Deep Vein Thrombosis Model Research Articles

The role of SERPIN citrullination in thrombosis

Aberrant protein citrullination is associated with many pathologies; however, the specific effects of this modification remain unknown. We have previously demonstrated that serine protease inhibitors (SERPINs) are highly citrullinated in rheumatoid arthritis (RA) patients. These citrullinated SERPINs include antithrombin, antiplasmin, and t-PAI, which regulate the coagulation and fibrinolysis cascades. Notably, citrullination eliminates their inhibitory activity. Here, we demonstrate that citrullination of antithrombin and t-PAI impairs their binding to their cognate proteases. By contrast, citrullination converts antiplasmin into a substrate. We recapitulate the effects of SERPIN citrullination using invitro plasma clotting and fibrinolysis assays. Moreover, we show that citrullinated antithrombin and antiplasmin are increased and decreased in a deep vein thrombosis (DVT) model, accounting for how SERPIN citrullination shifts the equilibrium toward thrombus formation. These data provide a direct link between increased citrullination and the risk of thrombosis in autoimmunity and indicate that aberrant SERPIN citrullination promotes pathological thrombus formation.

Platelet Cyclophilin D-Dependent Events Limit Venous Thrombotic Occlusion and Platelet Accretion

The role of platelets in arterial thrombosis is well established. Recent findings have reinforced their importance in venous thrombosis as well. Which of the platelet's many functions mediate its effects in the venous setting remains unclear, and how procoagulant platelet formation regulates venous thrombosis has not been investigated. Cyclophilin D (CypD) is a mitochondrial peptidylprolylisomerase that has the key function of regulation of mitochondrial permeability transition pore (mPTP) formation. In the absence of CypD, mPTP and procoagulant platelet formation are markedly impaired in response to physiologic agonists. The functional consequence of CypD's absence in vivo has been examined in photochemical injury models in photochemical injury models of both the carotid and mesenteric arteries. In mice with platelet CypD-deficiency, in both of these high shear settings, thrombotic vasoocclusion was accelerated implicating procoagulant platelet fragility and loss of adhesiveness as a critical factor limiting thrombus growth. Whether CypD deficiency would similarly impact thrombus growth in a venous setting has not been previously examined.To investigate the role of CypD and procoagulant platelets in the low shear setting of venous thrombosis, we utilized the inferior vena cava (IVC) stenosis ligature murine model of deep vein thrombosis (DVT). This model was chosen as it utilizes mechanical narrowing to induce thrombosis in the setting of venous flow. 24 hours following IVC ligation, thrombi were significantly larger (1.83 fold) in CypD-deficient mice than wild type. (p<0.05). Similarly, in mice with platelet-specific CypD-deficiency, thrombi were 1.54 fold larger than those found in WT mice (p<0.05). To begin to investigate the mechanism by which CypD-deficiency resulted in increased thrombus size, the composition of the venous thrombi was analyzed. Western blots for markers of platelets (CD41), neutrophils (Ly6G), and fibrin (59D8) revealed that their relative levels in thrombi were unchanged, but indicated that red blood cell (spectrin α1) accumulation increased. Histologic appearance in both CypD-deficient and wild-type thrombi was similar with a consistent pattern observed. A white cap area dominated the thrombus closest to the site of ligation, while red thrombus was more common in the remaining thrombus area. To investigate whether the observed pattern is due to more rapid occlusion, ultrasound was utilized to investigate the time course of venous vasoocclusion following ligature placement. Preliminary ultrasound studies suggest that CypD-deficient mice develop vasoocclusion more rapidly, perhaps thus accounting for their larger size.Previous studies have delineated the role of CypD-dependent events in limiting platelet accretion in vitro in arterial, but not venous, flow conditions. In vitro studies utilizing whole blood from CypD-deficient mice showed increased accretion of platelets on collagen under venous flow conditions. Similarly, platelet accretion increased when human whole blood was treated with cyclosporin A, an inhibitor of CypD, and flowed over collagen at venous rates. Interestingly, washed platelets from CypD-deficient mice also showed increased accumulation on a von Willebrand factor (VWF) - coated surface under venous flow conditions, a condition in which neither collagen nor coagulation-mediated procoagulant platelet formation would be expected to occur. Thus, these studies have identified an important role for CypD-dependent platelet events in the regulation of platelet accumulation in vitro and thus slowing the early stages of thrombus growth in vivo . The results here, in venous settings, and previously in arterial settings are consistent with a limiting role for procoagulant platelet formation in thrombus growth. This regulatory feedback role should be taken into consideration prior to modulation of procoagulant platelet formation in the setting of thrombosis or inflammation. DisclosuresNo relevant conflicts of interest to declare.

