A prospective cohort study to identify and evaluate endotypes of venous thromboembolism: Rationale and design of the Genotyping and Molecular Phenotyping in Venous ThromboEmbolism project (GMP-VTE).

Several clinical, genetic and acquired risk factors for venous thromboembolism (VTE) have been identified. However, the molecular pathophysiology and mechanisms of disease progression remain poorly understood. This is reflected by uncertainties regarding the primary and secondary prevention of VTE and the optimal duration of antithrombotic therapy. A growing body of literature points to clinically relevant differences between VTE phenotypes (e.g. deep vein thrombosis (DVT) versus pulmonary embolism (PE), unprovoked versus provoked VTE). Extensive links to cardiovascular, inflammatory and immune-related morbidities are testament to the complexity of the disease. The GMP-VTE project is a prospective, multi-center cohort study on individuals with objectively confirmed VTE. Sequential data sampling was performed at the time of the acute event and during serial follow-up investigations. Various data levels (e.g. clinical, genetic, proteomic and platelet data) are available for multi-dimensional data analyses by means of advanced statistical, bioinformatic and machine learning methods. The GMP-VTE project comprises n = 663 individuals with acute VTE (mean age: 60.3 ±â€¯15.9 years; female sex: 42.8%). In detail, 28.4% individuals (n = 188) had acute isolated DVT, whereas 71.6% subjects (n = 475) had PE with or without concomitant DVT. In the study sample, 28.9% (n = 129) of individuals with PE and 30.1% (n = 55) of individuals with isolated DVT had a recurrent VTE event at the time of study enrolment. The systems-oriented approach for the comprehensive dataset of the GMP-VTE project may generate new biological insights into the pathophysiology of VTE and refine our current understanding and management of VTE.

Direct thrombin inhibitors, but not the direct factor Xa inhibitor rivaroxaban, increase tissue factor-induced hypercoagulability in vitro and in vivo.

BACKGROUND: Increased hypercoagulability has been reported with low doses of direct thrombin inhibitors but not with direct factor Xa inhibitors. OBJECTIVES: To compare the effects of rivaroxaban with those of melagatran and dabigatran on thrombin generation (TG) and tissue factor-induced hypercoagulability and to explore the possible involvement of the thrombin-thrombomodulin/activated protein C system. METHODS: In normal human plasma and in protein C-deficient plasma, TG was investigated in vitro in the presence and absence of recombinant human soluble thrombomodulin (rhs-TM). TG was determined by calibrated automated thrombography and an ELISA for prothrombin fragments 1+2 (F1+2 ). In an in vivo rat model, hypercoagulability was induced by tissue factor; levels of thrombin-antithrombin (TAT) and fibrinogen and the platelet count were determined. RESULTS: Rivaroxaban inhibited TG in a concentration-dependent manner. In the absence of rhs-TM, melagatran and dabigatran also inhibited TG concentration dependently. However, in the presence of rhs-TM, lower concentrations of melagatran (119-474 nmol L(-1) ) and dabigatran (68-545 nmol L(-1) ) enhanced endogenous thrombin potential, peak TG, and F1+2 formation in normal plasma but not in protein C-deficient plasma. In vivo, rivaroxaban dose-dependently inhibited TAT generation, whereas melagatran showed a paradoxical effect, with an increase in TAT and a small decrease in fibrinogen and platelet count at lower doses. CONCLUSION: Low concentrations of the direct thrombin inhibitors melagatran and dabigatran enhanced TG and hypercoagulability, possibly via inhibition of the protein C system. In contrast, rivaroxaban reduced TG and hypercoagulability under all conditions studied, suggesting that it does not suppress this negative-feedback system.

Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94-9027) for haemophilia A.

