flow Device - Corrected Proof

Eric F. Grabowski, Marjorie A. Curran, Elizabeth M. Van Cott — 2012-01-24

Abstract: Background: A precise approach to the diagnosis of von Willebrand disease (vWD) remains elusive. One important reason is that vWD is a blood flow‐related disorder: a vW Factor‐platelet GPIb binding defect exists in this condition under the high shear‐rate (>1000sec‐1 in whole blood; >3000sec‐1 in PRP) conditions of physiologic blood flow which exist in the arterioles of mucous membranes, from which most bleeding in vWD occurs.Methods: We therefore studied 28 patients (mean 18.9yrs) with vWD, diagnosed according to the 2007 NHLBI clinical guidelines, and 26 healthy controls (mean 17.5yrs). Blood was collected into a plastic tube containing 4 U/ml FC dalteparin, 1.75μg/ml of the Tab (anti‐CD41) monoclonal antibody directed against platelet GPIIb, and 1.0μg/ml of an ALEXA 555‐conjugated rabbit anti‐mouse second antibody. Within 30–90min, the blood was then withdrawn at 667 and 1330sec−1 through a special flow chamber allowing for real-time epifluorescence digital videomicroscopy of platelets interacting with a microfibrillar collagen substrate. With MetaMorph software (Universal Imaging) we quantified the percent area (PA) covered by and total volume (TV) of adherent platelet aggregates within a 435μm×580μm field of view.Results: At 667sec−1 after 1min PA and TV were similar for patients and controls, but at 1330sec−1 PA was 9.32±4.21 (mean±SD) for patients, a value lower (p<0.001) than the 12.8±3.39 for controls. TV was (1.43±0.91)x104 for patients, a value also lower (p<0.001) than the (2.22±0.77)x104 for controls. PA or TV was below the 2.5th percentile for controls in 10 patients (36%) and both PA and TV were below the 2.5th percentile in eight.Conclusions: The novel flow device found that PA and TV were significantly reduced under high shear stress in vWD patients compared to normal controls. However, there was some overlap between the vWD and the control group, suggesting that some vWD patients had normal platelet adhesion/aggregation under the conditions studied. Further study with a higher shear rate appears indicated.

Thrombosis and cancer: 40years of research - Corrected Proof

Maria Benedetta Donati, Roberto Lorenzet — 2012-01-24

Abstract: We have taken here the task to go back to a brief history concerning the evolution of the concept of “cancer and thrombosis” according to the experience accumulated by our group in the last 40years. Since its first description by Armand Trousseau in 1865, the association between cancer and thrombosis only received attention again during the last decades of the XXth century: from scattered reports on experimental material (tumor extracts) or on animal models of tumor/metastasis growth, through the progress of cell biology and experimental pharmacology, the interest moved to clinical questions, such as: how to prevent and treat thrombosis, a frequent complication of both solid and hematologic malignancies? Has an occult cancer to be suspected in the majority of cases of idiopathic deep vein thrombosis? Do we need to prevent pharmacologically the occurrence of chemotherapy-associated thrombosis? Do anticoagulants have an impact on the natural history of some tumors? Why antiangiogenetic agents may be associated to a thrombotic risk? Presently, a continuous cross-talk between clinical results and experimental data is required to provide answers to these questions, taking advantage from a multidisciplinary approach to this still partially mysterious issue. If one would take a paradigm of the evolution of the subject during the past 40years, a symbol could be the knowledge accumulated on tissue factor, the “middleman” of the clotting cascade as well as of the interactions between thrombosis and cancer, as briefly reviewed at the end of this chapter.

Ticagrelor, but not clopidogrel and prasugrel, prevents ADP-induced vascular smooth muscle cell contraction: A placebo-controlled study in rats - Corrected Proof

2012-01-24

Abstract: Introduction: Off-target effects of novel antiplatelet agents due to their potential clinical benefits are currently an area of intensive investigation. We aimed to compare the effects of different P2Y12 antagonists on the reactivity of vascular smooth muscle cells.Materials and methods: Wistar rats (n=30) were pretreated with an investigated drug or placebo. Clopidogrel (50mg/kg, n=7), prasugrel (10mg/kg, n=7), ticagrelor (10mg/kg, n=7) or placebo (n=9) were administered orally 12 and 2hours before experiments. Constrictions of rat tail arteries induced with a stable analogue of adenosine diphosphate (2-MeS-ADP), phenylephrine and arginine vasopressin were measured as an increase in perfusion pressure. Effects of ticagrelor were assessed in the presence of ticagrelor (1μM/L) added to the perfusion solution as this drug reversibly inhibits the P2Y12 receptor.Results: Pretreatment with clopidogrel and prasugrel did not inhibit 2-MeS-ADP-induced contraction while ticagrelor did. Experiments employing endothelium-deprived arteries provided similar results. Clopidogrel and prasugrel did not influence concentration-response curves in the presence of neither phenylephrine nor arginine vasopressin. The curves obtained for both vasopressors in the presence of ticagrelor and 2-MeS-ADP were shifted to the right with a significant reduction in the maximal response.Conclusions: Oral administration of ticagrelor, in contrast to clopidogrel and prasugrel, prevents adenosine diphosphate-induced contraction of vascular smooth muscle cells in a rat model. Both the clinical significance and detailed mechanism of our findings warrant further investigation.

Bivalirudin is inferior to heparin in preservation of intraoperative autologous blood - Corrected Proof

Rolf C.G. Gallandat Huet, Vladimir Cernak, Adrianus J. de Vries, Ton Lisman — 2012-01-20

Abstract: Introduction: Bivalirudin is used as an alternative to heparin in cardiac surgery, and may be superior to heparin with regard to platelet function. Bivalirudin however, is prone to cleavage by thrombin resulting in coagulation in areas of stasis.Material and Methods: We compared the preservation of platelet function and the quality of anticoagulation in autologous blood of 26 cardiac surgical patients collected intraoperatively and anticoagulated ex vivo with either bivalirudin or heparin, with supplementation of bivalirudin over time and prevention of stasis.Results: We found in both preservatives a reduction in ADP-induced platelet aggregation response over a period of 105 minutes (median, IQR: 73–141) as measured by Multiplate®. Supplementation of additional bivalirudin (23±1.1μg/ml/hr) and prevention of stasis was not able to prevent thrombin generation. We found a 5-fold increase in levels of prothrombin fragment 1+2 in bivalirudin preserved autologous blood as compared to heparin preserved blood (F1+2 levels median 8.9 nM [quartile percentiles 4.2-12.4] vs 1.3 nM [0.6-2.1], P=0.001 Mann–Whitney, n=10).Conclusions: Our study suggests that preservation of platelet function in autologous blood anticoagulated with bivalirudin is not a suitable alternative to heparin.

