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.

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.

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.

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.

Deep Vein Thrombosis is Modulated by Inflammation Regulated via Sirtuin 1/NF-κB Signalling Pathway in a Rat Model.

BACKGROUND: Inflammation plays an important role in thrombus formation, and Sirtuin 1 (SIRT1) negatively regulates inflammation via deacetylating nuclear factor-kappa B. However, the relationship between SIRT1-regulated inflammation and deep vein thrombosis (DVT) is still unknown. OBJECTIVE: The aim of this study was to investigate whether SIRT1 plays a critical role in inferior vena cava (IVC) stenosis-induced DVT. MATERIALS AND METHODS: Thrombus weight and histopathologic analysis of IVC were evaluated at different time points after IVC stenosis in rats. Serum levels of inflammatory cytokines and protein expressions of SIRT1, acetylated p65 (Ace-p65), phosphorylated p65 (p-p65) and tissue factor (TF) in thrombosed IVC were assessed. Besides, the effects of resveratrol (RES, a SIRT1 agonist) and EX527 (a selective SIRT1 inhibitor) on DVT were evaluated. RESULTS: Thrombus weight was increased from 1 to 3 days after IVC stenosis, and then was decreased afterwards. Leukocytes infiltration appeared and serum levels of cytokines were significantly increased in rats of IVC stenosis. SIRT1 protein expression was significantly down-regulated at 1 hour and 1 day after stenosis, while p-p65, Ace-p65 and TF protein expressions appeared a contrary trend. RES reduced thrombus weight, leukocytes infiltration, levels of tumour necrosis factor-α and interleukin-1ß and protein expressions of Ace-p65 and TF as well. Moreover, RES significantly increased the protein and messenger ribonucleic acid expressions of SIRT1, while EX527 abolished the protective effects of RES. CONCLUSION: SIRT1 activation attenuated IVC stenosis-induced DVT via anti-inflammation in rats. Therefore, SIRT1 may be a potential therapeutic target that could ameliorate DVT.

Correlations between methylenetetrahydrofolate reductase gene polymorphisms and venous thromboembolism: A meta-analysis of 99 genetic association studies.

We performed this meta-analysis to better assess the relationship between methylenetetrahydrofolate reductase gene ( MTHFR) polymorphisms and the risk of venous thromboembolism. Eligible studies were searched in PubMed, Medline, Embase, and Web of Science. Odds ratios with 95% confidence intervals were used to assess associations of MTHFR polymorphisms with venous thromboembolism. A total of 99 genetic association studies were enrolled for analyses. Although no positive results were detected in overall analyses for the rs1801131 polymorphism. Further subgroup analyses according to ethnicity of participants and type of disease revealed that the rs1801131 polymorphism was significantly correlated with the risk of pulmonary embolism. For the rs1801133 polymorphism, significant association with the risk of venous thromboembolism was found in the dominant, recessive, and allele models. Further subgroup analyses according to ethnicity of participants revealed that the rs1801133 polymorphism was significantly associated with the risk of venous thromboembolism in Caucasians, East Asians, and West Asians. When we stratified available data according to type of disease, we found that the rs1801133 polymorphism was also significantly correlated with the risk of deep vein thrombosis and pulmonary embolism. In conclusion, our findings indicate that the MTHFR rs1801133 polymorphism may serve as a potential biological marker for venous thromboembolism in Caucasians, East Asians, and West Asians. Moreover, the MTHFR rs1801133 polymorphism may be implicated in the development of deep vein thrombosis and pulmonary embolism, while the MTHFR rs1801131 polymorphism may contribute to the development of pulmonary embolism.

CYP3A Activity and Rivaroxaban Serum Concentrations in Russian Patients with Deep Vein Thrombosis.