Patients with haemophilia (PWH) are usually monitored by the one-stage activated partial thromboplastin time (aPTT) factor VIII (FVIII) assay. Different aPTT activators may affect clotting time (CT) and FVIII:C levels in patients treated with PEGylated FVIII. To evaluate the characteristics of PEGylated FVIII (BAY 94-9027) in various aPTT clotting assays, and to identify suitable aPTT reagents for monitoring BAY 94-9027 during the treatment of PWH, BAY 94-9027 and World Health Organization (WHO) 8th FVIII standards (WHO-8) were spiked into pooled and individual severe haemophilia A plasma at 1.0, 0.25 and 0.05 IU mL(-1) . Five commercial aPTT reagents widely used in clinical laboratories were compared and evaluated for BAY 94-9027 activity in plasma from PWH. BAY 94-9027 and WHO-8 bestowed similar CT and excellent precision when ellagic acid (SynthAFax, Dade Actin, and Cephascreen) aPTT reagents were used. In contrast, BAY 94-9027 showed significantly prolonged CT and poor precision compared with WHO-8 using silica aPTT reagents (APTT-SP and STA PTT 5). Furthermore, free 60-kDa polyethylene glycol (PEG), used for the conjugation of FVIII, showed a dose-dependent prolongation of CT in the APTT-SP assay. There was no effect on the SynthAFax-APTT, prothrombin time, or FXIa-initiated thrombin generation assay, demonstrating that the PEG moiety on FVIII has no general effect on the coagulation cascade. In summary, ellagic aPTT reagents (SynthAFax, Dade Actin, and Cephascreen) are most suitable for evaluating potency of BAY 94-9027 and should be the preferred aPTT reagents used in clinical laboratories for monitoring FVIII activity after infusion of BAY 94-9027 to PWH.

Effects of rivaroxaban, acetylsalicylic acid and clopidogrel as monotherapy and in combination in a porcine model of stent thrombosis.

BACKGROUND: Despite standard dual antiplatelet therapy (DAT) (acetylsalicylic acid [ASA] and clopidogrel), there is a ≥ 1.4% incidence of in-stent thrombosis in patients with acute coronary syndrome. Factor Xa inhibitors are being investigated for secondary prevention after acute coronary syndrome. OBJECTIVE: To study the antithrombotic effects of the FXa inhibitor rivaroxaban alone or in combination with DAT. METHODS: Bare metal stents (12 per animal, three per intervention period) were deployed in a porcine ex vivo arteriovenous shunt and exposed to flowing arterial blood (shear rate: 1500 s(-1)). In-stent thrombus formation was analyzed under different treatments: vehicle (n = 7 animals); intravenous (i.v.) rivaroxaban (0.11, 0.33, and 1.0 µg kg(-1) min(-1)) (n = 8); rivaroxaban + ASA (1.0 mg kg(-1) i.v.) (n = 6); rivaroxaban + ASA (1.0 mg kg(-1) i.v.) + clopidogrel (0.5 mg kg(-1) i.v.) (n = 7); and ASA (1.0 mg kg(-1) i.v.) + clopidogrel (0.5 mg kg(-1) i.v.) (n = 6). RESULTS: Rivaroxaban dose-dependently reduced stent thrombus weight by ≤ 66% vs. vehicle (P < 0.05, all doses). Rivaroxaban + ASA further reduced thrombus weight vs. vehicle (86% at the highest rivaroxaban dose; P < 0.001). DAT reduced thrombus weight by ≤ 79%. However, rivaroxaban + ASA + clopidogrel almost completely abolished in-stent thrombus formation (98% reduction vs. vehicle at the highest rivaroxaban dose; P < 0.001). CONCLUSIONS: Our data on the inhibitory effect of rivaroxaban alone or with DAT are consistent with the ATLAS 2 trial findings, and support its potential use for preventing stent thrombosis after stent deployment.

Description of the chemical and pharmacological characteristics of a new hemisynthetic ultra-low-molecular-weight heparin, AVE5026.

