Resistance exercise affects catheter-related thrombosis in rats through miR-92a-3p, oxidative stress and the MAPK/NF-κB pathway.

BACKGROUND: MiR-92a-3p and oxidative stress are associated with catheter-related thrombosis (CRT). As a kind of physical intervention, resistance exercise can effectively promote blood circulation. In this study, we investigated the roles of miR-92a-3p, oxidative stress and the P38 mitogen-activated protein kinase/nuclear factor-κB (MAPK/NF-κB) pathway in CRT during resistance exercise. METHODS: The rat CRT model was used for resistance exercise intervention. Moreover, pathological changes from the right jugular vein to the right auricle were observed under an electron microscope. In addition, reactive oxygen species (ROS) production, malondialdehyde (MDA) activity and heme oxygenase (HO-1) level in rat serum were detected via ELISA. The expression levels of miR-92A-3p and HO-1 in the vascular tissues of the rats were determined via real-time quantitative PCR. Additionally, the expression levels of HO-1, NF-κB P65, p38MAPK and IκBa in the venous tissues of the rats were analysed by Western blot analysis. RESULTS: The pathological results showed that the thrombosis incidence rate in the CRT + RE group was lower than that in the CRT group. In the CRT group, the expression levels of ROS and MDA, which are markers related to oxidative stress in serum, significantly increased whilst the expression of HO-1 decreased. In the venous tissue, the expression of miR-92a-3p increased, the level of HO-1 decreased, the levels of p38MAPK and NF-κB p65 significantly increased but that of P-IκBa and IκBa significantly decreased. In the CRT + RE group, after administering the resistance exercise intervention, ROS production and MDA activity in serum significantly decreased, the expression level of HO-1 increased and the expression level of miR-92a-3p in the venous tissues significantly decreased and was negatively correlated with that of HO-1. The levels of p38MAPK and NF-κB p65 significantly decreased but that of P- IκBa and IκBa significantly increased. CONCLUSION: Resistance exercise intervention downregulated miR-92a-3p expression, repaired oxidative stress injury and prevented CRT formation.

Tuning the Thromboinflammatory Response to Venous Flow Interruption by the Ectonucleotidase CD39.

Objective- Leukocyte flux contributes to thrombus formation in deep veins under pathological conditions, but mechanisms that inhibit venous thrombosis are incompletely understood. Ectonucleotide di(tri)phosphohydrolase 1 ( ENTPD1 or Cd39), an ectoenzyme that catabolizes extracellular adenine nucleotides, is embedded on the surface of endothelial cells and leukocytes. We hypothesized that under venous stasis conditions, CD39 regulates inflammation at the vein:blood interface in a murine model of deep vein thrombosis. Approach and Results- CD39-null mice developed significantly larger venous thrombi under venous stasis, with more leukocyte recruitment compared with wild-type mice. Gene expression profiling of wild-type and Cd39-null mice revealed 76 differentially expressed inflammatory genes that were significantly upregulated in Cd39-deleted mice after venous thrombosis, and validation experiments confirmed high expression of several key inflammatory mediators. P-selectin, known to have proximal involvement in venous inflammatory and thrombotic events, was upregulated in Cd39-null mice. Inferior vena caval ligation resulted in thrombosis and a corresponding increase in both P-selectin and VWF (von Willebrand Factor) levels which were strikingly higher in mice lacking the Cd39 gene. These mice also manifest an increase in circulating platelet-leukocyte heteroaggregates suggesting heterotypic crosstalk between coagulation and inflammatory systems, which is amplified in the absence of CD39. Conclusions- These data suggest that CD39 mitigates the venous thromboinflammatory response to flow interruption.

Role of miR-92a-3p, oxidative stress, and p38MAPK/NF-κB pathway in rats with central venous catheter related thrombosis.