BACKGROUND: Rivaroxaban is metabolized in the liver via CYP3A4, the cytochrome involved in the metabolism of nearly 50% of all medications. Thus, its effective concentration depends on multiple pharmacologic parameters. METHODS: The primary goal of our research was to study the correlation between the CYP3A family activity and the safety and efficacy of anticoagulant therapy with rivaroxaban in patients with deep vein thrombosis (DVT). Thirty one patients with DVT aged 21-83 years, 18 men and 13 women, received rivaroxaban (Xarelto) 30 mg/day for 21 days after diagnosis and 20 mg/day for the follow-up period of 6 months. During the study period, Doppler ultrasound was performed weekly to assess the clot dynamics and recanalization time. RESULTS: We found a direct statistically reliable correlation between CYP3A4 activity and both peak and trough rivaroxaban levels. A correlation was also found between the initial clot length and the time to full recanalization r = 0.764 (0.554-0.883), p < 0.0001. No significant link was found between either the glomerular filtration rate and peak rivaroxaban concentrations or between CYP3A4 activity and the treatment effectiveness parameters. No connection between renal function and rivaroxaban concentration was established in our study, which agrees with the clinical trials data that allow unlimited rivaroxaban use in patients with glomerular filtration rate >30 mL/min. CONCLUSIONS: The direct link between the initial clot length and time to full recanalization that has been found means that patients with more advanced stages of thrombosis need more time to reach recanalization than their counterparts with a less severe condition.

Networks of enzymatically oxidized membrane lipids support calcium-dependent coagulation factor binding to maintain hemostasis.

Blood coagulation functions as part of the innate immune system by preventing bacterial invasion, and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca2+- and phosphatidylserine (PS)-dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) enhanced coagulation and restored normal hemostasis in clotting-deficient animals genetically lacking p12-LOX or 12/15-LOX activity. Murine platelets generated 22 eoxPL species, all of which were missing in the absence of p12-LOX. Humans with the thrombotic disorder antiphospholipid syndrome (APS) had statistically significantly increased HETE-PLs in platelets and leukocytes, as well as greater HETE-PL immunoreactivity, than healthy controls. HETE-PLs enhanced membrane binding of the serum protein ß2GP1 (ß2-glycoprotein 1), an event considered central to the autoimmune reactivity responsible for APS symptoms. Correlation network analysis of 47 platelet eoxPL species in platelets from APS and control subjects identified their enzymatic origin and revealed a complex network of regulation, with the abundance of 31 p12-LOX-derived eoxPL molecules substantially increased in APS. In summary, circulating blood cells generate networks of eoxPL molecules, including HETE-PLs, which change membrane properties to enhance blood coagulation and contribute to the excessive clotting and immunoreactivity of patients with APS.

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.

Prostaglandin-endoperoxide synthase-2 deletion affects the natural trafficking of Annexin A2 in monocytes and favours venous thrombosis in mice.

Deep-vein thrombosis (DVT) is a common condition that often leads to pulmonary thromboembolism (VTE) and death. The role of prostaglandin-endoperoxide synthase (PTGS)2 in arterial thrombosis has been well established, whereas its impact in venous thrombosis remains unclear. Here, we showed that PTGS2 deletion predisposes to venous thrombosis as suggested by greater clot firmness and clot elasticity, by higher plasma levels of functional fibrinogen, factor VIII and PAI-1 activity, and proved by bigger thrombi detected after inferior vena cava ligation (IVCL) compared to WT mice. PTGS2-/- thrombi have greater fibrin content, higher number of F4/80+, TF+ and ANXA2+ cells, and lower S100A10+ cells. Remarkably, monocyte depletion reduced thrombus size in mutant mice, suggesting an important role of PTGS2-/- monocytes in this experimental setting. Interestingly, PTGS2 deletion reduced membrane ANXA2, and total S100A10, promoted assembly of ANXA2/p50NF-kB complex and its nuclear accumulation, and induced TF in peritoneal macrophages, whereas ANXA2 silencing decreased dramatically TF. Finally, Carbaprostacyclin treatment prevented venous thrombus formation induced by IVCL in mutant mice, reduced the ANXA2 binding to p50NF-kB subunit and its nuclear trafficking, and decreased TF in PTGS2-/- macrophages. PTGS2 deletion, changing the natural distribution of ANXA2 in monocytes/macrophages, increases TF expression and activity predisposing to venous thrombosis. Interestingly, Carbaprostacyclin treatment, inhibiting nuclear ANXA2 trafficking, controls monocyte TF activity and prevents DVT occurrence. Our data are of help in elucidating the mechanisms by which PTGS2 inhibition increases DVT risk, and suggest a new role for ANXA2 in venous thrombosis.

γ-Glutamyl Transferase Is an Independent Biomarker of Splanchnic Thrombosis in Patients With Myeloproliferative Neoplasm.