BACKGROUND AND OBJECTIVES: AVE5026 is a novel, hemisynthetic, ultra-low-molecular-weight heparin (ULMWH), which is in clinical development for prevention of venous thromboembolism. Its unique structural features result from the highly selective depolymerization of heparin by the phosphazene base that protects the antithrombin (AT)-binding site from destruction. In the present paper, we describe the chemical and biological characteristics of AVE5026, as well as its effects on experimental thrombosis as compared to those of the low-molecular-weight heparin (LMWH) enoxaparin after a single subcutaneous (s.c.) administration in certain animal models. METHOD AND RESULTS: AVE5026 has a higher anti-factor Xa (anti-FXa) activity (approximately 160 U mg(-1)) along with a catalytic anti-thrombin (anti-FIIa) activity (approximately 2 U mg(-1)) as a result of its structure being strongly enriched in specific AT-binding oligosaccharides. In human plasma, potent inhibition of thrombin generation by AVE5026 was closely related to its anti-FXa activity. In a rat venous thrombosis model, AVE5026 showed a dose-dependent antithrombotic activity comparable to that of enoxaparin (ED50-AVE5026 = 1.6 mg kg(-1), ED50-enoxaparin = 2.8 mg kg(-1)). Interestingly, non-occlusive venous thrombosis in rabbits was inhibited by an ED50 of 0.1 mg kg(-1) AVE5026, whereas 0.316 mg kg(-1) enoxaparin was not active. In a canine model, similarly to enoxaparin (ED50 = 1.3 mg kg(-1)), AVE5026 dose-dependently inhibited arterial thrombosis (ED50 = 2.0 mg kg(-1)). At equipotent doses, AVE5026 did not affect bleeding parameters, whereas enoxaparin showed increased hemorrhage in rats, rabbits and dogs. CONCLUSION: These unique structural attributes distinguish AVE5026 from the LMWH class. Based on these data in well-established arterial and venous thrombosis models, AVE5026 could represent a valuable alternative in thrombosis prevention with an improved benefit-risk profile as compared to that of enoxaparin.

Correlation between dielectric and optical measurements in the smectic-C(*)(alpha) phase.

We present optical and dielectric measurements in the smectic-C(*)(alpha) (SmC(*)(alpha)) phase of three homologues of an alkoxy benzoate series. Two different behaviors are observed depending on the values and the temperature evolution of the azimuthal angle difference alpha between two adjacent layers. For moderate values of alpha, the Goldstone mode is predominant over the whole SmC(*)(alpha) phase. For large values of alpha, we can distinguish the soft mode near the SmA*-SmC(*)(alpha) phase transition and the Goldstone mode at lower temperatures. In this case, discontinuities are also observed at the SmC(*)(alpha)-SmA* phase transition. These dielectric features are correlated with optical properties using simulations based on the discrete phenomenological "clock model."

Static and dynamic electro-optic properties of a SmC* phase in surface stabilized geometry and dispersed in the polymer matrix.

Comparative electro-optical measurements have been made on a ferroelectric liquid crystal (FLC) in surface stabilized geometry and confined to an ellipsoidal cavity within a polymer matrix. The static and dynamic electro-optical characteristics were measured for both systems and show qualitatively similar behaviours. A fast switching and important bistability were observed and characterized as a function of the applied electric field strength. The switching time between the two stable states of the surface stabilized cell was found to be longer than that found for the composite films. We argue that the faster switching dynamic of the FLC in cavities is due to the enhance of the rotational mobility of the molecules, probably (and partly) because of the "soft" anchoring character of the molecules at the cavity walls. Using a collective switching model in the high field regime, which assume a linear coupling between the spontaneous polarization and the local cavity electric field, we give an estimate of the rotational viscosity of the FLC molecules in the droplets.

Treatment of chronic renal allograft rejection in rats with a low-molecular-weight heparin (reviparin).