BACKGROUND: miR-92a-3p and oxidative stress are reportedly associated with venous thrombosis. However, the role of miR-92a-3p and oxidative stress in catheter-related thrombosis (CRT) remains ambiguous. Herein, we studied the roles of miR-92a-3p, oxidative stress, and p38-mitogen-activated protein kinase/nuclear factor kappa-B (MAPK/NF-κB) pathway in CRT. METHODS: Forty-five male rats were randomly and equally divided into control, sham operation, and CRT groups. The rats were sacrificed after 10 days. Reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) levels in the serum were determined by enzyme-linked immunosorbent assay (ELISA). The expression levels of miR-92a-3p, heme oxygenase-1 (HO-1), NF-κB p65, and p38 MAPK in the venous tissues were detected with quantitative polymerase chain reaction (qPCR) and Western blot. RESULTS: Thrombosis was observed only in the CRT group. Compared with the levels in the control and sham operation groups, ROS and MDA significantly increased in the CRT group, but SOD significantly decreased. qPCR and Western blot results showed that miR-92a-3p, HO-1, p38 MAPK, and NF-κB p65 expression was significantly upregulated in the venous tissues of the CRT group. Moreover, miR-92a-3p was positively correlated with HO-1, which was positively correlated with p38 MAPK and NF-κB p65. CONCLUSION: miR-92a-3p was correlated with oxidative stress in CRT. miR-92a-3p and oxidative stress contributed to endothelial dysfunction and simultaneously was associated with CRT.

Downregulation of miR-103a-3p Contributes to Endothelial Progenitor Cell Dysfunction in Deep Vein Thrombosis Through PTEN Targeting.

OBJECTIVE: Bone-marrow-derived endothelial progenitor cells (EPCs) can accelerate the dissolution of thrombi. However, EPC functions are weakened in deep vein thrombosis (DVT), and miR-130a-3p is downregulated in DVT. As little is known about the function of miR-130a-3p in EPCs, we aimed to explore the effects of miR-130a-3p on EPC functions and the mechanisms of miR-130a-3p regulation of EPCs in DVT. METHODS: The EPCs were transfected with miR-130a-3p mimics or miR-130a-3p inhibitor. Migration and angiogenesis of EPCs were detected by wound healing, Transwell, and tube formation assays. Dual luciferase assay was used to test the relation of miR-130a-3p and phosphatase and tensin homolog (PTEN). Protein and mRNA levels of associated genes were measured by western blotting (WB) and qRT-PCR. RESULTS: miR-103a-3p could promote EPC migration and angiogenesis, and it was also downregulated in EPCs isolated from DVT patients. Moreover, PTEN was a target of miR-130a-3p. Upregulation of PTEN rescued the auxoaction of miR-130a-3p in EPC function. CONCLUSIONS: Downregulation of miR-103a-3p contributes to EPC dysfunction in DVT via targeting PTEN. Thus, miR-130a-3p may be a potential target for DVT treatment.

Should We Screen for Janus Kinase 2 V617F Mutation in Cerebral Venous Thrombosis?

BACKGROUND: The presence of Janus Kinase 2 (JAK2) V617F mutation represents a major diagnostic criterion for detecting myeloproliferative neoplasms (MPN) and even in the absence of overt MPN, JAK2 V617F mutation is associated with splanchnic vein thrombosis. However, the actual prevalence and diagnostic value of the JAK2 V617F mutation in patients with cerebral venous thrombosis (CVT) are not known. The aims of this study were to assess the prevalence of JAK2 V617F mutation in a large group of consecutive CVT patients, to detect clinical, biological, and radiological features associated with the mutation, and to determine the long-term venous thrombosis recurrence rate in CVT patients with JAK2 mutation but without overt MPN in order to recommend the best preventive treatment. METHODS: This was a prospective study conducted on consecutive patients with a first-ever radiologically confirmed CVT. JAK2 V617F mutation analysis was assessed in all the study subjects. JAK2 V617F-positive patients were followed up to detect new venous thrombotic events. RESULTS: Of the 125 included subjects, 7 were found to have JAK2 V617F mutation (5.6%; 95% CI 2.3-11.2). Older age (p = 0.039) and higher platelet count (p = 0.004) were independently associated with JAK2 V617F positivity in patients without overt MPN. During a mean follow-up period of 59 (SD 46) months, 2 JAK2 V617F-positive patients presented with 4 new venous thromboembolic events. CONCLUSIONS: Screening for the JAK2 V617F mutation in CVT patients seems to be useful even in the absence of overt MPN and/or in the presence of other risk factors for CVT because of its relatively high prevalence and the risk of thrombosis recurrence.

