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Background: Low-dose oral vitamin K decreases the international normalized ratio (INR) in overanticoagulated patients who receive warfarin therapy. Its effects on bleeding events are uncertain.Objective: To see whether low-dose oral vitamin K reduces bleeding events over 90 days in patients with warfarin-associated coagulopathy.Design: Multicenter, randomized, placebo-controlled trial. Randomization was computer-generated, and participants were allocated to trial groups by using sequentially numbered study drug containers. Patients, caregivers, and those who assessed outcomes were blinded to treatment assignment.Setting: 14 anticoagulant therapy clinics in Canada, the United States, and Italy.Patients: Nonbleeding patients with INR values of 4.5 to 10.0.Intervention: Oral vitamin K, 1.25 mg (355 patients randomly assigned; 347 analyzed), or matching placebo (369 patients randomly assigned; 365 analyzed).Measurements: Bleeding events (primary outcome), thromboembolism, and death (secondary outcomes).Results: 56 patients (15.8%) in the vitamin K group and 60 patients (16.3%) in the placebo group had at least 1 bleeding complication (absolute difference, -0.5 percentage point [95% CI, -6.1 to 5.1 percentage points]); major bleeding events occurred in 9 patients (2.5%) in the vitamin K group and 4 patients (1.1%) in the placebo group (absolute difference, 1.5 percentage points [CI, -0.8 to 3.7 percentage points]). Thromboembolism occurred in 4 patients (1.1%) in the vitamin K group and 3 patients (0.8%) in the placebo group (absolute difference, 0.3 percentage point [CI, -1.4 to 2.0 percentage points]). Other adverse effects were not assessed. The day after treatment, the INR had decreased by a mean of 1.4 in the placebo group and 2.8 in the vitamin K group (P < 0.001).Limitation: Patients who were actively bleeding were not included, and warfarin dosing after enrollment was not mandated or followed.Conclusion: Low-dose oral vitamin K did not reduce bleeding in warfarin recipients with INRs of 4.5 to 10.0.Funding: Canadian Institutes of Health Research and Italian Ministry of Universities and Research. [ABSTRACT FROM AUTHOR]

Background: Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation-dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll-like receptor 3), which recognizes (viral) single-stranded or double-stranded RNAs and self-RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism., Methods and Results: Thrombus formation and size in wild-type and TLR3 deficient (-/-) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic-polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release in vivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle-treated mice ( P <0.05). Polyinosinic-polycytidylic acid and eRNA treatments increased thrombus size in wild-type mice ( P <0.01 and P <0.05), but not in TLR3 -/- mice, by reinforcing neutrophil recruitment ( P <0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C-X-C motif chemokine 5) secretion ( P <0.001) and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation ( P <0.05). Finally, eRNA triggered plasma clot formation in vitro ( P <0.01)., Conclusions: We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.

Essentials Neutrophil extracellular traps (NETs) are generated during deep vein thrombosis (DVT). The role of interferon γ (IFNγ) and natural killer (NK) cells in NET formation was studied. IFNγ promote venous thrombosis through NET formation. NK cell depletion reduces DVT. SUMMARY: Background Neutrophils contribute to venous thrombosis through the release of neutrophil extracellular traps (NETs), but the mechanism triggering their formation remains unclear. In vitro data show that interferon (IFN)-γ induces the formation of NETs. Objectives To determine whether IFN-γ and the transcription factor T-box expressed on T cells (Tbet) promote venous thrombosis through neutrophil activation. Methods Venous thrombosis was induced by flow restriction in the inferior vena cava in IFN-γ -/- , Tbet -/- or wild-type (WT) mice. After 48 h, thrombus size was measured by the use of high-frequency ultrasound. NET formation was determined by immunofluorescence. Results and Conclusions Thrombus formation was reduced in Tbet -/- and IFN-γ -/- mice, suggesting that Tbet/IFN-γ-expressing cells are required for venous thrombosis. The number of NETs formed during thrombosis was significantly lower in Tbet -/- and IFN-γ -/- mice. NET formation was also decreased in WT mice treated with an IFN-γ-blocking antibody. Injection of recombinant IFN-γ into IFN-γ -/- mice rescued the phenotype. Natural killer (NK) cells were specifically depleted prior to venous thrombosis induction. NK cell depletion results in decreased NET formation and smaller thrombi, suggesting that NK cells are required for thrombus development. In depleted mice, adoptive transfer of WT NK cells induced a similar thrombosis burden as in WT mice. In contrast, adoptive transfer of IFN-γ -/- NK cells resulted in thrombi similar in size to those in depleted mice. In vitro, we showed that WT neutrophils released fewer NETs when they were cocultured with IFN-γ -/- NK cells. This study demonstrates that NK cell-dependent IFN-γ production is crucial for thrombus development by promoting the formation of NETs by neutrophils., (© 2018 International Society on Thrombosis and Haemostasis.)