Myeloproliferative neoplasms (MPNs) are associated with an increased risk of thrombotic events and constitute the major risk factor of splanchnic venous thrombosis (SVT) in Western countries. Although timely anticoagulation resolves SVT, unrecognized SVT frequently leads to portal hypertension and, potentially, variceal bleeding, which may render anticoagulation difficult. Thus, early identification of SVT development is clinically relevant in MPN patients.In this retrospective analysis, we included 126 patients with MPN and/or SVT referred to our hospital between 2009 and 2014. A total of 86 patients diagnosed with MPN formed the first cohort (PV n = 18, ET n = 16, and MF n = 40), whereas 40 patients who had SVT without adjunct MPN formed a control cohort. Median follow-up period was 960 days. Clinical and laboratory data were collected and analyzed for the identification of potential biomarkers applying descriptive statistics, nonparametric testing, Kaplan-Meier, and logistic regression analysis. The relevance of the identified biomarkers was evaluated in an independent 2nd cohort of 181 patients from the MPN registry of the Study Alliance of Leukemia (SAL-MPN).Thirty-three MPN patients (38%) in the 1st cohort had SVT. Elevated levels of aspartate aminotransferase, alanine aminotransferase, serum bilirubin, or γ-GT were significantly correlated to the presence of SVT. In multivariate testing, CRP and aspartate aminotransferase were predictors for survival and γ-GT remained the only significant variable associated with SVT in MPN patients (P < 0.05). These findings were confirmed in the 2nd cohort comprising 42% of patients with MPN suffering from SVT.Elevated γ-GT levels indicate SVT in MPN patients, whereas CRP levels are independent predictors of patient survival.

Possible Association Between the Methylenetetrahydrofolate Reductase Gene C677T Polymorphism and Preexisting Portal Vein Thrombosis in Liver Transplant Recipients.

OBJECTIVES: Liver transplant in patients with preexisting portal vein thrombosis involves complicated surgical procedures and increased blood loss, indicating the need for further surgical innovations to overcome these difficulties. Patients who are at high risk of developing portal vein thrombosis may benefit from prophylactic anticoagulant therapy while on the transplant wait list. Homozygosity for C677T polymorphism in the methylenetetrahydrofolate reductase gene has been associated with a high plasma homocysteine concentration, which is a risk factor for venous thrombosis. This study investigated the association between C677T polymorphism in the methylenetetrahydrofolate reductase gene and preexisting portal vein thrombosis in patients with liver cirrhosis undergoing liver transplant. MATERIALS AND METHODS: C677T polymorphism in the methylenetetrahydrofolate reductase gene was investigated in 48 patients who underwent liver transplant at Nagoya University. RESULTS: Of 48 patients, 7 (14.6%) had preexisting portal vein thrombosis confirmed at transplant. The frequency of methylenetetrahydrofolate reductase gene C677T genotype in recipients was significantly associated with preexisting portal vein thrombosis (P = .009, with P ≤ .013 considered significant). Logistic regression analysis showed that the TT genotype of the methylenetetrahydrofolate reductase gene was significantly associated with a higher incidence of preexisting portal vein thrombosis compared with the CC and CT genotypes (odds ratio of 14.6, 95% confidence interval, 1.86-115.21; P = .011). CONCLUSIONS: The TT genotype of the methylenetetrahydrofolate reductase gene may be associated with a higher incidence of preexisting portal vein thrombosis, as confirmed at liver transplant. Identification of this genotype in patients with liver cirrhosis at the time of placement on a wait list for liver transplant may enable preventive therapy for portal vein thrombosis in these patients, reducing the complexity of surgical procedures.

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.

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.

Neutrophil elastase-deficient mice form neutrophil extracellular traps in an experimental model of deep vein thrombosis.