Deficiency of superoxide dismutase impairs protein C activation and enhances susceptibility to experimental thrombosis.

OBJECTIVE: Clinical evidence suggests an association between oxidative stress and vascular disease, and in vitro studies have demonstrated that reactive oxygen species can have prothrombotic effects on vascular and blood cells. It remains unclear, however, whether elevated levels of reactive oxygen species accelerate susceptibility to experimental thrombosis in vivo. APPROACH AND RESULTS: Using a murine model with genetic deficiency in superoxide dismutase-1 (SOD1), we measured susceptibility to carotid artery thrombosis in response to photochemical injury. We found that SOD1-deficient (Sod1(-/-)) mice formed stable arterial occlusions significantly faster than wild-type (Sod1(+/+)) mice (P<0.05). Sod1(-/-) mice also developed significantly larger venous thrombi than Sod1(+/+) mice after inferior vena cava ligation (P<0.05). Activation of protein C by thrombin in lung was diminished in Sod1(-/-) mice (P<0.05 versus Sod1(+/+) mice), and generation of activated protein C in response to infusion of thrombin in vivo was decreased in Sod1(-/-) mice (P<0.05 versus Sod1(+/+) mice). SOD1 deficiency had no effect on the expression of thrombomodulin, endothelial protein C receptor, or tissue factor in lung or levels of protein C in plasma. Exposure of human thrombomodulin to superoxide in vitro caused oxidation of multiple methionine residues, including critical methionine 388, and a 40% decrease in thrombomodulin-dependent activation of protein C (P<0.05). SOD and catalase protected against superoxide-induced methionine oxidation and restored protein C activation in vitro (P<0.05). CONCLUSIONS: SOD prevents thrombomodulin methionine oxidation, promotes protein C activation, and protects against arterial and venous thrombosis in mice.

MMP3 -1171 5A/6A Promoter Genotype Influences Serum MMP3 Levels and Is Associated with Deep Venous Thrombosis.

BACKGROUND: The aim of this study was to investigate the roles of MMP3 (matrix metalloproteinase-3) gene polymorphism and protein expression in deep venous thrombosis (DVT) among Chinese Han population. METHODS: A total of 280 subjects were included in this study and categorized as case group (144 DVT patients) and control group (136 healthy individuals). Polymerase chain reaction-restriction fragment length polymorphism was used to detect MMP3 promoter -1171 5A>6A genotype and allele frequencies. MMP3 serum levels were measured by enzyme-linked immunosorbent assay. SPSS version 18.0 statistical software was used for data analysis. RESULTS: There was significant difference in genotype frequencies of MMP3 gene -1171 5A>6A between the case group and the control group (all P < 0.05). Furthermore, the 6A allele on MMP3 -1171 5A>6A may be associated with increased risk of DVT (odds ratio 1.961, 95% confidence interval 1.309-2.939, P < 0.01). The MMP3 serum level in DVT patients was markedly higher than the control group (case group: 28.45 ± 10.97 vs. CONTROL GROUP: 18.18 ± 9.03, P < 0.05). Serum MMP3 level in DVT patients carrying 5A/6A and 6A/6A genotypes was higher than the control group (P < 0.05). The bilateral calf circumference difference was significantly higher in DVT patients than the control group among all the genotypes at MMP3 gene -1171 5A>6A (all P < 0.05). CONCLUSION: MMP3 gene -1171 5A>6A polymorphism and upregulated protein expression may be associated with DVT risk in Chinese Han population.

Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice.