Sustained heparin effect contributes to reduced plasma thrombin generation capacity early after cardiac surgery - Corrected Proof

2012-01-20

Abstract: Introduction: Thrombin is a key component in the coagulation cascade, and impaired thrombin generation has been linked to increased bleeding after surgical procedures. The aim was to evaluate postoperative thrombin generation capacity in plasma after cardiac surgery, and its potential associations to activity of individual coagulation factors and heparin.Material and Methods: Forty-eight coronary artery bypass grafting patients were included in a prospective observational cohort study. Thrombin generation capacity was analysed in plasma with calibrated automated thrombogram with tissue factor as activator before (baseline), and 2h and 24h after surgery. In addition, plasma activity of coagulation factors II, V, VII, VIII, IX, X, XI, XIII, were determined. Heparin effect was assessed by anti-Xa activity, APTT and thrombin time.Results: Thrombin generation was markedly reduced 2h after surgery compared to baseline. Peak levels decreased with median 74% (interquartile range 52–90), p<0.001, and endogenous thrombin generation potential decreased with 65% (43–86), p<0.001. Postoperative changes in endogenous thrombin generation potential correlated inversely to changes in anti-Xa activity (r=−0.51, p=0.010) and to changes in thrombin time (r=−0.51, p=0.009), but there were no correlations to changes in individual coagulation factor activity.Conclusions: A marked reduction in thrombin generation potential was observed in the early postoperative phase after cardiac surgery. The decrease was independent of reductions in individual coagulation factor activity but correlated to heparin effects. The results indicate that a sustained heparin effect contributes to the postoperative reduction in thrombin generation capacity.

Novel management of anticoagulant resistant thrombotic hypodysfibrinogenaemia - Corrected Proof

C. Rea, B.J. Hunt — 2012-01-20

Abstract: Individuals with hypodysfibrinogenaemia and recurrent thrombosis are rarely encountered and there is no consensus regarding long-term management. Guidelines available suggest that oral anticoagulants should be sufficient treatment, but when this fails to control thrombotic disease no further management strategies are reported. A novel approach to treatment has been developed and used for two individuals who experienced life-threatening thrombosis despite anticoagulation adherent to current guidelines. These two affected individuals consented to receive infusions of exogenous fibrinogen concentrate thrice weekly in addition to continuing warfarin (target INR 3–4). Both have been thrombosis free 36 and 18months after starting this ongoing regime. This study suggests regular transfusions of fibrinogen concentrate may be a suitable treatment for anticoagulant resistant thrombotic hypodysfibrinogenaemia, but further research is required.

Mean platelet volume increases with aging in a large population study - Corrected Proof

Giuseppe Lippi, Tiziana Meschi, Loris Borghi — 2012-01-20

We read with interest the article of Demirin et al., who investigated the mean platelet volume (MPV) in a cohort of 326 healthy subjects by using a CELL-DYN 3700 SL analyzer (Abbott Diagnostics) . It was thereby concluded that no significant difference could be observed among age groups, and the mean MPV of the cohort was 8.9fL (95% confidence interval [95% CI] 7.2-11.7fL). In a very recent article, Tan et al. highlighted that although nearly all complete blood count parameters show excellent correlation coefficients when comparing different hematological analyzers, MPV values were substantially different using the Abbott Sapphire , which shares rather similar features with the CELL-DYN 3700. As such, we assessed the values of MPV on a large cohort of outpatients by employing the ADVIA 2120 hematological analyzer (Bayer Diagnostics, Newbury, Berkshire, UK).

Development and application of an automated chromogenic thrombin generation assay that is sensitive to defects in the protein C pathway - Corrected Proof

2012-01-20

Abstract: Background: Activated protein C resistance (APCR) within a thrombin generation test (TGT) system is associated with an increased risk of venous thromboembolism (VTE). However, application of the TGT is restricted by the analytical platforms used to monitor thrombin generation. Using a routine coagulation analyser we have developed an automated chromogenic TGT that is sensitive to defects in the protein C pathway.Method: The TGT was performed on a TOP500 analyser, in the presence and absence of Protac®. The reaction was monitored using a substrate with slow kinetics for thrombin (S-2444). Results were expressed as the area under the curve normalised ratio (AUCnr). Assay results were compared with Coatest APCR (expressed as APC-ratio [CoAPCr]).Patients: Samples were obtained from 35 healthy subjects and 91 patients with previous history of VTE. Of these patients, 19, 17, and 9 had heterozygous factor V Leiden (FVL), antiphospholipid syndrome (APS), and protein C/protein S deficiencies (PC/PS) respectively.Results: Inter-assay imprecision in the presence and absence of Protac® were 20% and <5% respectively. There was a significant difference between the AUCnr of normals (median [IQR]: 2.8 [2.4-4.7]) compared to: FVL (1.0 [0.7-1.2]); PC/PS (1.1 [0.9-1.2]); and APS (1.1 [0.8-1.4]); p<0.001 for each comparison. No significant difference was seen between the AUCnr of normals and other VTE patients. The detection rate of AUCnr and CoAPCr were: 100% and 56% for FVL; 88% and 44% for PC/PS; and 64% and 45% for APS respectively.Conclusion: The automated TGT exhibited good sensitivity to defects in the protein C pathway.

