308 research outputs found
Factor V Has Anticoagulant Activity in Plasma in the Presence of TFPI: Difference between FV1 and FV2
No full textBackground Activated factor V (FVa) is a potent procoagulant cofactor in the prothrombinase complex, whereas its precursor factor V (FV) stimulates the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-alpha (TFPI alpha), presumably by promoting TFPI alpha binding to phospholipids. Plasma FV comprises two glycosylation isoforms (FV1 and FV2) with low and high phospholipid-binding affinity, respectively. The FV1/FV2 ratio is increased in carriers of the FV R2 haplotype. Objective This article demonstrates the TFPI alpha-cofactor function of FV in plasma and compares FV1 and FV2. Materials and Methods Thrombin generation at low TF concentration was measured in FV-depleted plasma reconstituted with 0 to 100% FV, FV1 or FV2, and in 122 individuals genotyped for the R2 haplotype. The TFPI-cofactor activities of FV1 and FV2 were also investigated in a model system of TFPI alpha-mediated FXa inhibition. Results In the FV titration, thrombin generation first increased (up to 5% FV) and then progressively decreased at higher FV concentrations. This anticoagulant effect of FV, which was also observed with FV2 but not with FV1, was largely abolished by anti-TFPI alpha antibodies, suggesting that it reflects TFPI alpha-cofactor activity of FV. In the model system of TFPI alpha-mediated FXa inhibition, FV2 was a more potent TFPI alpha-cofactor than FV1, in line with their respective phospholipid affinities. Accordingly, FV R2 carriers had higher thrombin generation than non-carriers, even after correction for demographics and plasma levels of coagulation factors and inhibitors. Conclusion FV (and particularly its FV2 isoform) contributes to the TFPI alpha-dependent down-regulation of thrombin generation in plasma triggered with low TF
Marine-derived sulfated polysaccharides enhance hemocompatibility and endothelialization of nanofibrous PCL for vascular graft applications
No full textAntisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation
Full text linkAntisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 mu M) or a construct expressing antisense U7snRNA (0.25-2 mu g) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted
In vitro and ex vivo rescue of a nonsense mutation responsible for severe coagulation factor V deficiency
No full textBackground: Coagulation factor V (FV) deficiency is a rare bleeding disorder that is usually managed with fresh-frozen plasma. Patients with nonsense mutations may respond to treatment with readthrough agents. Objectives: To investigate whether the F5 p.Arg1161Ter mutation, causing severe FV deficiency in several patients, would be amenable to readthrough therapy. Methods: F5 mRNA and protein expression were evaluated in a F5 p.Arg1161Ter-homozygous patient. Five readthrough agents with different mechanisms of action, i.e. G418, ELX-02, PTC-124, 2,6-diaminopurine (2,6-DAP), and Amlexanox, were tested in in vitro and ex vivo models of the mutation. Results: The F5 p.Arg1161Ter-homozygous patient showed residual F5 mRNA and functional platelet FV, indicating detectable levels of natural readthrough. COS-1 cells transfected with the FV-Arg1161Ter cDNA expressed 0.7% FV activity compared to wild-type. Treatment with 0-500 μM G418, ELX-02, and 2,6-DAP dose-dependently increased FV activity up to 7.0-fold, 3.1-fold, and 10.8-fold, respectively, whereas PTC-124 and Amlexanox (alone or in combination) were ineffective. These findings were confirmed by thrombin generation assays in FV-depleted plasma reconstituted with conditioned media of treated cells. All compounds except ELX-02 showed some degree of cytotoxicity. Ex vivo differentiated megakaryocytes of the F5 p.Arg1161Ter-homozygous patient, which were negative at FV immunostaining, turned positive after treatment with all 5 readthrough agents. Notably, they were also able to internalize mutant FV rescued with G418 or 2,6-DAP, which would be required to maintain the crucial platelet FV pool in vivo. Conclusion: These findings provide in vitro and ex vivo proof-of-principle for readthrough-mediated rescue of the F5 p.Arg1161Ter mutation
Partial F8 gene duplication (Factor VIII Padua) associated with high factor VIII levels and familial thrombophilia
Full text linkHigh coagulation factor VIII (FVIII) levels are a common risk factor for venous thromboembolism (VTE), but the underlying genetic determinants are largely unknown. We investigated the molecular bases of high FVIII levels in two Italian families with severe thrombophilia. The proband of the first family had a history of recurrent VTE before the age of 50, with extremely and persistently elevated FVIII antigen and activity levels (>400%) as the only thrombophilic defect. Genetic analysis revealed a 23.4-kb tandem duplication of the proximal portion of the F8 gene (promoter, exon 1 and a large part of intron 1), which co-segregated with high FVIII levels in the family and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII levels ≥250% identified a second thrombophilic family with the same F8 rearrangement on the same genetic background, suggesting a founder effect. Carriers of the duplication from both families showed a ≥2-fold up-regulation of the F8 mRNA, consistent with the presence of open chromatin signatures and enhancer elements within the duplicated region. Testing of these sequences in a luciferase reporter assay pinpointed a 927-bp region of F8 intron 1 associated with >45-fold increased reporter activity in endothelial cells, potentially mediating the F8 transcriptional enhancement observed in carriers of the duplication. In conclusion, we report the first thrombophilic defect in the F8 gene (designated "FVIII Padua") associated with markedly elevated FVIII levels and severe thrombophilia in two Italian families
Similar hypercoagulable state and thrombosis risk in type Iand type III protein S-deficient individuals from families with mixed type I/IIIprotein S deficiency.