Deep vein thrombosis and pulmonary embolism are major health problems associated with high mortality. Recently, DNA-based neutrophil extracellular traps (NETs) resulting from the release of decondensed chromatin, were found to be part of the thrombus scaffold and to promote coagulation. However, the significance of nuclear decondensation and NET generation in thrombosis is largely unknown. To address this, we adopted a stenosis model of deep vein thrombosis and analyzed venous thrombi in peptidylarginine deiminase 4 (PAD4)-deficient mice that cannot citrullinate histones, a process required for chromatin decondensation and NET formation. Intriguingly, less than 10% of PAD4(-/-) mice produced a thrombus 48 h after inferior vena cava stenosis whereas 90% of wild-type mice did. Neutrophils were abundantly present in thrombi formed in both groups, whereas extracellular citrullinated histones were seen only in thrombi from wild-type mice. Bone marrow chimera experiments indicated that PAD4 in hematopoietic cells was the source of the prothrombotic effect in deep vein thrombosis. Thrombosis could be rescued by infusion of wild-type neutrophils, suggesting that neutrophil PAD4 was important and sufficient. Endothelial activation and platelet aggregation were normal in PAD4(-/-) mice, as was hemostatic potential determined by bleeding time and platelet plug formation after venous injury. Our results show that PAD4-mediated chromatin decondensation in the neutrophil is crucial for pathological venous thrombosis and present neutrophil activation and PAD4 as potential drug targets for deep vein thrombosis.

MMP-3-1612 polymorphism - a risk factor for deep venous thrombosis formation.

BACKGROUND: We investigated the association of the 5A/6A polymorphism in the promoter region at -1612 of the matrix metalloproteinase-3 gene (MMP-3-1612) and deep venous thrombosis (DVT). PATIENTS, MATERIALS AND METHODS: The distribution of the MMP-3 (-1612 5A/6A) polymorphism in the case and control groups was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Serum MMP-3 level of two groups was detected using enzyme-linked immunosorbent assay (ELISA). HepG2 cells containing MMP-3-1612 recombinant plasmid were cultured in vitro and the MMP-3 level was defined by luminescence intensity of luciferase. A DVT rat model was built. Serum MMP-3 level in the rats' wounded vein at different time points was detected by ELISA and recorded for investigation of the association between MMP-3 and DVT. Statistical data analysis was conducted with SPSS18.0. RESULTS: On the basis of the observation of MMP-3-1612 genotype frequency and allele frequency in the case and control groups, we identified significantly higher MMP-3-1612 5A allele frequency and higher serum MMP-3 level in the case group than in the control group (both P < 0.05). According to in vitro luciferase measurements, the 5A allele had higher transcriptional activity than the 6A allele. As observed in the rat model, serum MMP-3 level increased with time passing and thrombosis formation after modelling. CONCLUSIONS: The MMP-3-1612 5A/6A polymorphism may effect serum MMP-3 level and over-expression of serum MMP-3 level may be a risk factor for DVT formation.

The impact of sphingosine kinase 1 on the prognosis of hepatocellular carcinoma patients with portal vein tumor thrombus.

BACKGROUND: Though there is considerable evidence that sphingosine kinase 1(SPHK1) plays a key role in hepatocellular carcinoma(HCC) progression, the prognostic value of SPHK1 expression in HCC with portal vein tumor thrombus (PVTT) remains unclear. Aims. The purpose of this study was to investigate the relationship of SPHK1 expression with PVTT and HCC recurrence after hepatectomy. METHODS: After screening of gene expression profiling of tumor cell lines, real-time PCR and immunohistochemistry were used to investigate the SPHK1 expression in PVTT and HCC samples. The clinical data of 199 HCC patients with nonmain PVTT who underwent liver resection with curative intention were studied. RESULTS: We identified SPHK1 as the most over-expressed gene in PVTT via gene expression profiling of one human PVTT cell line (CSQT-2). SPHK1 expression was an independent factor affecting survival (hazard ratio [HR] 1.799, 95% confidence interval [CI] 1.337-2.368, P < 0.001) and tumor recurrence (HR 1.451, 95% CI 1.087-1.935, P = 0.011). Patients with SPHK1 over-expression had a poorer prognosis than those with SPHK1 under-expression (P < 0.001 and P = 0.011 for survival and tumor recurrence). CONCLUSIONS: SPHK1 might represent a novel and useful prognostic marker of HCC progression in patients with PVTT.