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma. Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

Objective: Coagulation and inflammation are inter-related. Gas6 (growth arrest-specific 6) promotes venous thrombosis and participates to inflammation through endothelial-innate immune cell interactions. Innate immune cells can provide the initiating stimulus for venous thrombus development. We hypothesize that Gas6 promotes monocyte recruitment during venous thrombosis., Approach and Results: Deep venous thrombosis was induced in wild-type and Gas6-deficient (-/-) mice using 5% FeCl 3 and flow reduction in the inferior vena cava. Total monocyte depletion was achieved by injection of clodronate before deep venous thrombosis. Inflammatory monocytes were depleted using an anti-C-C chemokine receptor type 2 (CCR2) antibody. Similarly, injection of an anti-chemokine ligand 2 (CCL2) antibody induced CCL2 depletion. Flow cytometry and immunofluorescence were used to characterize the monocytes recruited to the thrombus. In vivo, absence of Gas6 was associated with a reduction of monocyte recruitment in both deep venous thrombosis models. Global monocyte depletion by clodronate leads to smaller thrombi in wild-type mice. Compared with wild type, the thrombi from Gas6 -/- mice contain less inflammatory (CCR2 hi CX 3 CR1 lo ) monocytes, consistent with a Gas6-dependent recruitment of this monocyte subset. Correspondingly, selective depletion of CCR2 hi CX 3 CR1 lo monocytes reduced the formation of venous thrombi in wild-type mice demonstrating a predominant role of the inflammatory monocytes in thrombosis. In vitro, the expression of both CCR2 and CCL2 were Gas6 dependent in monocytes and endothelial cells, respectively, impacting monocyte migration. Moreover, Gas6-dependent CCL2 expression and monocyte migration were mediated via JNK (c-Jun N-terminal kinase)., Conclusions: This study demonstrates that Gas6 specifically promotes the recruitment of inflammatory CCR2 hi CX 3 CR1 lo monocytes through the regulation of both CCR2 and CCL2 during deep venous thrombosis., (© 2017 American Heart Association, Inc.)

Venous thromboembolism is a common complication of cancer. Based on recent evidence that (1) growth arrest-specific 6 (Gas6) regulates the expression of tissue factor during venous thrombosis, and (2) cancer promotes a procoagulant milieu, we hypothesize that Gas6 may be involved in cancer-induced coagulopathy. Venous thrombi were induced in both wild-type (WT) and Gas6-deficient ((-/-)) mice with cancer. WT mice with cancer developed larger thrombi than their healthy counterparts; these larger thrombi induced by cancer were not seen in Gas6(-/-) mice. Whole genome microarray analysis of differential gene expression in WT and Gas6(-/-) endothelial cells exposed to M27 murine lung carcinoma cells reveal that Gas6 increases prostaglandin E synthase (Ptges) expression in endothelial cells. This was confirmed using real-time polymerase chain reaction and immunofluorescence staining. Culture of WT endothelial cells with M27 increases the secretion of prostaglandin E2 (PGE2), the enzymatic product of Ptges, in WT but not in Gas6(-/-) endothelial cells. In WT endothelial cells, Ptges expression was regulated through extracellular signal-regulated kinase 1/2 phosphorylation (ERK1/2). In vitro, PGE2 activates platelets after binding to its receptor, EP3. In vivo, EP3 receptor antagonism reversed the effect of cancer-induced thrombosis in WT mice. These results show that Gas6, through upregulation of PGE2, contributes to cancer-induced venous thrombosis., (© 2016 by The American Society of Hematology.)

Background: Growth arrest-specific 6 (Gas6)-deficient mice are protected against venous thromboembolism (VTE), suggesting a role for Gas6 in this disorder. We previously demonstrated that Gas6 induces forkhead box O1 (FoxO-1) phosphorylation through the phosphoinositide 3-kinase-Akt pathway. FoxO-1 regulates the expression of vascular cell adhesion molecule-1 (VCAM-1), a molecule that has been implicated in VTE., Objectives: To assess the role of FoxO-1 in Gas6-dependent VCAM-1 expression., Methods: Thrombin was used to stimulate endothelial cells (ECs). Wild-type (WT) and Gas6(-/-) ECs were transfected with small interfering RNA targeting Axl or FoxO-1, a luciferase-coupled plasmid containing the FoxO-1 consensus sequence, and a phosphorylation-resistant FoxO-1 mutant, or treated with an Akt inhibitor. VCAM-1 mRNA expression was measured by real time-qPCR. VCAM-1 protein expression and FoxO-1 and Akt phosphorylation were assessed by western blot analysis. FoxO-1 localization was assessed by immunofluorescence. Adhesion of bone marrow mononuclear cells (BM-MCs) on ECs was assessed by fluorescence., Results and Conclusions: Thrombin induces both VCAM-1 expression and FoxO-1 phosphorylation and nuclear exclusion in WT ECs only. Silencing of FoxO-1 enhances VCAM-1 expression in both WT and Gas6(-/-) ECs. Inhibition of Akt or FoxO-1 phosphorylation prevents VCAM-1 expression in WT ECs. These data show that Gas6 induces FoxO-1 phosphorylation, leading to derepression of VCAM-1 expression. BM-MC-EC adhesion is increased by thrombin in WT ECs. BM-MC-EC adhesion is further increased when FoxO-1 is silenced, but decreased when FoxO-1 phosphorylation is inhibited. These results demonstrate that the Gas6-FoxO-1 signaling axis plays an important role in VCAM-1 expression in the context of VTE by promoting BM-MC-EC adhesion., (© 2015 International Society on Thrombosis and Haemostasis.)