Association between adiponectin gene polymorphisms and coronary artery disease across different populations - Corrected Proof

2012-01-19

Abstract: Objective: Many studies have suggested that adiponectin gene might be involved in the development of coronary artery disease (CAD). However, the results have been inconsistent. In this study, the authors performed a meta-analysis to assess the associations of +45T/G, +276G/T and −11377C/G polymorphisms in adiponectin gene with CAD susceptibility.Methods: Published literature from PubMed and EMBASE databases were searched. Pooled odds ratio (OR) and corresponding 95% confidence interval (CI) were calculated using fixed- or random-effects model.Results: Sixteen studies (4394 cases / 8187 controls) for +45T/G polymorphism, fifteen studies (3569 cases / 7463 controls) for +276G/T polymorphism, and thirteen studies (3531 cases / 7072 controls) for −11377C/G polymorphism were included in the meta-analysis. The overall results showed that there was a statistically significant association between −11377C/G polymorphism and CAD (G vs. C: OR=1.15, 95%CI 1.07-1.24).Similar results were observed among European (G vs. C: OR=1.11, 95%CI 1.02-1.20) and East Asian populations (G vs. C: OR=1.27, 95%CI 1.11-1.45). However, no significant association was found for +45T/G or +276G/T polymorphism with CAD susceptibility.Conclusions: The meta-analysis indicated the significant association of −11377C/G polymorphism, but not +45T/G or +276G/T polymorphism, with CAD susceptibility. However, large-scale studies with the consideration of gene-gene and gene-environment interactions should be conducted to investigate the associations in future.

A case series of five episodes of massive LMWH non-fatal self-induced overdose in a single patient - Corrected Proof

Jacob Odeberg, Anders Carlsson, Caroline Hällsjö-Sander, Anna Ågren — 2012-01-19

Low molecular weight heparins (LMWH) are frequently used for the prophylaxis and treatment of venous thromboembolic diseases (VTE). Their subcutaneous administration in fixed doses has made possible the treatment of thromboembolic diseases in outpatient settings. Self-induced massive overdoses have recently been described in case reports, with various treatments and outcomes . Thus, the management of such episodes is not clarified. Protamine is recommended by the American College of Chest Physicians for the reversal of LMWH in about the same doses that is established for reversal of the effect of heparin (unfractioned heparin, UFH). However, in contrast to heparin, LMWH is only partly neutralized by protamine . Furthermore, in massive overdose, the amounts of protamine needed to neutralize LMWH would far exceed 50mg, which is the maximal single dose recommended . In a recent case report on an overdose of 212,500IU of the LMWH dalteparin a bolus dose of 500mg reversed the LMWH effect only partially and transiently (measured as normalized APTT and anti-Xa) . However, no bleeding complication was noted, in accordance with other reports . This may indicate that a conservative approach in the management of LMWH overdose might be appropriate in patients without symptoms. Such an approach is supported by our present report describing very large overdoses of dalteparin in a single patient. However, our report also demonstrates that the presence of trauma or a locus minoris for bleeding should warrant a more active management.

The SISET incorrectly cited the JAAM DIC scoring system - Corrected Proof

2012-01-19

Dr Gando suggests to replace the JAAM score as currently reported in Table 1 of the Guidelines of the Italian Society for Haemostasis and Thrombosis (SISET) on the diagnosis and treatment of disseminated intravascular coagulation (DIC) with the revised version of the same score whose prognostic value has been evaluated in two publications .

Effect of Hawthorn (Crataegus aronia syn. Azarolus (L)) on Platelet Function in Albino Wistar Rats - Corrected Proof

2012-01-19

Abstract: Objective: This study was designed to investigate the possible antiplatelet effect of aqueous whole-plant C. aronia syn: Azarolus (L) extract using Wistar albino rats as a model.Materials and methods: Forty-two male albino Wistar rats weighing 200 to 250g were divided into seven groups with six rats in each group. Group 1 served as the control and received equal volumes of distilled water. Groups 2–6 served as the experimental groups and were given C. aronia extract at doses of 100, 200, 500, 1,000, and 2,000mg/kg, while group 7 served as a positive control and was given aspirin (25mg/kg). All the doses were administered orally once a day and the treatment was continued for seven days. In all groups, at the end of the experimental procedure, blood samples were obtained for platelet function measurements, including PFA-100, thromboxane B2 levels, platelet count, and haematocrit. The bleeding time was determined using a modified tail cutting method described previously.Results: The aqueous C. aronia syn. Azarolus (L) extract significantly altered the bleeding time and the closure time, as determined by the PFA-100 and thromboxane B2 levels, suggesting significant platelet function inhibition. These effects were observed with C. aronia doses between 100 – 500mg/kg, which yielded thromboxane B2 levels of 1,000mg/kg, whereas the higher dose (2,000mg/kg) produced opposite effects on these parameters.Conclusion: C. aronia syn. Azarolus (L) aqueous extract has antiplatelet effects in Wistar albino rats.

Platelet-leukocyte deregulated interactions foster sterile inflammation and tissue damage in immune-mediated vessel diseases - Corrected Proof

2012-01-16

Summary: Platelets and leukocytes co-localize and interact at sites of vessel injury, haemorrhage, thrombosis and inflammation. Recent studies have highlighted the role of local cues in the interaction between the two cell populations, including the exposure of anionic phospholipids and the release of Damage Associated Molecular Patterns (DAMPs) by activated platelets, the release of the prototypical tissue pentraxin PTX3 by neutrophils, as well as the generation of polarized clusters of neutrophil ß2 integrins. In turn, the reciprocal activatory cross-talk between platelets and leukocytes contributes to the generation of thrombo-inflammatory lesions and of vascular injury. Here we will discuss the implications of these results for the pathogenesis and the clinical features of self-sustaining immune-mediated vessel diseases.

PDE4 Regulates Tissue Plasminogen Activator Expression of Human Brain Microvascular Endothelial Cells - Corrected Proof

2012-01-16

Abstract: Introduction: Factors regulating brain tissue plasminogen activator (tPA) are pertinent for stroke. Recent observations have suggested a role for the phosphodiesterase-4 (PDE4) pathway in stroke pathogenesis, via an uncertain mechanism. We studied PDE4 regulation of tPA expression by human brain microvascular endothelial cells in a variety of conditions, including an in vitro model of ischemia.Materials and Methods: We analyzed tPA antigen and mRNA of human brain microvascular endothelial cells (HBECs) during normoxia and oxygen-glucose deprivation (OGD) following inhibition of PDE4 and PDE4D, using HBEC monocultures and co-cultures with astrocytes and pericytes, and analyzed relevant signal transduction pathways.Results: PDE4 inhibitor rolipram enhanced OGD effects on endothelial tPA release in endothelial monocultures and co-cultures with astrocytes; there was a 54±10% (p<0.001) reduction of tPA release in astrocyte-endothelial co-cultures under OGD. PDE4D siRNA reduced endothelial tPA mRNA to 40-55% of control (p<0.05). Use of Epac inducer mimicked, while use of Epac siRNA inhibited, these effects.Conclusions: Inhibition of PDE4 and PDE4D reduced expression of tPA by HBEC via Epac pathway.

