Murine Deep Vein Thrombosis Research Articles

Ferumoxytol-enhanced MRI assessment of venous Thrombus resolution and macrophage content in a murine deep vein thrombosis model

BackgroundImaging evaluation of acute deep vein thrombosis (DVT) or post-thrombotic syndrome (PTS) in animal or clinical models is limited to anatomical assessment of the location and extent of thrombi. We hypothesize that Fe-MRI, used to evaluate macrophage content in other inflammatory diseases, can be useful to evaluate the thromboinflammatory features after DVT over time. MethodsNineteen wild-type CD-1 mice underwent surgical IVC ligation to induce DVT. Mice received either saline or 5 mg/kg of 14E11, a Factor XI inhibitor, before the procedure. Fe-MRI was performed on days 6–7 after ligation to evaluate thrombus volume, perfusion, and macrophage content via T2-weighted images. Mice were euthanized at days 3–15 after surgery. The thrombi and adjacent vein walls were excised, weighed, formalin-fixed, and paraffin-embedded for immunohistological analysis. Specimens were stained with specific antibodies to evaluate macrophage content, collagen deposition, neovascularization, and recanalization. Significance was determined using the Mann-Whitney U or Student's t-test. ResultsAfter IVC-ligation in control mice, thrombus weights decreased by 59 % from day 3 to 15. Thrombus volumes peaked on day 5 before decreasing by 85 % by day 13. FXI inhibition led to reduced macrophage content in both thrombi (p = .008) and vein walls (p = .01), decreased thrombus volume (p = .03), and decreased thrombus mass (p = .01) compared to control mice. CCR2+ staining corroborated these findings, showing significantly reduced macrophage presence in the thrombi (p = .002) and vein wall (p = .002). ConclusionsFe-MRI T2 relaxation times can be used to characterize and quantify post-thrombotic changes of perfusion, macrophage content, and thrombus volume over time in a surgical mouse model of venous thrombosis. This approach could lead to better quantification of in vivo inflammation correlating monocyte and macrophage content within resolving thrombi and veins and may serve as a useful tool for research and clinically in the evaluation of the post-thrombotic environment.

The time-dependent expression of FPR2 and ANXA1 in murine deep vein thrombosis model and its relation to thrombus age.

Thrombus age determination in fatal venous thromboembolism cases is an important task for forensic pathologists. In this study, we investigated the time-dependent expressions of formyl peptide receptor 2 (FPR2) and Annexin A1 (ANXA1) in a stasis-induced deep vein thrombosis (DVT) murine model, with the aim of obtaining useful information for thrombus age timing. A total of 75 ICR mice were randomly classified into thrombosis group and control group. In thrombosis group, a DVT model was established by ligating the inferior vena cava (IVC) of mice, and thrombosed IVCs were harvested at 1, 3, 5, 7, 10, 14, and 21days after modeling. In control group, IVCs without thrombosis were taken as control samples. The expressions of FPR2 and ANXA1 during thrombosis were detected using immunohistochemistry and double immunofluorescence staining. Their protein and mRNA levels in the samples were determined by Western blotting and quantitative real-time PCR. The results reveal that FPR2 was predominantly expressed by intrathrombotic neutrophils and macrophages. ANXA1 expression in the thrombi was mainly distributed in neutrophils, endothelial cells of neovessels, and fibroblastic cells. After thrombosis, the expressions of FPR2 and ANXA1 were time-dependently up-regulated. The percentage of FPR2-positive cells and the level of FPR2 protein significantly elevated at 1, 3, 5 and 7 days after IVC ligation as compared to those at 10, 14 and 21 days after ligation (p < 0.05). Moreover, the mRNA level of FPR2 were significantly higher at 5 days than that at the other post-ligation intervals (p < 0.05). Besides, the levels of ANXA1 mRNA and protein peaked at 10 and 14 days after ligation, respectively. A significant increase in the mRNA level of ANXA1 was found at 10 and 14 days as compared with that at the other post-ligation intervals (p < 0.01). Our findings suggest that FPR2 and ANXA1 are promising as useful markers for age estimation of venous thrombi.

KLF11 Protects against Venous Thrombosis via Suppressing Tissue Factor Expression.