Full text linkBACKGROUND:
Protein S, which circulates in plasma in both free and bound forms, is an anticoagulant protein that stimulates activated protein C and tissue factor pathway inhibitor. Hereditary type I protein S deficiency (low total and low free protein S) is a well-established risk factor for venous thrombosis, whereas the thrombosis risk associated with type III deficiency (normal total and low free protein S) has been questioned.
DESIGN AND METHODS:
Kaplan-Meier analysis was performed on 242 individuals from 30 families with protein S deficiency. Subjects were classified as normal, or having type I or type III deficiency according to their total and free protein S levels. Genetic and functional studies were performed in 23 families (132 individuals).
RESULTS:
Thrombosis-free survival was not different between type I and type III protein S-deficient individuals. Type III deficient individuals were older and had higher protein S, tissue factor pathway inhibitor and prothrombin levels than type I deficient individuals. Thrombin generation assays sensitive to the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S revealed similar hypercoagulable states in type I and type III protein S-deficient plasma. Twelve PROS1 mutations and two large deletions were identified in the genetically characterized families.
CONCLUSIONS:
Not only type I, but also type III protein S deficiency is associated with a hypercoagulable state and increased risk of thrombosis. These findings may, however, be restricted to type III deficient individuals from families with mixed type I/III protein S deficiency, as these represented 80% of type III deficient individuals in our cohort
Development of a Plasma-Based Assay to Measure the Susceptibility of Factor V to Inhibition by the C-Terminus of TFPIα
No full textBackground Factor V (FV) is proteolytically activated to FVa, which assembles with FXa in the prothrombinase complex. The C-terminus of tissue factor pathway inhibitor-alpha (TFPI alpha) inhibits both the activation and the prothrombinase activity of FV(a), but the pathophysiological relevance of this anticoagulant mechanism is unknown. FV Leiden (FVL) is less susceptible to inhibition by TFPI alpha, while overexpression of FV splicing variants with increased affinity for TFPI alpha (FV-short) causes bleeding. Objective This study aims to develop a plasma-based assay that quantifies the susceptibility of FV(a) to inhibition by the TFPI alpha C-terminus. Materials and Methods FV in highly diluted plasma was preactivated with FXa in the absence or presence of the TFPI alpha C-terminal peptide. After adding prothrombin, thrombin formation was monitored continuously with a chromogenic substrate and prothrombinase rates were obtained from parabolic fits of the absorbance tracings. TFPI resistance was expressed as the ratio of the prothrombinase rates with and without peptide (TFPIr). Results The TFPIr (0.25-0.34 in 45 healthy volunteers) was independent of FV levels. The TFPIr increased from normal individuals (0.29, 95% confidence interval [CI] 0.28-0.31) to FVL heterozygotes (0.35, 95% CI 0.34-0.37) and homozygotes (0.39, 95% CI 0.37-0.40), confirming TFPI resistance of FVL. Two individuals overexpressing FV-short (Amsterdam) had markedly lower TFPIr (0.16, 0.18) than a normal relative (0.29), in line with the high affinity of FV-short for TFPI alpha. Conclusion We have developed and validated an assay that measures the susceptibility of plasma FV to the TFPI alpha C-terminus. Once automated, this assay may be used to test whether the TFPIr correlates with thrombosis or bleeding risk in population studies.</p
JAM-A: junctional adhesion molecule-A or Janus acting mediator in atherosclerosis
Full text linkPresently, atherosclerosis constitutes one of the leading causes for patients suffering morbidity and death around the world with no expected changes in frequency the next decades. A lot of efforts to find powerful therapeutic targets had been taken already, but the range of useful drugs is still comparably limited regarding the broad causalities of this multifaceted disease. Still plenty of crucial cellular and molecular mechanisms facilitating atherosclerosis are not understood completely. As atherosclerosis is a chronic inflammation of the vessel wall, leukocyte – endothelial cell interaction is one of the determining factors for atherosclerotic