Background: Gas6 has been shown to interact with Axl in endothelial cells and to induce several signaling pathways involved in cell survival and proliferation. However, the interaction of Gas6/Axl with lipid raft/caveolin-1 in endothelial cells and its role in thrombosis are unknown., Objectives: We tested whether Axl and/or caveolin-1 is involved in Gas6-induced Akt, ERK1/2, and c-Src activation leading to altered tissue factor expression in endothelial cells., Methods: Gas6-treated endothelial cells were transfected with small interfering RNA (siRNA) for Axl, caveolin-1, c-Src, and Akt or treated with pharmacological inhibitors of c-Src and ERK1/2. Sucrose gradient centrifugation and confocal microscopy were used to study lipid raft/caveolin-1-enriched fractions. Akt, ERK1/2, p38, and c-Src activation was analyzed by Western blot analysis. Tissue factor expression was assessed by real-time quantitative polymerase chain reaction and immunofluorescence., Results and Conclusion: Gas6 induced Axl and c-Src localization into lipid raft/caveolin-1-enriched fractions. Gas6 increased the phosphorylation of Akt, ERK1/2, and c-Src but not p38. Using siRNA, we demonstrated that Axl is required for Akt, ERK1/2, and c-Src activation after Gas6 stimulation. siRNA for caveolin-1 blocked Gas6-induced phosphorylation of Akt, ERK1/2, and c-Src. c-Src downregulation inhibited Gas6-induced Akt but not ERK1/2 phosphorylation. Finally, Gas6 increased tissue factor mRNA and protein expression in endothelial cells. Tissue factor expression was blocked by siRNA for Axl, caveolin-1, or Akt as well as c-Src inhibition. These data demonstrate that the signaling pathway Gas6/Axl/caveolin-1/c-Src/Akt is required for tissue factor expression in endothelial cells, providing mechanistic insight into how Gas6 exerts its prothrombotic role in the vasculature., (© 2013 International Society on Thrombosis and Haemostasis.)

Most fatalities from trauma occur due to severe blood loss. There is a need for improved hemostatic biomaterials that can address this problem. The aim of this study was to determine the in vivo efficacy of nanofibrous microspheres (NFM) loaded with hemostatic peptides, specifically ideal amphipathic peptides (IAP) that have been demonstrated to possess both procoagulant and antifibrinolyic activities. We demonstrate that IAP can be coupled to NFM (IAP-NFM) to form matrices that exhibit substantial hemostatic activity. IAP-NFM matrices were compared to a commercial zeolitic hemostatic biomaterial (QuikClot) and have superior efficacy in reducing bleeding in vivo. In both a murine tail transection and a murine hepatic injury model, bleeding times were significantly reduced (P<0.05) with the use of IAP-NFM as compared with equal masses of either QuikClot or NFM alone, or no treatment. Importantly, histological examination revealed no tissue injury when IAP-NFM or NFM were applied to hepatic lacerations. In contrast, QuikClot caused widespread hepatocyte degeneration and necrosis, with higher average injury zone thickness as determined by semiquantitative analysis. In summary, NFM was able to maintain the pro-coagulant properties of IAP in our preclinical model, caused no observable tissue damage at the site of application and had better performance than QuikClot controls., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Gas6 (growth-arrest specific gene 6) plays a role in thrombus stabilization. Gas6 null (-/-) mice are protected from lethal venous and arterial thromboembolism through platelet signaling defects induced only by 5 μM ADP and 10 μM of the thromboxane analog, U46619. This subtle platelet defect, despite a dramatic clinical phenotype, raises the possibility that Gas6 from a source other than platelets contributes to thrombus formation. Thus, we hypothesize that Gas6 derived from the vascular wall plays a role in venous thrombus formation. Bone marrow transplantation and platelet depletion/reconstitution experiments generating mice with selective ablations of Gas6 from either the hematopoietic or nonhematopoietic compartments demonstrate an approximately equal contribution by Gas6 from both compartments to thrombus formation. Tissue factor expression was significantly reduced in the vascular wall of Gas6(-/-) mice compared with WT. In vitro, thrombin-induced tissue factor expression was reduced in Gas6(-/-) endothelial cells compared with wild-type endothelium. Taken together, these results demonstrate that vascular Gas6 contributes to thrombus formation in vivo and can be explained by the ability of Gas6 to promote tissue factor expression and activity. These findings support the notion that vascular wall-derived Gas6 may play a pathophysiologic role in venous thromboembolism.