Evaluation of the Stratus® CS Acute Care™ D-dimer assay (DDMR) using the Stratus® CS STAT Fluorometric Analyzer: A prospective multisite study for exclusion of pulmonary embolism and deep vein thrombosis - Corrected Proof

2012-01-16

Abstract: Background: D-dimer testing is an integral part of the diagnostic algorithm in excluding patients with venous thromboembolism. In this study, we prospectively evaluated the Stratus DDMR D-dimer test in patients suspected of pulmonary embolism (PE) and deep vein thrombosis (DVT).Methods: Patients suspected of venous thromboembolism were prospectively enrolled at four different clinical sites, with sodium citrate and lithium heparin plasma was tested using the DDMR D-dimer test on the Stratus CS analyzer.Results: 1,012 patients were enrolled for analysis, with 85/603 (14.1%) patients with PE and 80/443 (18.1%) with DVT, and four of the patients (0.4%) with PE and DVT. For the samples collected in 3.2% sodium citrate, the DDMR method had a sensitivity, specificity, and negative predictive value for VTE of 98.0%, 38.1%, and 99.1%, respectively. For the samples collected in lithium heparin, the DDMR method had a sensitivity, specificity, and negative predictive value (NPV) for VTE of 98.9%, 28.8%, and 99.4%, respectively. In PE, DDMR testing on citrate plasma had a sensitivity, specificity, and NPV of 98.8%, 39.5%, and 99.6%, respectively, while heparin samples had a sensitivity, specificity, and NPV for PE of 98.0%, 28.4%, and 99.1%, respectively. In DVT, citrate plasma had a sensitivity, specificity, and NPV for DVT of 97.5%, 32.0%, and 98.3%, respectively, while heparin samples had a sensitivity, specificity, and NPV for DVT of 100%, 27.8%, and 100%, respectively.Conclusion: The Stratus CS DDMR D-dimer can be used in those patients with non-high clinical pre-test probability for the exclusion of PE.

Characteristics of Fibrin Formation and Clot Stability in Individuals with Congenital Type IIb Protein C Deficiency - Corrected Proof

J.H. Foley, L. Ferris, K.E. Brummel-Ziedins — 2012-01-16

Abstract: Many studies have shown that unregulated or excessive thrombin formation is potentially a cause of thrombosis; however, studies examining processes that contribute to fibrin stabilization in individuals predisposed to thrombosis are limited. In this study, we investigate a family with familial thrombosis via type IIb protein C (PC) deficiency. Using contact pathway inhibited whole blood, thrombin generation, fibrin clot formation and factor (f)XIII activation were monitored over time in control (n=5) and PC deficient (n=4, 34 – 69% PC by activity) subjects. The dynamics of thrombin generation varied significantly with the time required to reach the maximal rate of thrombin-antithrombin formation being much shorter in PC deficiency (5.8±0.4 minutes) than in controls (9.7±0.4 minutes; p<0.001). PC deficient clots were significantly heavier than control clots (p<0.001) and this difference could not be contributed exclusively to differences in fibrinogen levels between groups. FXIII was consumed faster in PC deficient subjects (23.7±2.0 nM/minute) than in controls (5.1±1.5 nM/minute; p<0.0001) suggesting increased fXIII activation and incorporation of fXIIIa substrates into the clot. In plasma, the clot lysis time was increased in PC deficiency by both TAFIa dependent and independent mechanisms. Since PC deficient clots are both denser and show a greater degree of resistance to fibrinolysis, these clots would likely resist fibrinolysis and potentiate fibrin deposition observed in thrombosis.

The effect of total hip/knee replacement surgery and prophylactic dabigatran on thrombin generation and coagulation parameters - Corrected Proof

2012-01-16

Abstract: Introduction: Total hip/knee replacement surgery (THR/TKR respectively) is associated with an increased risk of venous thromboembolism. Dabigatran is recommended as a thromboprophylactic agent post orthopaedic surgery. The aim of this study was to assess the post-operative (Day-1 and Day-2) effect of prophylactic Dabigatran on: the thrombin generation (TG) assay; prothrombin fragment 1.2 (F1.2); thrombin-antithrombin complexes (TAT); D-dimer (D-D); and other coagulation parameters.Methods and samples: Nineteen patients (12 THR, 7 TKR) who received 110mg dabigatran 4hours post-operatively, then 220mg the following day, were recruited. Blood was collected: pre-operatively (Pre-); peri-operatively (Peri-); 19hours after 110mg dabigatran (Day-1); and 17hours after 220mg dabigatran (Day-2). The TG assay was measured using the Calibrated Automated Thrombogram and a low concentration of tissue factor. Other coagulation parameters measured included activated partial thromboplastin time (APTT), thrombin-time (TT), ecarin-clotting time (ECT) and Hemoclot tests.Results: From Pre- to Peri-, ETP/peak-thrombin, F1.2, TAT and D-D increased significantly. From Peri- to Day-1 and Day-2: TAT reduced progressively; D-D increased; F1.2 did not change significantly; lag-time and time-to-peak prolonged; ETP/Peak-thrombin increased spuriously, due to Dabigatran interfering with the α-2 macroglobulin:thrombin complex in the TG assay. APTT, TT, ECT and Hemoclot increased progressively post-operatively; good correlations were seen between these tests.Conclusion: The effect of dabigatran on the TG assay, showed a spurious increase in ETP and Peak-thrombin due to its interference with the TG assay. Dabigatran reduced TAT, but not F1.2, suggesting that thrombin was still being generated after surgery, but was blocked by Dabigatran.