In Vivo Platelet Detection Using a Glycoprotein IIb/IIIa-Targeted Near-Infrared Fluorescence Imaging Probe.

Platelets play a prominent role in multiple diseases, in particular arterial and venous thrombosis and also in atherosclerosis and cancer. To advance the in vivo study of the biological activity of this cell type from a basic experimental focus to a clinical focus, new translatable platelet-specific molecular imaging agents are required. Herein, we report the development of a near-infrared fluorescence probe based upon tirofiban, a clinically approved small-molecule glycoprotein IIb/IIIa inhibitor (GPIIb/IIIa). Through in vitro experiments with human platelets and in vivo ones in a murine model of deep-vein thrombosis, we demonstrate the avidity of the generated probe for activated platelets, with the added benefit of a short blood half-life, thereby enabling rapid in vivo visualization within the vasculature.

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.

A PSGL-1 glycomimetic reduces thrombus burden without affecting hemostasis

Visual Abstract [Display omitted]

E-selectin inhibitor is superior to low-molecular-weight heparin for the treatment of experimental venous thrombosis

BackgroundThis study evaluated E-selectin inhibition with GMI-1271 (Uproleselan [GMI]) alone and in combination with the standard of care low-molecular-weight heparin (LMWH) to improve vein recanalization, decrease vein wall inflammation and protect against adverse bleeding in a primate model. We sought to examine this novel treatment of venous thrombosis. MethodsUsing a well-documented primate animal model, iliac vein thrombosis was induced by balloon occlusion of the iliac vein for 6 hours. Starting on day 2 after thrombosis, animals began treatment in two phases. In phase one, nontreated controls received no treatment (n = 5) vs animals treated with the E-selectin inhibitor GMI, 25 mg/kg, subcutaneous (SC), once daily (n = 4) for 21 days (previously published data). In phase two, animals were treated with GMI plus a combination of LMWH 1.5 mg/kg or 40 mg (GMI + LMWHc) SC once daily (n = 8) for 19 days; and animals treated with LMWH 1.5 mg/kg or 40 mg (LMWHc) SC once daily (n = 6) for 19 days. Animals were evaluated by magnetic resonance venography for vein recanalization and inflammation by gadolinium extravasation, duplex ultrasound, coagulation tests (thromboelastography, bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen) and complete blood count at baseline, days 2, 7, 14, and 21 at euthanasia. Statistical analysis included using unpaired t test with Welch's correction for direct comparisons and one-way analysis of variance for comparison between the groups. ResultsPercent vein recanalization by magnetic resonance venography was highest in the GMI alone group followed by GMI + LMWHc, both significantly different from control. On ultrasound examination, animals treated with GMI alone had no decrease in open vein lumen by day 21, whereas decreases were observed in groups GMI + LMWHc (–26%), LMWHc (–27%), and controls (–80%). Vein wall inflammation decreased significantly in all treated groups. Intimal fibrosis and intimal thickness was best preserved in the GMI alone group. An analysis of total vein wall collagen revealed a trend in all treatment groups of decreasing vein wall collagen. No clinically significant bleeding events were noted in any group. The LMWH groups trended to have prolonged coagulation test values, whereas E-selectin inhibition with GMI did not cause clinically significant changes in coagulation measures. ConclusionsTreatment with E-selectin inhibition results in improved vein recanalization, a decrease in vein wall inflammation and vein wall intimal thickness and fibrosis, with no changes in markers of coagulation. E-selectin inhibition with GMI alone is superior to E-selectin inhibition combined with LMWH, LMWH alone, and no treatment in this deep vein thrombosis model of iliac vein thrombosis.