UNLABELLED: ESSENTIALS: Neutrophil elastase (NE) plays a role in extracellular trap formation (NETosis) triggered by microbes. The contribution of NE was evaluated in mouse NETosis models of sterile inflammation and thrombosis. NE is not required for mouse neutrophil NET production in vitro with non-infectious stimuli. NE deficiency had no significant effect on thrombosis in the inferior vena cava stenosis model. BACKGROUND: Neutrophil serine proteases have been implicated in coagulation and neutrophil extracellular trap (NET) formation. In human neutrophils, neutrophil elastase (NE) translocates to the nucleus during NETosis and cleaves histones, thus aiding in chromatin decondensation. NE(-/-) mice were shown not to release NETs in response to microbes. However, mouse studies evaluating the role of NE in NET formation in sterile inflammation and thrombosis are lacking. OBJECTIVE: We wished to establish if neutrophils from NE(-/-) mice have a defect in NETosis, similar to peptidylarginine deiminase 4 (PAD4(-/-)) mice, and how this might have an impact on venous thrombosis, a model where NETs are produced and are crucial to thrombus development. METHODS: We performed in vitro NET assays using neutrophils from wild-type (WT), NE(-/-), SerpinB1 (SB1)(-/-) and NE(-/-) SB1(-/-) mice. We compared WT and NE(-/-) animals using the inferior vena cava stenosis model of deep vein thrombosis (DVT). RESULTS: Neutrophil elastase deficiency resulted in a small reduction in ionomycin-induced NET formation in vitro without affecting histone citrullination. However, NET production in response to phorbol 12-myristate 13-acetate or platelet activating factor was normal in neutrophils from two independent NE-deficient mouse lines, and in NE(-/-) SB1(-/-) as compared with SB1(-/-) neutrophils. NE deficiency or inhibition did not prevent NETosis in vivo or DVT outcome. CONCLUSIONS: Neutrophil elastase is not required for NET formation in mice. NE(-/-) mice, which form pathological venous thrombi containing NETs, do not phenocopy PAD4(-/-) mice in in vitro NETosis assays or experimental venous thrombosis. Our study suggests that NET-targeted therapies need to be highly effective to have an impact on DVT.

Methylenetetrahydrofolate Reductase C677T: Hypoplastic Left Heart and Thrombosis.

The incidence of congenital heart defects is higher in infants with mutation of methylenetetrahydrofolate reductase (MTHFR) gene. The MTHFR C677T gene decreases the bioavailability of folate and increases plasma homocysteine, a risk factor for thrombosis. There have been no reported cases in the literature on the clinical implications of this procoagulable state in the setting of cyanotic heart disease, which itself has prothrombotic predisposition. Two patients with hypoplastic left heart syndrome developed postoperative thrombotic complications, both were homozygous for MTHFR C677T. We present these cases and highlight the implications of MTHFR mutation in the management of complex congenital heart disease.

Matrix Metalloproteinase 9 (MMP-9) Regulates Vein Wall Biomechanics in Murine Thrombus Resolution.

OBJECTIVE: Deep venous thrombosis is a common vascular problem with long-term complications including post-thrombotic syndrome. Post-thrombotic syndrome consists of leg pain, swelling and ulceration that is related to incomplete or maladaptive resolution of the venous thrombus as well as loss of compliance of the vein wall. We examine the role of metalloproteinase-9 (MMP-9), a gene important in extracellular remodeling in other vascular diseases, in mediating thrombus resolution and biomechanical changes of the vein wall. METHODS AND RESULTS: The effects of targeted deletion of MMP-9 were studied in an in vivo murine model of thrombus resolution using the FVB strain of mice. MMP-9 expression and activity significantly increased on day 3 after DVT. The lack of MMP-9 impaired thrombus resolution by 27% and this phenotype was rescued by the transplantation of wildtype bone marrow cells. Using novel biomechanical techniques, we demonstrated that the lack of MMP-9 significantly decreased thrombus-induced loss of vein wall compliance. Biomechanical analysis of the contribution of individual structural components showed that MMP-9 affected the elasticity of the extracellular matrix and collagen-elastin fibers. Biochemical and histological analyses correlated with these biomechanical effects as thrombi of mice lacking MMP-9 had significantly fewer macrophages and collagen as compared to those of wildtype mice. CONCLUSIONS: MMP-9 mediates thrombus-induced loss of vein wall compliance by increasing stiffness of the extracellular matrix and collagen-elastin fibers during thrombus resolution. MMP-9 also mediates macrophage and collagen content of the resolving thrombus and bone-marrow derived MMP-9 plays a role in resolution of thrombus mass. These disparate effects of MMP-9 on various aspects of thrombus illustrate the complexity of individual protease function on biomechanical and morphometric aspects of thrombus resolution.

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.