Background: The most important complication of atrial fibrillation (AF) is thromboembolic stroke. Although AF-related remodeling is considered important in atrial thrombogenesis, its role never has been directly tested. This study assessed effects of AF-related remodeling on the atrial thrombogenic milieu by using radiofrequency ablation (RFA) to create a quantifiable prothrombotic nidus., Methods and Results: We studied normal control dogs (control, n=16) and 3 canine AF-models: (1) atrial tachycardia remodeling (ATR; n=16) induced by atrial tachypacing (400 bpm for 1 week, with atrioventricular block and ventricular pacing at 80 bpm); (2) congestive heart failure (CHF; n=14) attributable to ventricular tachypacing (240 bpm for 2 weeks); and (3) chronic AF (CAF; n=8) induced by atrial tachypacing (35±3 days) without atrioventricular block. CAF dogs had AF for 13±1 days until euthanization. After remodeling was established, RFA lesions were created in both atria. Half the ATR and CHF dogs were subjected to atrial tachypacing during 7-day post-RFA follow-up. Electrophysiological and echocardiographic studies were performed before RFA and 7 days after RFA, and then hearts were removed and atrial thrombi were quantified by histomorphometry. Burst-pacing-induced AF duration was significantly greater in ATR, CHF, and CAF groups versus control group. The atrial effective refractory period shortened in ATR and CAF groups. Left atrial diameter was significantly larger with CHF, but not with ATR. Neither total thrombus volume nor thrombus volume per lesion differed significantly among groups. Table.Properties of Ablation Lesions and Atrial Thrombi Experimental GroupControl (n=16)ATR (n=16)CHF (n=14)CAF (n=8)N of ablation lesions per dog6.9±0.36.6±0.27.2±0.26.9±0.4Ablation lesion area, mm(2)53.1±3.558.3±4.857.7±4.944.3±3.7Ablation lesion depth, mm5.2±0.25.1±0.35.3±0.25.2±0.2Ablation lesion volume, mm(3)205.2±17.8211.6±17.6231.5±29.0176.8±22.2N of thrombi per dog5.4±0.44.7±0.35.6±0.46.5±0.4Presence of thrombus, %80±572±577±695±3Mean thrombus volume in both atria, mm(3)20.8±3.414.9±2.212.2±2.622.5±5.6Mean thrombus volume in left atria, mm(3)8.2±1.54.0±0.95.5±1.68.1±3.3Mean thrombus volume in right atria, mm(3)30.1±5.422.7±4.317.9±4.132.8±8.3Total thrombus volume in both atria, mm(3)140.5±21.399.7±16.886.1±17.5131.1±22.7Total thrombus volume in left atria, mm(3)22.8±5.311.8±3.317.0±3.723.3±6.4Total thrombus volume in right atria, mm(3)117.7±21.587.8±17.269.1±16.1107.8±23.3Thrombus volume normalized to ablation lesion area in both atria, mm(3)/mm(2)0.5±0.10.4±0.11.5±1.10.8±0.3Thrombus volume normalized to ablation lesion volume in both atria0.2±0.10.1±0.00.5±0.40.3±0.1 ATR indicates atrial tachycardia remodeling; CAF, chronic atrial fibrillation; and CHF, congestive heart failure. There were no statistically significant differences for any groups vs control group for any of these variables studied., Conclusions: None of the AF substrates tested, including sustained atrial tachycardia/AF itself, enhanced post-RFA atrial thrombus formation. Indices of electrical and structural remodeling did not predict post-RFA thrombogenic potential. Contrary to widely held but previously untested notions, we were unable to demonstrate prothrombotic effects of AF-related remodeling.

Gas6 (growth arrest-specific 6) belongs structurally to the family of plasma vitamin K-dependent proteins. Gas6 has a high structural homology with the natural anticoagulant protein S, sharing the same modular composition. Interestingly, despite the presence of a γ-carboxyglutamic acid domain in its structure, no role in the coagulation cascade has been identified for gas6. Gas6 has been shown to be involved in vascular homeostasis and more precisely is involved in proliferation, apoptosis, efferocytosis, leukocyte migration, and sequestration and platelet aggregation. It is also involved in the activation of different cell types, from platelets to endothelial and vascular smooth muscle cells. Thus, it has been shown to play a role in several pathophysiological processes such as atherosclerosis, cancer, and thrombosis. Interestingly, studies using gas6 null mice highlighted that gas6 may represent a novel potential target for anticoagulant therapy, because these animals are protected from lethal venous thromboembolism without excessive bleeding. However, the mechanism in thrombus occurrence remains to be further explored. In the present review, we will focus on the role of gas6 in innate immunity, atherosclerosis, thrombosis, and cancer-related events.

Background: Venous thrombosis (VT) is an important cause of morbidity and mortality in clinical medicine. Animal models studying venous thrombosis are scarce and, in most cases, very crude and rely on sacrificing the animals to excise formed thrombi. Developing an in vivo murine model of venous thrombosis can be a powerful tool for studying venous thrombosis., Objectives: We sought to use a high-frequency ultrasound system (HFUS) to dynamically and non-invasively monitor thrombus formation in the inferior vena cava (IVC) of mice., Methods: We developed a murine model of venous thrombosis using, for detection, the Vevo 770(®), a micro-imaging HFUS. Two different thrombosis models were used to generate thrombi in the IVC of C57Bl/6NCr mice: (i) ligation and (ii) application of ferric chloride (FeCl(3)). We then assessed venous thrombosis by HFUS., Results: In both models, measurements of the clot pathologically correlated favorably with measurements acquired with HFUS. Thrombus develops less than an hour after ligation or FeCl(3) -induced injury of the IVC and the size of the clot increases over time for up to 24 h. Importantly, we demonstrate that HFUS can be used to monitor the effect of an anticoagulant such as dalteparin until complete resolution of the thrombus., Conclusions: These data show that HFUS assesses venous thrombosis in mice reliably and non-invasively. Developing a murine model of thrombosis using more accurate, and clinically more relevant, techniques such as ultrasonography, is a step towards a better understanding of the pathophysiology of venous thromboembolism., (© 2011 International Society on Thrombosis and Haemostasis.)

In this study, we validated a thromboelastography (TEG) method to evaluate the hemostatic effects of 3 peptides. The first peptide is an ideal amphipathic peptide composed of 22 leucine and lysine in a ratio of 2:1. At a very low concentration, the peptide had a procoagulant effect shown by decreases in reaction time (R) and coagulation time (K) but was impaired by a decrease in maximum amplitude (MA). At higher concentrations, the peptide had an anticoagulant effect. The α angle was minimally affected by the peptide. The second peptide is melittin derived from bee venom. Melittin showed procoagulant effects reflected by a decrease in clotting time but led to lower MA. The third peptide derived from fibrinogen γ chain promoted hemostasis only at an optimal concentration and became anticoagulant at a higher concentration. The hemostatic mechanisms of each peptide were discussed. Our study would facilitate further development of peptides for either hemorrhage control or thrombosis treatment.