Interleukin-10 promoter microsatellite polymorphisms influence the immune response to heparin and the risk of heparin-induced thrombocytopenia - Corrected Proof

2012-01-13

Abstract: Introduction: Heparin-induced thrombocytopenia (HIT) results from an atypical immune response with synthesis of IgG antibodies (Abs) to platelet factor 4/heparin complexes (PF4/H), and probably involves both B and T cells. We investigated whether 3 single nucleotide polymorphisms (SNPs), rs1800896 (−1082G/A), rs1800871 (−819C/T) and rs1800872 (−592C/A) and the polymorphic CA repeat microsatellites IL10R [5325CA(11_15)] and IL10G [8134CA(14_29)] are associated with the synthesis of Abs to PF4/heparin and HIT.Materials and methods: Eighty-two patients with definite HIT and two control groups were studied. The first control group (Abneg) consisted of 85 patients without Abs to PF4/heparin after cardiopulmonary bypass (CPB). The second control group (Abpos) consisted of 84 patients who had developed significant levels of PF4-specific antibodies after CPB, but without HIT.Results: Allele frequencies of the 3 SNPs were similar in HIT patients and controls. Fourteen alleles in IL10G (G16 to G29) and 3 alleles in IL10R (R13 to R15) were defined. The short G20 allele of IL10G was more frequent in Abneg patients (8.2%) than in Abpos (2.9%) and HIT patients (3%). It thereby appeared to protect against developing Abs to PF4/heparin (OR 0.29; 95% CI [0.12-0.70], p=0.006). Combined haplotypes cH1/cH8 comprising the short G20+R13 alleles were less frequent in HIT (OR 0.33; 95% CI [0.11-0.97], p=0.036), and levels of Abs to PF4 in Abpos patients were lower in cH1/cH8 subjects (p=0.019).Conclusion: These results suggest that IL10 promoter microsatellite polymorphisms might influence the immune response against PF4/heparin and the risk of HIT.

Endothelial progenitor cells and thrombosis - Corrected Proof

Eugenia Rosa Nuzzolo, Maria Grazia Iachininoto, Luciana Teofili — 2012-01-12

Abstract: The remodelling of existing vessels (i.e. angiogenesis) and the “de novo” vessel formation (i.e. vasculogenesis) occur not only during the embryonic development but also over the entire postnatal life. In 1997, the Asahara group first reported that endothelial progenitor cells circulate in peripheral blood and that they are recruited at sites of ischemia, thus proving that these cells are able to promote vasculogenesis. Since then, several different approaches have been set up to investigate endothelial progenitor cells. This review summarizes the different modalities utilized to enumerate these cells, delineates their involvement in the haemostatic pathways, and depicts their altered trafficking during cardiovascular diseases. Finally, recent observations suggesting a primary role for endothelial progenitors in particular situations such as pulmonary arterial hypertension or Philadelphia negative-myeloproliferative neoplasms are discussed.

Blood platelet production and morphology - Corrected Proof

Alessandro Malara, Alessandra Balduini — 2012-01-09

Abstract: Circulating platelets are highly specialized cells produced by megakaryocytes (Mks) that participate in hemostatic and inflammatory functions. Despite their critical role little is known about the molecular mechanisms involved in their production from megakaryocytes, or about the pathogenesis of platelet disorders. Megakaryopoiesis occurs in a complex microenvironment within the bone marrow. The underlying relationships between Mk maturation and bone marrow components are key factors in this process. Mk interactions with extracellular matrices (ECM) via specific surface receptors control many functions, with chemistry, physical parameters and membrane elasticity as fundamental elements of the processes involved. Alteration of Mk–ECM interactions in the bone marrow environment may lead to pathophysiologic situations, such as myelofibrosis and congenital thrombocytopenia. Searching the mechanisms related to Mks–bone marrow environment interactions, will provide novel insight into fundamental control of Mk function, leading to new concepts in the study of Mk-related disease states and future modes for therapeutic inquiry.

Concomitant use of medication with antiplatelet effects in patients receiving either rivaroxaban or enoxaparin after total hip or knee arthroplasty - Corrected Proof

2012-01-09

Abstract: Introduction: The RECORD programme compared oral rivaroxaban with enoxaparin for prevention of venous thromboembolism after elective total hip or knee replacement. This analysis compared the safety of concomitant use of specified medications with rivaroxaban and enoxaparin by evaluating postoperative bleeding rates from the pooled RECORD1–4 data.Materials and methods: The co-medications were non-steroidal anti-inflammatory drugs and platelet function inhibitors, including acetylsalicylic acid (no dose restriction). The endpoints evaluated were the composite of major and non-major clinically relevant bleeding and any bleeding occurring after first oral study drug intake. The time relative to surgery was stratified into three time periods: day 1–3, day 4–7 and after day 7. Relative bleeding rate ratios for co-medication use versus non-use were derived using stratified Mantel–Haenszel methods and compared between rivaroxaban and enoxaparin groups.Results: Co-medication use with rivaroxaban or enoxaparin resulted in non-significant increases in bleeding events. Respective rate ratios were not significantly different between rivaroxaban and enoxaparin for all bleeding endpoints with concomitant use of non-steroidal anti-inflammatory drugs (any bleeding, 1.22 vs 1.22; major and non-major clinically relevant bleeding, 1.28 vs 0.90) and with concomitant use of platelet function inhibitors/acetylsalicylic acid (any bleeding, 1.32 vs 1.40; major and non-major clinically relevant bleeding, 1.11 vs 1.13).Conclusions: This explorative analysis indicates that there is no significant increase in bleeding risk for rivaroxaban compared with enoxaparin when co-administered with non-steroidal anti-inflammatory drugs or acetylsalicylic acid, although, because of low usage, the experience with platelet function inhibitors (except acetylsalicylic acid) was limited.

Critical role of factors II and X during coumarin anticoagulation and their combined measurement with a new Fiix-prothrombin time - Corrected Proof

2012-01-06

Abstract: Vitamin K antagonists (VKA) are monitored with prothrombin time (PT) based assays that are equally sensitive to reductions in factors II, VII or X. We compared the effect of vitamin K dependent (VKD) coagulation factors on PT and also on rotational thromboelastometric (ROTEM) parameters. The PT was equally sensitive to reductions in factors II, VII or X but ROTEM parameters correlated poorly with the PT in anticoagulated patients´ plasmas. ROTEM parameters were more affected by mild and moderate reductions in FII or FX than by FVII or FIX which had little influence except at very low coagulant activity. We developed a modified PT that was sensitive only to reductions in factors II and X. The Fiix-PT (Fiix-INR) correlated well with PT (INR) but the Fiix-INR fluctuated less than the INR in an anticoagulated patient reflecting its insensitivity to FVII. The ROTEM results suggest that mild to moderate reductions in factors II or X are more important during clot formation than factors VII or IX. Reductions in FII and X may better reflect anticoagulation with VKA than FVII or IX. The new Fiix-PT may more accurately reflect the degree of therapeutic anticoagulation in patients treated with VKA than the current PT which is subject to a confounding variation caused by FVII.