BACKGROUND: Low-molecular-weight heparin (LMWH) has been shown to prolong survival of rat cardiac allografts independently from immunosuppressive treatment. Furthermore, long-term treatment reduces the development of chronic graft vascular disease after experimental heart transplantation. The aim of the present study was to determine whether treatment with the LMWH reviparin has a beneficial effect on chronic rejection in a rat renal allograft model. METHODS: Kidneys of Fisher (F344) rats were transplanted into unilaterally nephrectomized Lewis (LEW) recipients. LEW-->LEW isografts served as controls. Animals were treated with cyclosporine (5 mg/kg/d) for the first 10 days. Nephrectomy of the remaining kidney was performed after 10 days. Allografted animals were treated either with reviparin (2 mg/kg/d subcutaneously) for 24 weeks (Allo-24), from week 12 to 24 (Allo-12), or with vehicle for 24 weeks. Proteinuria was determined at regular intervals. Kidneys were harvested after 24 weeks for histomorphological and immunohistochemical evaluation. RESULTS: No major bleeding complications were observed in reviparin-treated animals. Proteinuria was significantly reduced in allografted animals both by early as well as by late-onset treatment with reviparin. Transplant glomerulopathy was diminished in Allo-24 and in Allo-12 groups compared to vehicle-treated animals, whereas tubulointerstitial inflammation was influenced only in animals immediately treated with reviparin. Immunohistochemical studies demonstrated a marked reduction of renal monocyte and T-cell infiltration as well as expression of MHC II by treatment with reviparin. CONCLUSIONS: Treatment with the LMWH reviparin significantly improved chronic renal allograft rejection in the F344-to-LEW rat model, both after early and late start of therapy. Although the exact mechanisms of this beneficial effect remain unclear, our data offer a potential new therapeutical approach for prevention of chronic allograft nephropathy.

Antithrombotic efficacy in a rat model of the low molecular weight heparin, reviparin sodium, administered by the oral route.

Previous studies in rats show that unfractionated heparin and the low molecular weight heparin logiparin have a dose-dependent antithrombotic effect and are found in endothelium and plasma when administered orally. Objectives of the present study were to determine if similar evidence of absorption could be observed with oral reviparin sodium. Thrombosis incidence was determined 4 h after application of 10% formalin in methanol to the exposed jugular vein. A dose-dependent antithrombotic effect was observed when 0.01 to 7.5 mg/kg (20 rats/group) was administered by stomach tube immediately following thrombus initiation. Thrombotic incidence was also significantly reduced when 0.025 mg/kg was given 4 and 2 h prior to, immediately after, and 2 and 3 h following thrombus initiation. Reviparin was recovered from endothelium and plasma in trace amounts at all doses. At 0.025 mg/kg, peak aortic endothelial reviparin concentrations were found at 1 and 2 h and peak plasma anti-Xa activity was detected at 2 h. Trace amounts of plasma TFPI were found only at 8 h after administration. Dose-dependent antithrombotic activity and recovery from endothelium and plasma support the hypothesis that orally administered reviparin sodium is absorbed.

Magnetic resonance imaging studies on the effect of the fibrinogen-lowering agent ancrod on cerebral lesions in two rat models of acute stroke.

In vivo magnetic resonance imaging was used to study the effect of ancrod (CAS 9046-56-4, Arwin), a plasma fibrinogen level lowering agent, on brain lesion in two rat models of acute focal cerebral ischaemia. Total lesion volume was determined by multislice T2-weighted magnetic resonance imaging 24 h after permanent middle cerebral artery occlusion. Intravenous infusion of ancrod starting 30 min after middle cerebral artery occlusion at dosages of 10, 30, 50 or 70 IU/kg (n = 9/group) significantly diminished cerebral lesion volume by 20 to 33% as compared to vehicle-infused controls (n = 12). None of the ancrod-treated rats showed evidence of intracerebral bleeding on T2-weighted magnetic resonance images taken after 24 h. In photochemically induced (rose bengal) unilateral thrombotic cortical infarction brain damage was displayed by multislice diffusion-weighted magnetic resonance imaging after 24 h. Again, post treatment with ancrod reduced total volume of cerebral lesion dose-dependently from 142 +/- 28 mm3 in the controls (n = 10) to 121 +/- 28 mm3 (n = 10) and 111 +/- 20 mm3 (n = 11, p < 0.05) in rats treated with 10 and 30 IU/kg ancrod, respectively (means +/- S.D.). These results suggest cerebroprotection in focal cerebral ischaemia by improvements in the cerebral microcirculation which may offer a potential and safe approach for therapy of acute stroke.