Heyde's syndrome is characterized by iron deficiency anemia due to gastrointestinal bleeding and calcific aortic stenosis. Patients with this syndrome have a bleeding diathesis due to a loss of the largest multimers of von Willebrand factor (vWF). Here we present a case of Heyde's syndrome diagnosed with abnormal closure times and normal vWF Ristocetin cofactor activity (vWF:Rco). In this case, a 79-year-old man with known aortic stenosis and recurrent gastrointestinal bleeding was cured of a life-threatening hemorrhage after the replacement of his stenotic aortic valve. Factor VIII activity (1.53  IU/ml), vWF antigen (1.26  IU/ml), vWF:Rco (1.11  IU/ ml), and the ratio of vWF antigen/vWF:Rco (0.88) were all within normal limits. Instead, to prove a defect in platelet aggregation, closure times measured with collagen/ADP and collagen/epinephrine were abnormal (>300  s). Postoperatively, these closure times normalized. What is unique about our current report is that we measured both vWF:Rco and closure times, the two readily available assays in most coagulation laboratories. vWF:Rco is a standard assay for measuring platelet activity but may miss defects in platelet aggregation that are only seen under high shear stress. As the closure times can detect such defects, it is perhaps more representative than traditional assays, and in situations such as our case, closure times may be the only method by which subtle abnormalities in vWF function could be detected.

Growth arrest-specific 6 (gas6), a novel vitamin K-dependent protein, has been demonstrated to have a role in thrombus stabilization as gas6 null mice are resistant to lethal venous and arterial thrombosis. However, the association between gas6 and venous thromboembolism has not been elucidated in humans. The present study aims to assess the role of gas6 in human venous thromboembolic (VTE) disease. Using a highly specific ELISA method, we measured plasma levels of gas6 in plasma samples obtained from 279 patients with VTE and 79 healthy volunteers. Medication history, comorbid conditions and VTE characteristics were documented. Mean gas6 levels were higher in patients with VTE as compared to healthy volunteers, being 46 ±11 ng/ml and 35 ±6.4 ng/ml respectively (P < 0.001). Odds ratios (OR) for VTE given elevated (≥90th percentile of healthy volunteers) gas6 levels were estimated in regression models in the whole study population. After adjustment for age, sex, medications and comorbidity, subjects with elevated gas6 had an increased risk of VTE (OR of 16.3 (95% CI 5.8-45.7, P < 0.001) compared to those with lower levels of gas6. This association remains significant even among patients with a comparable age distribution. Among patients with VTE, mean gas levels showed a trend of higher levels in those with more extensive thrombi. There was no correlation between elevated gas6 levels and recurrent VTE. In conclusion, we demonstrate an association between VTE and elevated gas6 levels consistent with in vivo murine models of thrombosis. This constitutes a potential novel mechanism for thrombosis in humans and may aid in the understanding of the pathophysiology of VTE.

Hyperhomocysteinemia (HHcy) has been shown to induce endothelial dysfunction in part as a result of enhanced oxidative stress. Function and survival of endothelial progenitor cells (EPCs, defined as sca1(+) c-kit(+) flk-1(+) bone marrow-derived cells), which significantly contribute to neovascularization and endothelial regeneration, depend on controlled production of reactive oxygen species (ROS). Mice heterozygous for the gene deletion of methylenetetrahydrofolate reductase (Mthfr(+/-)) have a 1.5- to 2-fold elevation in plasma homocysteine. This mild HHcy significantly reduced the number of circulating EPCs as well as their differentiation. Mthfr deficiency was also associated with increased ROS production and reduced nitric oxide (NO) generation in Mthfr(+/-) EPCs. Treatment of EPCs with sepiapterin, a precursor of tetrahydrobiopterin (BH(4)), a cofactor of endothelial nitric oxide synthase (eNOS), significantly reduced ROS and improved NO production. mRNA and protein expression of eNOS and the relative amount of eNOS dimer compared with monomer were decreased by Mthfr deficiency. Impaired differentiation of EPCs induced by Mthfr deficiency correlated with increased senescence, decreased telomere length, and reduced expression of SIRT1. Addition of sepiapterin maintained cell senescence and SIRT1 expression at levels comparable to the wild type. Taken together, these results demonstrate that Mthfr deficiency impairs EPC formation and increases EPC senescence by eNOS uncoupling and downregulation of SIRT1.

Gene therapy for hemophilia B and other hereditary plasma protein deficiencies showed great promise in pre-clinical and early clinical trials. However, safety concerns about in vivo delivery of viral vectors and poor post-transplant survival of ex vivo modified cells remain key hurdles for clinical translation of gene therapy. We here describe a 3D scaffold system based on porous hydroxyapatite-PLGA composites coated with biomineralized collagen 1. When combined with autologous gene-engineered factor IX (hFIX) positive mesenchymal stem cells (MSCs) and implanted in hemophilic mice, these scaffolds supported long-term engraftment and systemic protein delivery by MSCs in vivo. Optimization of the scaffolds at the macro-, micro- and nanoscales provided efficient cell delivery capacity, MSC self-renewal and osteogenesis respectively, concurrent with sustained delivery of hFIX. In conclusion, the use of gene-enhanced MSC-seeded scaffolds may be of practical use for treatment of hemophilia B and other plasma protein deficiencies., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Unanticipated elevation of the INR is common in patients receiving warfarin. We performed a prospective cohort study of 107 warfarin-treated patients with INR values of more than 10 who received a single 2.5 mg dose of oral vitamin K. During the first week, one patient experienced major bleeding, and one died. In the first 90 days after enrolment four patients had major bleeding (3.7%, 1.0% to 9.3%), eight patients (7.5%, 3.3% to 14.2%) died and two had objectively confirmed thromboembolism. Based on our low rate of observed major bleeding we conclude that 2.5 mg of oral vitamin K is a reasonable treatment for patients with INR values of more than 10 who are not actively bleeding.