Intrathrombotic appearances of AQP-1 and AQP-3 in relation to thrombus age in murine deep vein thrombosis model.

Aquaporins (AQPs) are membrane-bound proteins for water transportation and are useful for diagnosing drowning and wound vitality in forensic pathology. Here, we examined intrathrombotic expression of AQP-1 and AQP-3 using deep vein thrombosis models in mice. To perform immunohistochemical analyses, we used anti-AQP-1 and anti-AQP-3 antibodies. In thrombus samples with the post-ligation intervals of 1 to 5 days, AQP-1+ areas were over 70%. At 7 days after the IVC ligation, AQP-1+ areas became less than 50%, eventually decreasing to 11% at 21 days. At 3 days after the IVC ligation, AQP-3+ cells started to appear from the peripheral area. Thereafter, the positive cell number progressively increased and reached to a peak at 10 days after the IVC ligation. When the intrathrombotic AQP-1+ area was as large as the intrathrombotic collagen area or smaller, it would indicate a thrombus age of ≥ 10 days. AQP-3+ cell number of > 30 would indicate a thrombus age of 10–14 days. Collectively, our study implied that the detection of AQP-1 and AQP-3 would be useful for the determination of thrombus age.

Neutralizing the pathological effects of extracellular histones with small polyanions

Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9–1.4 kDa) that interact electrostatically with histones, neutralizing their pathological effects. In vitro, SPAs inhibited the cytotoxic, platelet-activating and erythrocyte-damaging effects of histones, mechanistic studies revealing that SPAs block disruption of lipid-bilayers by histones. In vivo, SPAs significantly inhibited sepsis, deep-vein thrombosis, and cardiac and tissue-flap models of ischemia-reperfusion injury (IRI), but appeared to differ in their capacity to neutralize NET-bound versus free histones. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings. Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, thus destabilising the structure. Finally, based on our work, a non-toxic SPA that inhibits both NET-bound and free histone mediated pathologies was identified for clinical development.

Von Willebrand Factor Is a Critical Mediator of Deep Vein Thrombosis in a Mouse Model of Diet-Induced Obesity.

Obesity is characterized by chronic low-grade inflammation and consequentially a hypercoagulable state, associating with an increased incidence of venous thromboembolism. Increased VWF (von Willebrand factor) plasma concentration and procoagulant function are independent risk factors for venous thromboembolism and are elevated in obese patients. Here, we explore the pathobiological role of VWF in obesity-associated venous thrombosis using murine models. Approach and Results: We first showed that diet-induced obese mice have increased VWF plasma levels and FVIII (factor VIII) activity compared with littermate controls. Elevated VWF levels appeared to be because of both increased synthesis and impaired clearance. Diet-induced obesity-associated venous thrombosis was assessed using the inferior vena cava-stenosis model of deep vein thrombosis. Diet-induced obese mice developed larger venous thrombi that were rich in VWF, erythrocytes, and leukocytes. Administering a polyclonal anti-VWF antibody or an anti-VWF A1 domain nanobody was protective against obesity-mediated thrombogenicity. Delayed administration (3 hours post-inferior vena cava stenosis) similarly reduced thrombus weight in diet-induced obese mice. This study demonstrates the critical role of VWF in the complex, thrombo-inflammatory state of obesity. It adds to the growing rationale for targeting VWF-specific interactions in thrombotic disease.

Shedding light on venous thromboembolism

A preclinical deep vein thrombosis model was developed in mice using blood flow restriction and illumination.

Synergic effect of GPIBA and von Willebrand factor in pathogenesis of deep vein thrombosis.