The JAK2 46/1 haplotype (GGCC) in myeloproliferative neoplasms and splanchnic vein thrombosis: a pooled analysis of 26 observational studies.

Numbers of observational studies suggest that the JAK2 46/1 (GGCC) haplotype may increase the risk of myeloproliferative neoplasms (MPNs) and splanchnic vein thrombosis (SVT), but the results remain controversial. We aimed to examine the association between the JAK2 46/1 haplotype and risk of MPNs and SVT by conducting a meta-analysis. PubMed, EMBASE, Cochrane Library, CBM, and CNKI databases were searched to identify eligible studies without restrictions and by reviewing reference lists of obtained articles. Both fixed and random-effects models were used to calculate the summary risk estimates. We identified 26 observational studies of the JAK2 46/1 haplotype and risk of MPNs and SVT involving 8,561 cases and 7,434 participants. In the overall analysis, it was found that the JAK2 46/1 haplotype significantly elevated the risk of MPNs (rs10974944: C vs T: odds ratio (OR) = 2.19, 95 % confidence interval (CI) = 1.86-2.57, P < 0.0001; CC vs TT: OR = 4.63, 95 % CI = 3.32-6.47, P < 0.0001; CT vs TT: OR = 2.49, 95 % CI = 2.11-2.95, P < 0.0001; (CC + CT) vs TT: OR = 2.92, 95 % CI = 2.51-3.39, P < 0.0001; rs12343867: C vs T: OR = 1.88, 95 % CI = 1.59-2.22, P < 0.0001; CC vs TT: OR = 3.16, 95 %CI = 2.14-4.65, P < 0.0001; CT vs TT: OR = 2.04, 95 % CI = 1.51-2.74, P < 0.0001; (CC + CT) vs TT: OR = 2.25, 95 % CI = 1.73-2.95, P < 0.0001) and SVT (C vs T: OR = 1.27, 95 % CI = 1.06-1.52, P = 0.011; CC vs TT: OR = 2.33, 95 % CI = 1.42-3.81, P = 0.001; (CC + CT) vs TT: OR = 1.25, 95 % CI = 1.02-1.53, P = 0.034). There was no evidence of a significant association between the rs12343867 and the risk of SVT in the genetic model (CT vs TT: OR = 1.01, 95 % CI = 0.80-1.29, P = 0.906). This meta-analysis provides new evidence supporting the conclusion that the JAK2 46/1 haplotype enrichment is significantly associated with the development of MPNs and SVT in these patients.

The effect of matrix metalloproteinase 2 and matrix metalloproteinase 2/9 deletion in experimental post-thrombotic vein wall remodeling.

BACKGROUND: Vein wall fibrotic injury following deep venous thrombosis (VT) is associated with elevated matrix metalloproteinases (MMPs). Whether and by what mechanism MMP2 contributes to vein wall remodeling after VT is unknown. METHODS: Stasis VT was produced by ligation of the inferior vena cava and tissue was harvested at 2, 8, and 21 days in MMP2 -/- and genetic wild type (WT) mice. Tissue analysis by immunohistochemistry, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and zymography was performed. RESULTS: Thrombus resolution was less at 8 days in MMP2 -/- compared with WT, evidenced by a 51% increase in VT size (P < .01), and threefold fewer von Willebrand's factor positive channels (P < .05). In MMP2 -/- mice, the main phenotypic fibrotic differences occurred at 8 days post-VT, with significantly less vein wall collagen content (P = .013), fourfold lower procollagen III gene expression (P < .01), but no difference in procollagen I compared with WT. Decreased inflammation in MMP2 -/- vein walls was suggested by ∼ threefold reduced TNFα and IL-1ß at 2 days and 8 days post-VT (P < .05). A fourfold increase in vein wall monocytes (P = .03) with threefold decreased apoptosis (P < .05), but no difference in cellular proliferation at 8 days was found in MMP2 -/- compared with WT. As increased compensatory MMP9 activity was observed in the MMP2 -/-mice, MMP2/9 double null mice had thrombus induced with VT harvest at 8 days. Consistently, twofold larger VT, a threefold decrease in vein wall collagen, and a threefold increase in monocytes were found (all P < .05). Similar findings were observed in MMP9 -/- mice administered an exogenous MMP2 inhibitor. CONCLUSIONS: In stasis VT, deletion of MMP2 was associated with less midterm vein wall fibrosis and inflammation, despite an increase in monocytes. Consideration that VT resolution was impaired with MMP2 (and MMP2/9) deletion suggests direct inhibition will likely also require anticoagulant therapy.