An ideal amphipathic peptide (IAP), composed of simply lysine and leucine residues in a 1:2 ratio (K(7)L(15)), specifically prolongs in vitro coagulation assays that use phospholipids, such as the activated partial thromboplastin time (APTT). The main hypothesis of the present work is that IAP's anticoagulant effect occurs by competing with phospholipid membranes in in vitro coagulation reactions. We verified this hypothesis by employing different phospholipid-dependent coagulation assays, such as the APTT, the dilute prothrombin time (dPT) and the dilute Russell viper venom time (dRVVT) with both low and high amounts of phospholipids. We show that coagulation times are prolonged by IAP in a concentration-dependent manner, and that this prolongation is abrogated by adding excess phospholipid, demonstrating a phospholipid dependence for this inhibition. Using an ELISA-based binding assay, we show IAP inhibits the binding of one of the vitamin K-dependent coagulation factors, factor X, to phospholipid membranes. This is further confirmed with fluorescence spectroscopy, where the interaction of IAP and factor X is inhibited by phospholipid. In summary, this work demonstrates that IAP can act as an anticoagulant by impairing the interaction of coagulation factors with phospholipid membranes and provides a paradigm for the development of novel anticoagulants., (Copyright 2009 Elsevier Inc. All rights reserved.)

The fabrication of a novel in situ forming hydrogel composed of a multifunctional poly(ethylene glycol) (PEG) N-hydroxysuccinimide ester (NHS) and poly(allylamine hydrochloride) (PAA) was investigated. FTIR confirmed that PAA formed the hydrogel matrix (i.e., the formation of a PAA-like hydrogel). A factorial experiment was conducted to identify the key parameters that controlled gelation time, gel content, and swelling properties. The type of PEG (e.g., 4- and 6-arm) appeared to play a major role in determining all three performance parameters, with the greatest effect on gelation time. Other influencing factors include (a) the PEG concentration, which contributes to the gelation time and gel content; (b) pH of the buffer used for dissolving each polymer, which can affect the gelation time; and (c) PAA molecular weights, which contribute to the gel content and swelling. The concentration of PAA solution had no significant effects on hydrogel formation and properties within the investigated range, presumably due to negligible changes in the crosslinking density of the hydrogels. The PAA buffer pH influenced the gel content as well. Finally, thromboelastography was used to examine the effects of each polymer and their in situ gelation on blood coagulation in vitro. All individual polymers tested reduced clot strength, while the gelation of the polymers enhanced overall procoagulant effects. These results suggest that the biomaterial can be optimized to provide a combination of rapid gelation and swelling properties suitable for hemorrhage control and thus warrant further studies in animal bleeding models.

Background: Growth Arrest Specific gene product 6 (gas6) is a gamma-carboxylated protein that protects endothelial cells against apoptosis. Gas6 has previously been shown to induce phospatidyl-3-inositol-kinase (PI3K)/Akt signaling. Other studies have demonstrated a link between PI3K/Akt signaling and forkhead transcription factors in endothelial cells., Objective: To test the hypothesis that gas6 promotes cell survival via a forkhead-dependent pathway., Results and Conclusions: Treatment of serum-starved human umbilical vein endothelial cells (HUVECs) with gas6 induced time-dependent phosphorylation and nuclear exclusion of FOXO1a. This effect was suppressed by the PI3K inhibitor wortmannin, demonstrating that FOXO1a phosphorylation is PI3-kinase dependent. Transduction of HUVECs with a phosphorylation-resistant form of FOXO1a [triple mutant (TM)-FOXO1a] abrogated the pro-survival effect of gas6 on serum-starved endothelial cells. Finally, treatment of serum-starved HUVECs with gas6 resulted in a reduction of FOXO1a transcriptional activity and downregulation of the pro-apoptotic gene, p27(kip1). Taken together, these findings suggest that gas6 protects endothelial cells from apoptosis by a mechanism that involves PI3K-Akt-dependent inactivation of FOXO1a.

The modification of glutamic acid residues to gamma-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent enzyme gamma-glutamylcarboxylase. Despite ubiquitous expression of the gamma-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-containing proteins after the liver, we sought to identify Gla proteins secreted by bone marrow-derived mesenchymal stromal cells (MSCs). We used a proteomics approach to screen the secretome of MSCs with a combination of two-dimensional gel electrophoresis and tandem mass spectrometry. The most abundant Gla-containing protein secreted by MSCs was identified as periostin, a previously unrecognized gamma-carboxylated protein. In silico amino acid sequence analysis of periostin demonstrated the presence of four consensus gamma-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation and purification of the recombinant protein. Carboxylation of periostin could be inhibited by warfarin in MSCs, demonstrating its dependence on the presence of vitamin K. We were able to demonstrate localization of carboxylated periostin to bone nodules formed by MSCs in vitro, suggesting a role in extracellular matrix mineralization. Our data also show that another fasciclin I-like protein, betaig-h3, contains Gla. In conclusion, periostin is a member of a novel vitamin K-dependent gamma-carboxylated protein family characterized by the presence of fasciclin domains. Furthermore, carboxylated periostin is produced by bone-derived cells of mesenchymal lineage and is abundantly found in mineralized bone nodules in vitro.