In cardiovascular disease, deep vein thrombosis is one of the vital symptoms causing pulmonary thromboembolism. However, the pathogenesis of deep vein thrombosis is still not clear. One of the critical factors leading to deep vein thrombosis is the platelet aggregation that is mediated by a set of key genes including platelet membrane protein coded by platelet glycoprotein Ib alpha chain (GPIBA). Deep vein thrombosis model was established according to the previous protocol, and venous blood and thrombi were collected for further analysis. The dynamic changes of GPIBA and coagulation factor, von Willebrand factor, were observed in deep vein thrombosis models. Meanwhile, critical proteins participating in adhesion and binding of platelets such as epithelial membrane protein 2 (EMP2), vascular cell adhesion protein 1 (VCAM1), immunoreceptor tyrosine-based activation motif 1 (ITAM1), integrin subunit alpha M (ITGAM), or fibronectin were also differentially expressed in deep vein thrombosis models. Application of heparin could reverse these dynamic changes in deep vein thrombosis models. Thus, we explained the potential synergic role of GPIBA and von Willebrand factor in regulating the occurrence of deep vein thrombosis and provide therapeutic target against cardiovascular disease.

The application of autophagy to thrombus age estimation in murine deep vein thrombosis model.

We immunohistochemically examined the intrathrombotic dynamics of autophagy during thrombogenesis using murine deep vein thrombosis (DVT) models. To perform the immunohistochemical analyses, we used anti-LC3 antibody and anti-p62 antibody for detecting the intrathrombotic autophagic functions. We estimated dynamics of the intrathrombotic autophagy as LC3+ cell number (×1000, five fields) with the thrombus ages (each group n = 5). The number of LC3+ cells was once decreased at 3 days, and then increased until 10 days. On the contrary, the number of p62+ cells progressively increased until 10 days after the inferior vena cava (IVC) ligation, and then gradually decreased. Especially, in all of thrombus samples with the postligation intervals of 5-10 days, both numbers were larger than 10. Subsequently, we compared the number of LC3+ cells to that of p62+ cells. Although, at 1 day after the IVC ligation, LC3+ cell number significantly exceeded p62+ cell number, the former was significantly or relatively less than the latter at 3 days or more after the IVC ligation. Thus, positive cells of > 10 in both LC3 and p62 indicated the thrombus age of 5-10 days. Upon comparison of immunopositive cells in LC3 and p62, the p62/LC3 ratio was > 1.0 in 29 out of 30 thrombus samples aged 3-21 days, and all of 1-day-old thrombus had the p62/LC3 ratio of < 0.5. Thus, the ratio of > 1.0 and that of < 0.5 could indicate thrombus age of 3 days or more and that of 1 day, respectively. Collectively, our study implied that the detection of autophagy-related molecules such as LC3 and p62 would be useful for the determination of thrombus age.

Resolution of thrombus burden by targeting inflammatory mediators to treat DVT.

Treatment with resolvin D4, a specialized pro-resolving mediator, significantly attenuated thrombus burden in a mouse model of deep vein thrombosis.

Enhanced Thrombolysis by Ultrasound-Assisted Catheter-Directed Thrombolysis and Microbubbles in an In Vitro Model of Iliofemoral Deep Vein Thrombosis.

There is a need to improve the efficacy and safety of catheter-directed thrombolysis (CDT) for thrombo-occlusive diseases, and ultrasound-assisted CDT (USAT) is a promising approach. We tested if thrombolysis efficacy of USAT can be improved by adding gaseous microbubbles (MB). We developed an in vitro dynamic overflow model for iliofemoral deep vein thrombosis, and added MB to an USAT system with ultrasound energy and dose of tissue plasminogen activator according to clinical practice. A total of 64 clots (mean baseline weight of 8.23 ± 1.12 g, generated from citrated human whole blood from 7 healthy male volunteers) were randomly assigned to 1 of 4 study protocols of 30 minutes' duration: negative control, CDT, USAT, and USAT + MB.Thrombolysis efficacy was assessed by measuring the change in D-dimer levels in the overflow liquid and the percentage of clot weight reduction. Compared to negative control, change in D-dimer increased by 62% (p = 0.017), 128% (p = 0.002), and 177% (p < 0.001) in the CDT, USAT, and USAT + MB groups, respectively. D-dimer increase was greater in the USAT than in the CDT group (p = 0.014), and greater in the USAT + MB than in the USAT group (p = 0.033). Compared to negative control, percentage of clot weight reduction increased by 123% (p = 0.016), 154% (p = 0.002), and 233% (p < 0.001) in the CDT, USAT, and USAT + MB groups, respectively. Percentage of clot weight reduction was greatest in the USAT + MB group (p < 0.05 compared with all other groups). In conclusion, our in vitro study suggests that the thrombolytic efficacy of USAT in human whole blood clots can be improved by local administration of MB.