A Randomized Controlled Trial of Empiric Warfarin Dose Reduction with the Initiation of Doxycycline Therapy - Corrected Proof

Mary Beth Dowd, Kellie A. Kippes, Daniel M. Witt, Thomas Delate, Kerri Martinez — 2012-01-06

Abstract: Background: When interacting medications, such as doxycycline, are initiated during warfarin therapy, one method to correct for non-therapeutic international normalized ratio (INR) is adjusting the warfarin dose, if necessary. Another approach is preemptive warfarin dose adjustment. This study's objective was to evaluate the utility of preemptive warfarin dose adjustment for preventing non-therapeutic INR following doxycycline-warfarin co-administration.Methods: Patients were randomized to either a 10% to 20% preemptive warfarin dose reduction (intervention) or reactive warfarin dose adjustment (control) within 72hours of warfarin-doxycycline co-administration. Subjects received a follow-up INR within 7days (index INR). Primary outcome was the occurrence of index INR≥1 point over the INR goal range upper limit. Secondary outcomes included INR control, purchases of prescription vitamin K, and warfarin-associated adverse events in the 30days after doxycycline initiation.Results: Twenty and 17 patients comprised the intervention and control groups. The intervention group's warfarin dose was reduced by a median of 11%. More control patients (n=2) experienced an INR≥1 point over the INR goal range upper limit compared to intervention (n=0); however, the difference (12% vs. 0%) was not statistically significant (p=0.20). A higher percentage of intervention patients had subtherapeutic index INRs compared to control (35% vs. 6%, p<0.05). One patient from each group experienced warfarin-associated bleeding. No thromboembolic complications or vitamin K use were observed.Conclusions: For warfarin patients initiating doxycycline therapy, preemptive warfarin dose reduction did not result in supratherapeutic INRs but increased the likelihood of subtherapeutic INRs compared to INR monitoring with reactive warfarin dose adjustment.

Pioglitazone inhibits platelet function and potentiates the effects of aspirin: A prospective observation study - Corrected Proof

2012-01-06

Abstract: Background: Thiazolidinediones (TZDs) are agonists of PPARγ and exert beneficial metabolic effects in patients with diabetes. They may also affect platelet function.Objectives: To characterize potential platelet inhibitory effect of pioglitazone alone and in the presence of aspirin.Methods: 20 normal and 20 diabetic subjects were enrolled in a prospective study. On day 1, a blood sample was obtained at baseline and a second one after ingestion of 30mg of pioglitazone. PRP was prepared and platelet aggregation and release were evaluated using ADP, collagen and arachidonic acid as agonists. Subjects returned at 6–9days later after ingesting a single 81mg dose of aspirin and a third blood sample was obtained. The subjects then again ingested 30mg of pioglitazone and a fourth and final blood sample was obtained. Platelet aggregation and release were measured. PRP was incubated with thrombin to activate platelets, and the serum was separated and assayed for thromboxane B2, TGFβ and CD40LResults: Pioglitazone alone did not affect aggregation with arachidonic acid. However, following ingestion of both aspirin and pioglitazone aggregation was significantly decreased compared to aspirin alone (P<0.0001). Pioglitazone also potentiated aspirin-induced inhibition of ATP release using either arachidonic acid or collagen. Following pioglitazone alone, TXB2 release was 32,719±3,585pg/ml which was significantly reduced compared to baseline (42,075±4,479, P=0.0004). Pioglitazone also potentiated the inhibition of TXB2 release by aspirin.Conclusion: Pioglitazone inhibits platelet function and potentiates the inhibitory effects of aspirin.

Global haemostasis assays, from bench to bedside - Corrected Proof

Mark van Geffen, Waander L. van Heerde — 2012-01-05

Abstract: Bleeding and thrombosis are the ultimate clinical outcomes of aberrations in the haemostatic process. Haemostasis prevents excessive blood loss due to the effort of various compartments like the vasculature, blood cells, coagulation and fibrinolysis. The complexity of all processes involved makes the diagnosis of aberrations difficult, cumbersome and expensive. A single assay to detect any factor disturbing this haemostatic balance with high sensitivity and specificity would be of great value, especially if the outcome of this assay correlates well with clinical outcome. Despite years of research, such an assay is not yet available; however, some interesting candidates are under development and combine the effects of various compartments. This review describes the development of global haemostasis assays and summarizes the current state of the art of these haemostasis assays covering thrombin and plasmin generation, turbidity and thromboelastography/thromboelastometry. Finally, we discuss the applicability of global assays in clinical practice and we provide a future perspective on the ongoing development of automation and miniaturisation as it is our belief that these developments will benefit the standardization of global haemostasis assays.

Prognostic significance of adhesion molecules (sICAM-1, sVCAM-1) and VEGF in colorectal cancer patients - Corrected Proof

2012-01-04

Abstract: Introduction: Adhesion molecules take part in the interaction between host cells and cancer cells. In the current study the relationship between the soluble adhesion molecules sICAM-1 and sVCAM-1 and proangiogenic factor VEGF in colorectal cancer progression were measured.Materials and methods: The study group consisted of 46 patients with colorectal carcinoma (classified due to TNM classification) and 40 controls. sICAM-1, sVCAM-1 and VEGF plasma concentration were measured by enzyme-linked immunosorbent assay (ELISA).Results: All measured parameters levels were increased significantly in patients with colorectal cancer in comparison to controls (p<0.001). sICAM-1, sVCAM-1 and VEGF increased significantly due to colorectal cancer progression. There was a positive correlation between sICAM-1 and sVCAM-1 in all study groups.Conclusions: Our results demonstrated in CRC patients significantly increased levels of soluble adhesion molecules (VCAM-1 and sICAM-1) and angiogenic factor (VEGF) as compared to control group. The dynamics of these molecules showed the growing tendency along with tumor size and metastasis formation.