IL-6-174 G > C and MMP-9-1562 C > T polymorphisms are associated with increased risk of deep vein thrombosis in cancer patients.

BACKGROUND: A growing body of evidence shows an increased risk of deep vein thrombosis (DVT) among cancer patients. Novel markers are needed to identify patients prone to develop DVT. The aim of the present study was to determine whether IL-6-174 G > C and MMP-9-1562 C > T polymorphisms may influence the development of DVT in cancer patients. METHODS: Polymorphisms of IL-6 and MMP-9 were analyzed in 320 DNA samples from cancer patients (DVT+ and DVT-) and in 215 healthy donors. IL-6 and MMP-9 plasma levels were also measured by ELISA. RESULTS: Distribution of -174 IL-6 genotype and -1562 MMP-9 were similar between healthy controls and DVT- cancer cases (OR = 0.98 and 1.04, respectively). Different results were obtained by compared healthy controls with DVT+ cancer patients. -174 IL-6 GG polymorphism was associated to DVT (OR = 2.07; 95% CI: 1.30-3.30), as well as -1562 MMP-9 CC polymorphism (OR = 2.60; 95% CI: 1.48-4.57). CONCLUSION: The results of the present study support a model in which the GG and CC genotypes, respectively for IL-6-174 G > C and MMP-9-1562 C > T polymorphisms, are associated with a risk of DVT in cancer patients by inducing the release of IL-6 with subsequent increment of MMP-9. Overall, these findings may contribute to the management of DVT in cancer patients.

Successful liver transplantation in a patient with splanchnic vein thrombosis and pulmonary embolism due to polycythemia vera with Jak2v617f mutation and heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a rare complication of heparin treatment resulting in a severe acquired thrombophilic condition with an associated mortality of about 10 %. We report the first case of successful urgent liver transplantation (LT) in a patient with end-stage liver disease due to a Budd-Chiari syndrome, portal vein thrombosis and pulmonary embolism due to acquired thrombophilia associated to polycythemia vera carrying JAK2V617F gene mutation and HIT in the acute phase. Lepirudin was used to provide anticoagulation in the LT perioperative period that was performed without haemorrhagic and thrombotic complications despite the donor received heparin during liver explantation.

Abdominal contouring procedures increase activity of the coagulation cascade.

One of the most serious complications in plastic surgery is a thromboembolic event. However, little physiologic evidence exists to support the observed hypercoagulable state seen in contouring procedures. Twenty-one consecutive patients were enrolled prospectively to assess thrombin generation, which measures activity of the coagulation cascade, at baseline, intraoperative, and 24 hours after surgery. Compared with preoperative values, total thrombin generation increased by a mean of 997 nM intraoperatively (1.3-fold, P<0.004) and 1406 nM postoperatively (1.4-fold, P<0.001) in 9 patients undergoing abdominoplasty without deep venous thrombosis (DVT) chemoprophylaxis. The mean thrombin generation did not significantly change during or after surgery in 12 patients who received heparin for DVT prophylaxis (P=0.3). Thrombin generation was significantly less in patients receiving chemoprophylaxis compared with those who received no prophylaxis (P<0.01). This suggests abdominal contouring procedures induce a significant increase in the activity of the coagulation cascade that can be prevented by DVT chemoprophylaxis.

Acadesine inhibits tissue factor induction and thrombus formation by activating the phosphoinositide 3-kinase/Akt signaling pathway.