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.

Correlation between expression and mechanism of imterleukin-1β and related markers in deep venous thrombosis of lower extremities

Objective Verification the changes in expression and mechanism of serum inflammatory factor IL-1 beta and related biomarkers by establishing an animal model of deep vein thrombosis (DVT) in lower extremity of rats. Methods Seventy male SPF SD rates, weighing (200±20) g, randomly divided into groups of ten and made each group of rats equal in weight. Recorded as control group, sham operation group, model experiment 2, 8, 24, 48, 72 h group. The control group was fed normally without any treatment; the sham operation group, laparotomy was performed without vein ligation; the model experimental group was divided into different groups according to different time periods. The model of venous thrombosis of lower extremities was established through venous ligation surgery in rats, they were sacrificed at the 2 hours, 8 hours, 24 hours, 48 hours and 72 hours after the modeling, and blood samples and tissues were collected. The expression levels of IL-1β, tissue factor and xanthine oxidase (XOD) in blood samples of 7 groups of rats were measured by ELISA. DVT morphology were analyzed by Pathology, flow cytometry were used to count peripheral blood circulating endothelial cells in rats separated after injury. The expressions of IL-1β, tissue factor, XOD, pathological and flow results were compared between the normal control group, the sham operation group and the lower extremity deep vein thrombosis model group at different time periods. All data were represented by mean standard deviation (Mean±SD). One-way anova was performed on the measurement data, and the LDS method was used to compare the two pairs. The test level was α=0.05. Results IL-1β, tissue factor, XOD showed no significant difference between the control group and the sham operation group, P>0.05. The model experimental group showed an upward trend in the process of 2 h-24 h and reached a peak value at 24 h. The histopathology showed that red thrombus and mixed thrombus could be seen within 2 h-48 h in the thrombosis model experiment group. The blood vessel wall was accompanied by inflammatory cell infiltration. After 72 h, the thrombus was obviously organized. The CD31 concentration of the control group and the sham operation group were (4.04±1.00), (4.82±1.12), and the difference between the two difference between the two groups was not statistically significant (P<0.05); the CD31 concentration of model experimental group had significant differences from 2 h, (5.188±0.895); 8 h, (5.614±1.243); 24 h, (9.785±1.996); 48 h, (14.198±2.172); 72 h, (18.118±1.025), it continued to increase, P<0.05. Conclusion High expression of inflammatory IL-1β and related markers tissue factor, XOD confirmed the mechanism of injury of deep venous endothelial cells in lower extremities caused by IL-1β and the mechanism of further aggravation of thrombosis after injury. Key words: Venous thrombosis; Interleukins; Inflammation; Circulating enelothelinal cells

Tspan18 is a novel regulator of the Ca2+ channel Orai1 and von Willebrand factor release in endothelial cells.

Ca2+ entry via Orai1 store-operated Ca2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific ‘partner proteins’ and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca2+ signaling and von Willebrand factor release in response to inflammatory stimuli.