On the basis of previous evidence that amphipathic helical peptides accelerate Factor IXa activation of Factor X [Blostein, Rigby, Furie, Furie and Gilbert (2000) Biochemistry 39, 12000-12006], the present study was designed to assess the procoagulant activity of an IAP (ideal amphipathic peptide) of Lys(7)Leu(15) composition. The results show that IAP accelerates Factor X activation by Factor IXa in a concentration-dependent manner and accelerates thrombin generation by Factor Xa with a comparable peptide- and substrate-concentration-dependence. A scrambled helical peptide with the same amino acid composition as IAP, but with its amphipathicity abolished, eliminated most of the aforementioned effects. The Gla (gamma-carboxyglutamic acid)-rich domain of Factor X is required for IAP activity, suggesting that this peptide behaves as a phospholipid membrane. This hypothesis was confirmed, using fluorescence spectroscopy, by demonstrating direct binding between IAP and the Gla-rich domain of Factor X. In addition, the catalytic efficiencies of the tenase and prothrombinase enzymatic complexes, containing cofactors Factor VIIIa and Factor Va respectively, are enhanced by IAP. Finally, we show that IAP delays clot lysis in vitro. In summary, these observations demonstrate that IAP not only enhances essential procoagulant reactions required for fibrin generation, but also inhibits fibrinolysis, suggesting a potential role for IAP as a haemostatic agent.

Gas6 is a gamma-carboxylated ligand for the receptor tyrosine kinase Axl. Gas6-Axl interactions can rescue endothelial cells from apoptosis, and this study examined the intracellular signaling mechanisms responsible for this phenomenon. Using flow cytometry, we first confirmed that Gas6 can abrogate apoptosis induced by serum starvation of primary cultures of human umbilical vein endothelial cells (HUVECs). This effect is mediated through phosphorylation of the serine-threonine kinase Akt, with maximal phosphorylation observed after 4 h of treatment with 100 ng/ml Gas6. Inhibition of Akt phosphorylation and abrogation of gas6-mediated survival of HUVECs by wortmannin implicated phosphatidylinositol 3-kinase as the mediator of Akt phosphorylation. Dominant negative Akt constructs largely abrogated the protective effect of Gas6 on HUVECs, underscoring the importance of Akt activation in Gas6-mediated survival. Several downstream regulators of this survival pathway were identified in HUVECs, namely, NF-kappaB as well as the antiapoptotic and proapoptotic proteins Bcl-2 and caspase 3, respectively. We showed that NF-kappaB is phosphorylated early after Gas6 treatment as evidenced by doublet formation on Western blotting. As well, the level of Bcl-2 protein increased, supporting the notion that the Bcl-2 antiapoptotic pathway is stimulated. The levels of expression of the caspase 3 activation products p12 and p20 decreased with Gas6 treatment, consistent with a reduction in proapoptotic caspase 3 activation. Taken together, these experiments provide new information about the mechanism underlying Gas6 protection from apoptosis in primary endothelial cell cultures.

During blood coagulation factor IXa binds to factor VIIIa on phospholipid membranes to form an enzymatic complex, the tenase complex. To test whether there is a protein-protein contact site between the gamma-carboxyglutamic acid (Gla) domain of factor IXa and factor VIIIa, we demonstrated that an antibody to the Gla domain of factor IXa inhibited factor VIIIa-dependent factor IXa activity, suggesting an interaction of the factor IXa Gla domain with factor VIIIa. To study this interaction, we synthesized three analogs of the factor IXa Gla domain (FIX1-47) with Phe-9, Phe-25, or Val-46 replaced, respectively, with benzoylphenylalanine (BPA), a photoactivatable cross-linking reagent. These factor IX Gla domain analogs maintain native tertiary structure, as demonstrated by calcium-induced fluorescence quenching and phospholipid binding studies. In the absence of phospholipid membranes, FIX1-47 was able to inhibit factor IXa activity. This inhibition is dependent on the presence of factor VIIIa, suggesting a contact site between the factor IXa Gla domain and factor VIIIa. To demonstrate a direct interaction we did cross-linking experiments with FIX1-479BPA, FIX1-4725BPA, and FIX1-4746BPA. Covalent cross-linking to factor VIIIa was observed primarily with FIX1-4725BPA and to a much lesser degree with FIX1-4746BPA. Immunoprecipitation experiments with an antibody to the C2 domain of factor VIIIa indicate that the factor IX Gla domain cross-links to the A3-C1-C2 domain of factor VIIIa. These results suggest that the factor IXa Gla domain contacts factor VIIIa in the tenase complex through a contact site that includes phenylalanine 25 and perhaps valine 46.