A microarray study on the effect of four hormone therapy regimens on gene transcription in whole blood from healthy postmenopausal women - Corrected Proof

2012-01-04

Abstract: Background: Postmenopausal hormone therapy is associated with many diseases and conditions, e.g., cardiovascular diseases and asthma, but the underlying molecular mechanisms are incompletely understood. The aim of the current study was to investigate the effect of four different postmenopausal hormone therapy regimens on gene transcription.Materials and methods: Twenty-four healthy postmenopausal women (six women in four groups) were randomly allocated to conventional-dose 17β-estradiol/norethisterone acetate (NETA), low-dose 17β-estradiol/NETA, tibolone, or raloxifene hydrochloride. RNA was isolated from whole blood before and after 6weeks of treatment. The changes in mRNA were assessed with a microarray chip.Results: The genes FKBP5, IL13RA1, TPST1, and TLR2 were up-regulated and among the most significantly changed genes in the groups treated with conventional-dose 17β-estradiol/NETA and tibolone. Up-regulation of TPST1 was associated with reduction of tissue factor pathway inhibitor in plasma. Nine biological pathways were associated with conventional-dose 17β-estradiol/NETA, most significantly the pathways for asthma, toll-like receptor signaling, cell adhesion molecules, and MAPK signaling. Transcriptional changes with false discovery rate below 0.10 were found in 10 genes in the conventional-dose 17β-estradiol/NETA group, 7 genes in the tibolone group, and zero genes in the women on low-dose 17β-estradiol/NETA. No genes or pathways were associated with raloxifene treatment.Conclusions: The difference between low-dose and conventional-dose17β-estradiol/NETA indicates an effect of dose on transcriptional response. Several genes and pathways related to cell adhesion molecules and immunity related cell surface receptors were influenced by conventional-dose 17β-estradiol/NETA.

Platelet Oxidative Stress and Thrombosis - Corrected Proof

Francesco Violi, Pasquale Pignatelli — 2012-01-03

Abstract: Experimental and clinical studies support the pivotal role played by reactive oxidant species in the mechanism of platelet activation. This effect is achieved via multiple pathways, including enhanced formation of isoprostanes. Platelet NADPH oxidase seems to be the main source of platelet reactive oxidant species and isoprostanes. Pharmacologic approach that reduces platelet isoprostane formation may represent a future target of antiplatelet drugs. At this regard polyphenols would be of potential interest in virtue of their antioxidant property but interventional trials are necessary to see if they actually inhibit platelet aggregation in vivo and eventually prevent atherosclerotic progression.

ABCB1 C3435T polymorphism and risk of adverse clinical events in clopidogrel treated patients: A meta-analysis - Corrected Proof

Man Luo, Jiaoxing Li, Xiaowei Xu, Xunsha Sun, Wenli Sheng — 2012-01-03

Abstract: Introduction: The ABCB1 C3435T polymorphism limits oral bioavailability of clopidogrel and may influence prognosis of patients treated with clopidogrel. Several studies have examined the association between the C3435T polymorphism and risk of adverse clinical events in clopidogrel treated patients, but the results were inconsistent. To assess the role of the C3435T polymorphism in the impact on clinical outcomes, a meta-analysis was conducted.Methods: 6 studies with 10,153 subjects were included in this meta-analysis. Fixed- or random-effects model was chosen according to heterogeneity. Publication bias was evaluated by fail-safe numbers.Results: The association of the C3435T polymorphism with risk of overall recurrent ischemic events in clopidogrel treated patients was not statistically significant for all genetic models (OR=1.13, 95%CI: 0.78–1.64, P=0.51; OR=1.15, 95%CI: 0.99–1.33, P=0.07; OR=1.19, 95%CI: 0.81–1.76, P=0.37). Significant association was identified between the C3435T polymorphism and risk of short-term recurrent ischemic events (OR=1.55, 95% CI: 1.09-2.20, P=0.01; OR=1.41, 95% CI: 1.06-1.87, P=0.02; OR=1.77, 95% CI: 1.19-2.63, P=0.005). No statistically significant association between the C3435T polymorphism and stent thrombosis (OR=0.79, 95% CI: 0.47-1.32, P=0.37) or bleeding (OR=0.98, 95% CI: 0.79-1.21, P=0.82) was identified. The results may be affected by publication bias.Conclusions: This meta-analysis failed to show an association between the ABCB1 C3435T polymorphism and risk of overall recurrent ischemic events, stent thrombosis or bleeding in clopidogrel treated patients. However, the association between TT homozygotes of the C3435T polymorphism and risk of short-term recurrent ischemic events may exist, but needs more studies to confirm.

Non-fatal cardiovascular disease, malignancies, and other co-morbidity in adult haemophilia patients - Corrected Proof

2012-01-03

Abstract: Introduction: With increasing life expectancy, more haemophilia patients will be confronted with age-related problems. To ensure optimal care, it is important to know the occurrence of both fatal and non-fatal cardiovascular disease, malignancies and other types of co-morbidity in these patients. Our aim was to retrospectively assess the occurrence of co-morbidity and causes of death in a substantial birth-cohort of haemophilia patients.Methods: Data on all types of co-morbidity were collected from medical records of 408 haemophilia patients (204 severe, 204 non-severe) born before 1971, and compared with the Dutch age-matched general male population.Results: Ten patients had 11 myocardial infarctions, none of which were fatal. The cumulative incidence of non-fatal myocardial infarction was significantly lower in patients with severe haemophilia than in the general population (0.5% versus 4.8%), but was not decreased in patients with non-severe haemophilia (4.4%). Intracranial bleeding occurred significantly more often in haemophilia patients. The occurrence of non-virus related malignancies, and other non-virus related co-morbidities was similar in haemophilia patients and the general population. HIV infection was present in 12% of patients, and hepatitis C infection in 56%. Seventy-eight patients (19%) were deceased. Main causes of death were malignancies, AIDS, hepatitis C, and intracranial bleeding.Conclusions: Our results showed a decreased occurrence of myocardial infarction in patients with severe haemophilia, suggesting a protective effect of very low clotting factor levels on thrombotic cardiac events. No differences were found between haemophilia patients and the general population in the occurrence of any other type of non-virus related co-morbidity.

Diversity of Human Plasma Protein C Inhibitor - Corrected Proof

Akio Saito — 2011-12-30

Abstract: Protein C inhibitor was purified from human plasma by use of a dermatan sulfate or heparin column, followed by hydroxyapatite, gel filtration and ion exchange columns. A dimer of protein C inhibitor was detected by SDS-PAGE under reducing conditions, in addition to two forms of monomer species. One of the monomers, 52-kDa PCI, formed a stable complex with activated protein C, urokinase, plasma and tissue kallikrein, but the dimer species and 48-kDa PCI were inactive. When the monomer and dimer forms of protein C inhibitor were applied to 2D-PAGE, more than 20 spots were observed by Western blot analysis and were confirmed to be protein C inhibitor by MALDI-TOF mass spectrometry. The heterogeneity of the protein C inhibitor species was not due to glycosylation or phosphorylation.