lesion formation. Thus, investigating mechanisms underlying leukocyte recruitment is important for development of novel therapeutic strategies as they still are not explored to a satisfying degree. One of the involved adhesion molecules, at which attention had be drawn to already years ago, is JAM-A. Over the years a rag rug of roles for JAM-A expressed by differing cell types in different inflammatory setups was generated. For example the role of JAM-A in injury models of liver, carotid artery and heart had been investigated, sometimes with contrary results, but a conclusive study directly comparing the role of JAM-A from differing cellular origin on atherogenesis is still missing. With this study, we aimed to unravel the influence of the adhesion molecule JAM-A expressed from different cell types on atherosclerotic lesion formation and progression. Meanwhile, identified new concepts of leukocyte extravasation and were able to verify established ones. In detail, we found that:1. JAM-A from differing cellular origin exerts diverse effects on atherogenesis2. Whereas somatic deficiency has no effect, endothelial JAM-A deficiency reduces plaque formation via decreasing leukocyte recruitment3. Leukocytes deficient for JAM-A lack active transmigratory capacity and damage the vessel wall, thus enhancing plaque formation4. Endothelial JAM-A is more abundant on the cell surface under hyperlipidemic and altered shear stress conditions5. Normal flow conditions exert atheroprotective function by reducing JAM-A expression via increased miR-145 levels6. Lovastatin completely abolishes oxidized LDL-mediated JAM-A re-localization and increase in leukocyte infiltratio
TFPI-dependent activities of Protein S
No full textProtein S is an essential anticoagulant protein that acts as a cofactor for full length tissue factor pathway inhibitor (TFPI) and activated protein C (APC) in the down regulation of coagulation. Protein S enhances APC-mediated inactivation of the coagulation factors Va and VIIIa, and it stimulates inhibition of factor (F) Xa by TFPI. Because TFPI is a tight binding, but slow inhibitor of FXa, the TFPI/protein S system fails to regulate FXa generation at high tissue factor/FVIIa concentrations. In this review, we explain how TFPI/protein S can regain its activity at high tissue factor concentrations in the presence of APC, resulting in an intertwinement of TFPI- and APC-cofactor activities of protein S, and making TFPI a major determinant of APC-anticoagulant activity in plasma
The role of tissue factor pathway inhibitor in atherosclerosis and arterial thrombosis
No full textTissue factor pathway inhibitor (TFPI) is the main inhibitor of tissue factor (TF)-mediated coagulation. In atherosclerotic plaques TFPI co-localizes with TF, where it is believed to play an important role in attenuating TF activity. Findings in animal models such as TFPI knockout models and gene transfer models are consistent on the role of TFPI in arterial thrombosis as they reveal an active role for TFPI in attenuating arterial thrombus formation. In addition, ample experimental evidence exists indicating that TFPI has inhibitory effects on both smooth muscle cell migration and proliferation, both which are recognized as important pathological features in atherosclerosis development. Nonetheless, the clinical relevance of these antithrombotic and atheroprotective effects remains unclear. Paradoxically, the majority of clinical studies find increased instead of decreased TFPI antigen and activity levels in atherothrombotic disease, particularly in atherosclerosis and coronary artery disease (CAD). Increased TFPI levels in cardiovascular disease might result from complex interactions with established cardiovascular risk factors, such as hypercholesterolemia, diabetes and smoking. Moreover, it is postulated that increased TFPI levels reflect either the amount of endothelial perturbation and platelet activation, or a compensatory mechanism for the increased procoagulant state observed in cardiovascular disease. In all, the prognostic value of plasma TFPI in cardiovascular disease remains to be established. The current review focuses on TFPI in clinical studies of asymptomatic and symptomatic atherosclerosis, coronary artery disease and ischemic stroke, and discusses potential atheroprotective actions of TFPI
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