The role of the Gla domain of human prothrombin in interaction with the prothrombinase complex was studied using a peptide with the sequence of the first 46 residues of human prothrombin, PT-(1-46). Intrinsic fluorescence measurements showed that PT-(1-46) undergoes a conformational alteration upon binding calcium; this conclusion is supported by one-dimensional (1)H NMR spectroscopy, which identifies a change in the chemical environment of tryptophan 41. PT-(1-46) binds phospholipid membranes in a calcium-dependent manner with a K(d) of 0.5 microm and inhibits thrombin generation by the prothrombinase complex with a K(i) of 0.8 microm. In the absence of phospholipid membranes, PT-(1-46) inhibits thrombin generation by factor Xa in the presence but not absence of factor Va, suggesting that PT-(1-46) inhibits prothrombin-factor Va binding. The addition of factor Va to PT-(1-46) labeled with the fluorophore sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (PT-(1-46)AMCA) caused a concentration-dependent quenching of AMCA fluorescence, providing direct evidence of a PT-(1-46)-factor Va interaction. The K(d) for this interaction was 1.3 microm. These results indicate that the N-terminal Gla domain of human prothrombin is a functional unit that has a binding site for factor Va. The prothrombin Gla domain is important for interaction of the substrate with the prothrombinase complex.

Blood coagulation factor IXa gains proteolytic efficiency upon binding to a phospholipid membrane. We have found that an amphipathic, membrane-binding peptide from the C2 domain of factor VIII, fVIII(2303)(-23), enhances proteolytic efficiency of factor IXa in the absence of phospholipid membranes. This enhancement is the result of a reduction in the K(M) for the substrate, factor X, with little effect on the k(cat). Enhanced function requires interaction of the gamma-carboxyglutamic acid (Gla) domains of factor IXa and factor X since (i) a synthetic peptide comprising the Gla domain of factor IXa and antibodies directed to the Gla domain of factor IXa inhibit this acceleration, (ii) the acceleration is Ca(II) dependent, and (iii) conversion of Gla-domainless factor X is not affected by the presence of fVIII(2303)(-23). The effect of fVIII(2303)(-23) on factor IXa parallels the enhanced function produced by phosphatidylserine-containing bilayers, and fVIII(2303)(-23) does not further enhance function of factor IXa when phospholipid vesicles are present. The critical feature of fVIII(2303)(-23) is apparently its amphipathic helix-forming structure [Gilbert, G. E., and Baleja, J. D. (1995) Biochemistry 34, 3022-3031] because other alpha-helical peptides such as a homologous peptide from the C2 domain of factor V and melittin have similar effects. Diastereomeric analogues of fVIII(2303)(-23) and melittin, which have reduced helical content, do not support factor IXa activity. A truncated peptide of fVIII(2303)(-23) with three C-terminal residues deleted retains alpha-helical content but loses capacity to enhance factor X cleavage, suggesting that a minimum length of alpha-helix is required. Although these results probably do not illuminate the physiologic function of the factor VIII peptide corresponding to fVIII(2303)(-23), they demonstrate a novel, membrane-mimetic role of amphipathic helical peptides in supporting function of factor IXa.

The blood coagulation and regulatory proteins that contain gamma-carboxyglutamic acid are a part of a unique class of membrane binding proteins that require calcium for their interaction with cell membranes. Following protein biosynthesis, glutamic acids on these proteins are converted to gamma-carboxyglutamic acid (Gla) in a reaction that requires vitamin K as a cofactor. The vitamin K-dependent proteins undergo a conformational transition upon metal ion binding, but only calcium ions mediate protein-phospholipid interaction. To identify the site on Factor IX that is required for phospholipid binding, we have determined the three-dimensional structure of the Factor IX Gla domain bound to magnesium ions by NMR spectroscopy. By comparison of this structure to that of the Gla domain bound to calcium ions, we localize the membrane binding site to a highly ordered structure including residues 1-11 of the Gla domain. In the presence of Ca2+, Factor IX Gla domain peptides that contain the photoactivatable amino acid p-benzoyl-L-phenylalanine at positions 6 or 9 cross-link to phospholipid following irradiation, while peptides lacking this amino acid analog or with this analog at position 46 did not cross-link. These results indicate that the NH2 terminus of the Gla domain, specifically including leucine 6 and phenylalanine 9 in the hydrophobic patch, is the contact surface on Factor IX that interacts with the phospholipid bilayer.

Two major studies have established clinical criteria for the diagnosis of veno-occlusive disease of the liver (VOD) after bone marrow transplantation (BMT). McDonald and co-workers defined VOD as the onset of two of the following occurring before day 30 post-BMT: (a) jaundice (bilirubin > 27 mmol/l), (b) tender hepatomegaly, and (c) ascites or weight gain. In contrast, Jones and co-workers defined VOD as the onset, before day 21 post-BMT, of hyperbilirubinemia (bilirubin > 34 mmol/l) as well as two of the following: (a) hepatomegaly, (b) ascites, and (c) weight gain. We retrospectively reviewed the occurrence of VOD in 101 patients transplanted primarily for hematologic malignancies between 1979 and 1990, applying both sets of criteria. Of the 101 patients, eight (7.9%) fulfilled the Jones criteria whereas 32 (31.7%) had VOD according to the McDonald criteria (p < 0.001). Early mortality (prior to 50 days post-BMT) was 75% (6/8) in patients who fulfilled the Jones criteria but only 28.1% (9/32) in the McDonald group (p < 0.005). Overall, mortality in each group was 75% (6/8) and 65.6% (21/32), respectively. All of the six patients with VOD according to the Jones criteria who died had evidence of hepatic failure. Of the 32 patients who fulfilled the McDonald criteria, eight have also fulfilled the Jones criteria and are described above. Of the remaining 24 patients, 22 had complete resolution of VOD as defined by these criteria within 50 days of BMT, none developed hepatic failure, and 15 died.(ABSTRACT TRUNCATED AT 250 WORDS)