Hematologic malignancies and thrombosis - Corrected Proof

F. Elice, F. Rodeghiero — 2011-12-26

Abstract: Patients with hematologic malignancies have an increased risk of venous thromboembolism (VTE), particularly at diagnosis and during the treatment with chemotherapy, asparaginase or immunomodulatory drugs (IMiDs). A disease-dependent hypercoagulable condition associated with other risk factors like drugs, central venous catheter (CVC), immobility and infections are responsible for this high VTE rate. Thrombotic complications have a significant impact on morbidity and in some cases also on mortality of patients with onco-hematologic diseases, therefore thromboprophylaxis to prevent VTE in this setting is needed. However, thrombocytopenia and hemorrhagic complications pone many difficulties in the management of an anticoagulant or antiaggregant treatment in these patients. Recommendations from current guidelines are limited to multiple myeloma patients treated with thalidomide or lenalidomide associated with dexamethasone or chemotherapy, but hematological clinical departments should implement a policy for prevention and treatment of thromboembolic complications in hematologic malignancies.

Factor XIII, clot structure, thrombosis - Corrected Proof

Zsuzsa Bagoly, Zsuzsa Koncz, Jolán Hársfalvi, László Muszbek — 2011-12-26

Abstract: Blood coagulation factor XIII (FXIII) is a tetrameric protein consisting of two catalytic A (FXIII-A) and two carrier/inhibitory B (FXIII-B) subunits. It is a zymogen, which becomes transformed into an active transglutaminase (FXIIIa) in the final phase of coagulation cascade by thrombin and Ca2+. FXIII is essential for hemostasis, its deficiency results in severe bleeding diathesis. FXIIIa mechanically stabilizes fibrin by cross-linking its α-, and γ-chains. It also protects newly formed fibrin from fibrinolysis, primarily by cross-linking α2-plasmin inhibitor to fibrin. Beside the above prothrombotic effects, it is involved in limiting thrombus growth by down-regulating platelet adhesion to fibrin. Elevated FXIII level seems to be a gender-specific risk factor of both coronary artery disease and peripheral arterial disease, it represents an increased risk only in females. The association of FXIII level with the risk of ischemic stroke and venous thromboembolism was investigated only in a few studies from which no clear conclusion could be drawn. Among the FXIII subunit polymorphisms, concerning their effect on the risk of thrombotic diseases, only FXIII-A p.Val34Leu was investigated extensively. Meta-analyses of reported data suggest that this polymorphism provides a moderate protection against coronary artery disease and venous thromboembolism, but not against ischemic stroke. Gene-gene and gene-environmental interactions might modify its effect. Further studies are required to explore the effect of other FXIII subunit polymorphism on the risk of thrombotic diseases.

Tissue factor/TFPI and blood cells - Corrected Proof

Bjarne Østerud — 2011-12-26

Abstract: Vascular injury-induced access of blood to tissue factor (TF) leads to the formation of a TF-FVII/FVIIa complex and the triggering of blood coagulation. The activated TF-dependent pathway is regulated by Tissue Factor Pathway Inhibitor (TFPI), which binds and inhibits FXa, but more importantly forms an inactive quaternary complex with TF-FVIIa-FXa, effectively shutting off the TF activity. The old view of TF residing in extravascular sites exclusively has recently been challenged by several reports on TF expression in various blood cells. The latter arena has unfortunately been marred by many contradictions, apparently related to inferior tools and/or study design, notably the widespread use of antibodies with inferior and misleading specificity and TF activity assays of low sensitivity/specificity. Our own studies along with many other reports, compels the conclusion that in blood of healthy individuals TF is exclusively associated with and expressed in circulating monocytes. In this short review the distribution of TF and TFPI in blood is discussed.

Factor XI: Hemostasis, Thrombosis, and Antithrombosis - Corrected Proof

Rong He, Dong Chen, Shilin He — 2011-12-26

Abstract: Coagulation factor FXI (FXI), a plasma serine protease zymogen, has important roles in both intrinsic and extrinsic coagulation pathways and bridges the initiation and amplification phases of plasmatic hemostasis. Recent studies have provided new insight into the molecular structure and functional features of FXI and have demonstrated distinct structural and biological differences between activated factor XII (FXIIa)–mediated FXI activation and tissue factor/thrombin–mediated FXI activation. The former is important in thrombosis; the latter is more essential in hemostasis. Activated partial thromboplastin tine (aPTT) artificially reflects FXIIa-initiated intrinsic coagulation pathway in vitro. Conversely, FXIIa-inhibited diluted thromboplastin time assay may reflect tissue factor/thrombin–mediated FXI activation in vivo. Further explication of the genetic mutations of FXI deficiency has improved the understanding of the structure-function relationship of FXI. Besides its procoagulant activity, the antifibrinolytic activity of FXI was well documented in a wealth of literature. Finally, the new emerging concept of inhibiting FXI as a novel antithrombotic approach with an improved benefit-risk ratio has been supported through observations from human FXI deficiency and various animal models. Large- and small-molecule FXI inhibitors have shown promising antithrombotic effects. The present review summarizes the recent advancements in the molecular physiology of FXI and the molecular pathogenesis of FXI deficiency and discusses the evidence and progress of FXI-targeting antithrombotics development.

Diabetes mellitus and thrombosis - Corrected Proof

Natale Vazzana, Paola Ranalli, Chiara Cuccurullo, Giovanni Davì — 2011-12-26

Abstract: Atherothrombosis is the leading cause of morbidity and mortality in patients with diabetes mellitus. Several mechanisms contribute to the diabetic prothrombotic state, including endothelial dysfunction, coagulative activation and platelet hyper-reactivity. In particular, diabetic platelets are characterised by dysregulation of several signaling pathways leading to enhanced adhesion, activation and aggregation. These alterations result from the interaction among hyperglycemia, insulin resistance, inflammation and oxidative stress. This review will provide an overview of the current status of knowledge on mechanisms of accelerated atherothrombosis in patients with diabetes mellitus.