OBJECTIVE: Acadesine, an adenosine-regulating agent and activator of AMP-activated protein kinase, has been shown to possess antiinflammatory activity. This study investigated whether and how acadesine inhibits tissue factor (TF) expression and thrombus formation. METHODS AND RESULTS: Human umbilical vein endothelial cells and human peripheral blood monocytes were stimulated with lipopolysaccharide to induce TF expression. Pretreatment with acadesine dramatically suppressed the clotting activity and expression of TF (protein and mRNA). These inhibitory effects of acadesine were unchanged for endothelial cells treated with ZM241385 (a specific adenosine A(2A) receptor antagonist) or AMP-activated protein kinase inhibitor compound C, and in macrophages lacking adenosine A(2A) receptor or alpha1-AMP-activated protein kinase. In endothelial cells and macrophages, acadesine activated the phosphoinositide 3-kinase/Akt signaling pathway, reduced the activity of mitogen-activated protein kinases, and consequently suppressed TF expression by inhibiting the activator protein-1 and NF-kappaB pathways. In mice, acadesine suppressed lipopolysaccharide-mediated increases in blood coagulation, decreased TF expression in atherosclerotic lesions, and reduced deep vein thrombus formation. CONCLUSION: Acadesine inhibits TF expression and thrombus formation by activating the phosphoinositide 3-kinase/Akt pathway. This novel finding implicates acadesine as a potentially useful treatment for many disorders associated with thrombotic pathology, such as angina pain, deep vein thrombosis, and sepsis.

Prevalence of Janus Kinase 2 mutations in patients with unusual site venous thrombosis.

We aimed to study patients with splanchnic vein thrombosis (SVT) and cerebral vein thrombosis (CVT) searching for JAK2 mutations. We evaluated 14 patients (median age: 41.5 years) with portal vein thrombosis (PVT) = 7; mesenteric vein thrombosis (MVT) = 3; and CVT = 4. JAK2 V617F was assessed by allele specific PCR of peripheral blood DNA. In addition, DNA was sequenced for other JAK2 mutations. Other inherited and acquired thrombophilia risk factors were evaluated. JAK2 V617F was positive in four out of seven patients with PVT and in one CVT patient. These five patients had a diagnosis of myelo-proliferative disorder (MPD) at the moment of the occurrence of thrombosis (n = 2) or later (n = 2). Patients with MVT and CVT were negative for JAK2 V617F, except one patient with CVT and a diagnosis of essential thrombocythemia. No other JAK2 mutations were found in this cohort. Besides MPD, other thrombophilia risk factors were identified in five patients. One patient had MPD as well as thrombophilia risk factor. In this group, 4 out of 7 of the patients with PVT carried the JAK2 V617F mutation with or without overt MPD. However, the investigation of other JAK2 mutations may not be necessary in patients with thrombosis at unusual sites.

[Thrombin-activated inhibitor of fibrinolysis and efficacy and safety of long-term warfarin treatment in patients with venous thromboembolic complications].

AIM: To study effects of thrombin-activated fibrinolysis inhibitor (TAFI) on efficacy and safety of long-term anti-coagulant treatment in patients with venous thromboembolic complications (VTEC). MATERIAL AND METHODS: A total of 111 patients with a history of an episode of deep vein thrombosis (DVT) and/or pulmonary artery thromboembolism (PATE) entered the study. All the patients received unfractionated or low-molecular heparin for at least 5 days than switch on warfarin (target values of INR 2.0-3.0). Baseline blood levels of TAFI were measured. The patients were followed up for 18 months. Recurrent (DVT/TAFI and hemorrhagic complications (HC) were endpoints. Also, frequency of complete lysis of deep vein thrombi was assessed after 12 months of treatment. RESULTS: A TAFI level varied from 50 to 217% (median 106%, interquartile rage 90-133%). TAFI concentration positively correlated with fibrinogen and thromb size. The patients were divided into two groups depending on TAFI content: group 1 patients had low TAFI (under 25th percentile; < 90%); patients of group 2 had high TAFI (above 25th percentile; > 90%). Group 1 patients were characterized by less stable anticoagulation. This association did not depend on genetic characteristics which determine sensitivity to warfarin (CYP2C9 and VKORC1). Low TAFI was associated with reduced risk of DVT for 18 months and higher probability of complete lysis of the thrombi after 12 months of anticoagulant therapy compared to VTEC patients with high TAFI. No differences were found by TAFI level in patients with HC and without HC, but in HC patients low TAFI was associated with spontaneous hemorrhages and bleeding in therapeutic INR values. CONCLUSION: The results of this pilot study evidence that a TAFI level can be one of the factors influencing efficacy and safety of long-term anticoagulant therapy in patients with VTEC on warfarin treatment.