Near-infrared off-on fluorescence probe activated by NTR for in vivo hypoxia imaging

Selective and efficient detection and imaging of nitroreductase (NTR) overexpressed in hypoxic tissues is of great importance for better understanding its biological functions. The effective optical probes equipped for NTR detection in vivo is still lacking, so we developed an innovative near infrared (NIR) fluorescent on-off probe (Cy-NO2) composed of fluorescence reporting unit (an aminocyaine dye) and recognition unit (p-nitrobenzylcarbamate group). The response and mechanism of the NTR activated reduction of Cy-NO2 were evaluated in vitro through kinetic optical studies, mass spectra analysis and docking calculations. The results demonstrated that Cy-NO2 could rapidly recognize NTR with high selectivity and sensitivity. The efficient NTR detection ability of Cy7-NO2 was further validated by fluorescence imaging of hypoxic cells (A549, PC-12 and HUVEC cell). Furthermore, the in vivo hypoxia imaging by Cy-NO2 were evaluated on tumor-bearing mouse, cerebral ischemia (CIS) and deep vein thrombosis models. The results indicated a rapid and distinct enhancement of its NIR fluorescence highly appropriate for in vivomonitoring of NTR in all of the three animal models, which revealed aspiring clinical value of this NIR fluorescent hypoxia probe.

Comparison of the serum metabolic signatures based on 1H NMR between patients and a rat model of deep vein thrombosis

Deep vein thrombosis (DVT) and pulmonary embolism (PE) have high morbidity, reduce quality of life, and can cause death. Biomarkers or genetic risk factors have not been identified in patients with DVT. In present study, serum of 61 patients suffering from DVT and a rat DVT model (n = 10) were assayed by a proton nuclear magnetic resonance (1H NMR) metabolomics technique combing with multivariate statistical analysis to identify the metabolites. The MetPA platform was used to identify differences in the metabolic pathways between the rat model and patients. The metabolomics results discovered that 11 different metabolites in rats and 20 different metabolites in DVT patients. Seven metabolites both altered in the rats and patients. Moreover, we observed changes in the metabolic pathways, including carbohydrate metabolism, lipid metabolism, and amino acid metabolism that were induced immediately by the thrombosis. Pathway of aminoacyl-tRNA biosynthesis perturbed only in the patients which was associated with the genetic risk factor of DVT. The study demonstrated that serum 1H NMR metabolomics can be used to diagnose DVT in the clinic. The altered pathways related to thrombosis and genetics will provide a foundation and new strategies for understanding the pathological mechanism and pharmacological targets of DVT.

Abstract 523: The Effects of Spontaneous Exercise on Murine Model of Deep Vein Thrombosis

Introduction: Recent studies suggest physical activity may have beneficial effects on deep vein thrombosis (VT). Long-term moderate exercise has been shown to reduce inflammatory cytokines and attenuate plasma fibrinogen compared to uninterrupted sitting. However, the effect exercise may have in VT is unknown. Our objective is to quantify the effects of prophylactic exercise on the thrombotic process in a murine model of VT. Methods: C57BL6 male mice were either provided in-cage running wheels (E: n=6) starting 2 weeks prior to and continuously following VT induction, or no wheel exposure (C: n=4). VT was induced with the electrolytic inferior vena cava model (EIM), which induces thrombosis in the continuous presence of blood flow. MR images were acquired days 2 (acute) or 6 (chronic) post VT using a T2-weighted sequence sensitive to edema-related fluid, then the thrombi were harvested for histology. Results: Four of six mice engaged in exercise. Three of four mice continued running after thrombus induction, with reduced activity (88 ± 11% reduction). While thrombus volume was not significantly different between groups, the volume of thrombus exhibiting high T2 signal was reduced by ~57% at day 6 in exercising mice, suggesting a decreased inflammatory response with exercise (C 0.81 ± 0.13 mm 3 vs. E 0.35 ± 0.07 mm 3 , p =0.08). Fibrin content, assessed by Picro-Mallory staining, was reduced by 60% at day 2 in exercising mice (C 2.08 ± 0.29 um 2 vs. E 0.83 ± 0.45 um 2 , p =0.09). Conclusion: Our results suggest that exercise may reduce inflammation in VT and lead to less fibrin-rich clots. Future studies will increase the number of animals, investigate inflammatory components, and extend to more chronic time points. Understanding the impact of exercise on specific thrombotic components could lead to better recommendations for patients with DVT.