Inhibitory effects of pentoxifylline on LPS-induced leukocyte adhesion and macromolecular extravasation in the microcirculation.

Pentoxifylline (PTX) has been shown to combat effectively endotoxin induced symptoms of shock or inflammation by reducing both leukocyte activation and endogenous cytokine formation. With regard to blood perfusion, inflammation is defined as a local reaction to injury of the living microvasculature and its content. Leukocyte margination, rolling, adhesion, and emigration is mediated by adhesion molecules along the endothelium of postcapillary venules and is considered to be an important step in the inflammatory response. Changes in the vascular integrity can be estimated in terms of increased extravasation of macromolecules. Using intravital microscopy with the help of an analogous video image processing system we measured the effect of PTX on lipopolysaccharide (LPS, 15 mg/kg i.v.) induced leukocyte adhesion and extravasation of FITC-rat serum albumin (FITC-RSA) in rat mesenteric venules. The changes in vascular permeability correlates significantly (r = 0.75) with a locally increased number of adherent leukocytes. PTX significantly inhibits both leukocyte adhesion and extravasation of FITC-RSA dose dependently. Our results indicate that PTX effectively preserves vascular integrity in the microcirculation by acting primarily on LPS-induced leukocyte adhesion.

Mediator-induced changes in macromolecular permeability in the rat mesenteric microcirculation.

An intravital fluorescence microscopic method for measurement of changes in macromolecular permeability has been established in the mesenterial microcirculation of the rat. After exteriorization of the fat-free distal part of the ileal mesentery, a 1-hr period of stabilization was followed by the injection of FITC-labeled macromolecules. Five minutes later, histamine, leukotriene B4, or leukotriene C4 was topically applied to the tissue by means of a micromanipulator. Areas of 1 mm2 were videotaped with a SIT camera. The fluorescence intensity of these areas was measured by an analogous video image processing system and displayed as gray value histograms. The shift of the frequency of gray levels from lower to upper regions could be attributed to an increase in light intensity in the mesentery, indicating an increase in vessel wall permeability. The sites of action of histamine and leukotriene C4 were very similar. Both mediators affected mainly the larger collecting venules. In contrast, leukotriene B4 exerted its effect at postcapillary venules. Moreover, leukotriene B4-induced extravasation was inhibited by superoxide dismutase, suggesting an involvement of oxygen radicals. The studies with histamine alone and with H1- and H2-antagonists demonstrated that histamine-induced extravasation in the rat mesentery was mediated by H1-histamine receptors. The present study introduces an experimental model for the measurement of changes in macromolecular permeability, which is useful for studying mediator effects and their pharmacological inhibition in the microcirculation of the rat mesentery.

Platelet function in the dorsal skin fold chamber of the rat.

The majority of the preparations described for studying platelet functions in vivo require the administration of anesthetic agents and surgery. Some anesthetic which are frequently used in microvascular research (such as ketamine hydrochloride and pentobarbital) are reported to exert an effect on platelet properties themselves. Using a modified chamber preparation situated in the rat's dorsal skin fold, it is possible to study platelet function in the same rat with or without anesthesia. Thrombus formation is induced in arterioles and venules by argon-laser irradiation with a local capacity of 25mW and an exposure time of 1/30 s. Platelet function was studied for ten successive days in the same group of rats to examine the model's aptitude for long-term investigations. There was no significant alteration in platelet response during the experimental period. To evaluate the potential antithrombotic effects of the anesthetics, the animals were studied before and after the application of ketaminehydrochloride (130 mg/kg b.w., pentobarbital (25 mg/kg b.w.), or urethane (1,25 g/kg i.m.). The first two drugs caused significant increase in the number of laser injuries in arterioles and venules, although the effect of pentobarbital was weaker especially in venules. The influence of thrombus formation while using these anesthetics could influence the testing of antithrombotic drugs. Urethane had no effect on laser-induced thrombus formation. Furthermore, special attention was paid to spontaneous and laser-induced vasomotion with respect to thrombus formation.