NLRP3 Inflammasome Assembly in Neutrophils Is Supported by PAD4 and Promotes NETosis Under Sterile Conditions.

Neutrophil extracellular trap formation (NETosis) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome assembly are associated with a similar spectrum of human disorders. While NETosis is known to be regulated by peptidylarginine deiminase 4 (PAD4), the role of the NLRP3 inflammasome in NETosis was not addressed. Here, we establish that under sterile conditions the cannonical NLRP3 inflammasome participates in NETosis. We show apoptosis-associated speck-like protein containing a CARD (ASC) speck assembly and caspase-1 cleavage in stimulated mouse neutrophils without LPS priming. PAD4 was needed for optimal NLRP3 inflammasome assembly by regulating NLRP3 and ASC protein levels post-transcriptionally. Genetic ablation of NLRP3 signaling resulted in impaired NET formation, because NLRP3 supported both nuclear envelope and plasma membrane rupture. Pharmacological inhibition of NLRP3 in either mouse or human neutrophils also diminished NETosis. Finally, NLRP3 deficiency resulted in a lower density of NETs in thrombi produced by a stenosis-induced mouse model of deep vein thrombosis. Altogether, our results indicate a PAD4-dependent formation of the NLRP3 inflammasome in neutrophils and implicate NLRP3 in NETosis under noninfectious conditions in vitro and in vivo.

Matrix Metalloproteinase-9 Levels are Associated with Brain Lesion and Persistent Venous Occlusion in Patients with Cerebral Venous Thrombosis.

BACKGROUND: Elucidating mechanisms of brain damage in cerebral venous thrombosis (CVT) would be instrumental to develop targeted therapies and improve prognosis prediction. Matrix metalloproteinase-9 (MMP-9), a gelatinase that degrades major components of the basal lamina, has been associated to blood-brain barrier disruption. We aimed to assess, in patients with CVT, the temporal change in serum concentrations of MMP-9 and its association with key imaging and clinical outcomes. METHODS: Pathophysiology of Venous Infarction-PRediction of InfarctiOn and RecanalIzaTion in CVT (PRIORITy-CVT) was a multicenter prospective cohort study of patients with newly diagnosed CVT. Serial collection of peripheral blood samples performed on day 1, 3, and 8, and standardized magnetic resonance imaging on day 1, 8, and 90. MMP-9 was quantified using enzyme-linked immunosorbent assay in 59 patients and 22 healthy controls. Primary outcomes were parenchymal brain lesion, early evolution of brain lesion, early recanalization, and functional outcome on day 90. RESULTS: CVT patients with parenchymal brain lesion had higher baseline concentrations of MMP-9 compared with controls (adjusted p = 0.001). The area under receiver operating characteristic curve value for MMP-9 for predicting brain lesion was 0.71 (95% confidence interval [CI]: 0.57-0.85, p = 0.009). Patients with venous recanalization showed early decline of circulating MMP-9 and significantly lower levels on day 8 (p = 0.021). Higher MMP-9 on day 8 was associated with persistent venous occlusion (odds ratio: 1.20 [per 20 ng/mL], 95% CI: 1.02-1.43, p = 0.030). CONCLUSION: We report a novel relationship among MMP-9, parenchymal brain damage, and early venous recanalization, suggesting that circulating MMP-9 is a dynamic marker of brain tissue damage in patients with CVT.