Krüppel-like factors (KLFs) play essential roles in multiple biological functions, including maintaining vascular homeostasis. KLF11, a causative gene for maturity-onset diabetes of the young type 7, inhibits endothelial activation and protects against stroke. However, the role of KLF11 in venous thrombosis remains to be explored. Utilizing stasis-induced murine deep vein thrombosis (DVT) model and cultured endothelial cells (ECs), we identified an increase of KLF11 expression under prothrombotic conditions both in vivo and in vitro. The expression change of thrombosis-related genes was determined by utilizing gain- and loss-of-function approaches to alter KLF11 expression in ECs. Among these genes, KLF11 significantly downregulated tumor necrosis factor-α (TNF-α)-induced tissue factor (TF) gene transcription. Using reporter gene assay, chromatin immunoprecipitation assay, and co-immunoprecipitation, we revealed that KLF11 could reduce TNF-α-induced binding of early growth response 1 (EGR1) to TF gene promoter in ECs. In addition, we demonstrated that conventional Klf11 knockout mice were more susceptible to developing stasis-induced DVT. These results suggest that under prothrombotic conditions, KLF11 downregulates TF gene transcription via inhibition of EGR1 in ECs. In conclusion, KLF11 protects against venous thrombosis, constituting a potential molecular target for treating 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.

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.

Influence of Circadian Rhythm on Thrombus Formation of Murine Deep Vein Thrombosis Model

Influence of Circadian Rhythm on Thrombus Formation of Murine Deep Vein Thrombosis Model

Detection of intrathrombotic endothelial progenitor cells and its application to thrombus age estimation in a murine deep vein thrombosis model.

Endothelial progenitor cells (EPCs), a newly identified cell type, are bone marrow-derived progenitor cells that co-express stem cell markers and Flk-1 (one of the receptors for vascular endothelial growth factor). In this study, double-color immunofluorescence analysis was performed using anti-CD34 and anti-Flk-1 antibodies in order to examine the time-dependent intrathrombotic appearance of EPCs, using the thrombi of DVT model mice with different thrombus ages (1-21days). In thrombus cross-section specimens with an age of less than 3days, CD34+/Flk-1+ EPCs were not detected. EPCs were initially observed in wounds aged 5days, and their number was increased in thrombi with the advance of thrombus ages. The number of EPCs was the largest in the 10-day thrombus. Moreover, all 15 samples aged 7-14days had an EPC number of more than 10, and, in 9 of them, the number of intrathrombotic EPCs was over 20. In contrast, in all thrombus samples aged 21days, the number of intrathrombotic EPCs was less than 20. However, in three of them, the intrathrombotic EPC number was ≥10. These observations suggested that an intrathrombotic EPC number exceeding 20 would indicate a thrombus age of approximately 7-14days.

Immunohistochemical detection of intrathrombotic fibrocytes and its application to thrombus age estimation in murine deep vein thrombosis model.

Deep vein thrombi are dissolved after fibrosis process along with an increase of thrombus age. Fibrocytes are circulating bone marrow-derived cells with mesenchymal features that potentially have a unique and critical function in fibrosis. In this study, a double-color immunofluorescence analysis was carried out by using anti-CD45 and anti-collagen type I antibodies to examine the time-dependent appearance of fibrocytes in the murine model of stasis-induced deep vein thrombosis. The thrombus ages were 1, 3, 5, 7, 10, 14, and 21days. In a thrombus age of less than 5days, CD45+ and collagen type I+ fibrocytes were never detected. The intrathrombotic fibrocytes were initially observed in thrombi aged 7days, and their number increased with advances in thrombus age. In a quantitative morphometrical analysis, the average number of intrathrombotic fibrocytes was highest in 14-day-old thrombi, and all of the five samples aged 14days had the fibrocyte number of more than 25, and in three out of them, the number of intrathrombotic fibrocytes was over 30. On the contrary, in all of thrombus samples with the postligation intervals of 10 and 21days, the number of intrathrombotic fibrocytes was less than 25. These observations imply that thrombi containing fibrocytes are at least 7days old and that a fibrocyte number exceeding 30 would indicate the thrombus age of approximately 14days. Our observations indicate that the detection of fibrocytes could be useful for thrombus age determination.

138: Absence of IL-6 accelerates thrombus formation through reduced MMPs and uPA expression in murine deep vein thrombosis model

Deep vein thrombosis (DVT) is a major cause of pulmonary thromboembolism, a leading cause of death in individuals with DVT. And DVT is multifactorial and often results from a combination of risk factors such as genetic conditions, obesity, drugs, pregnancy, aging, trauma, and malignancy. We demonstrated the pathophysiological roles of cytokines such as IFN-g and TNF-α in the processes of thrombus resolution using genetically engineered mice. In this study, we examined the pathophysiological roles of IL-6 in the resolution of DVT by the use of Il6 KO mice. Upon the ligation of the inferior vena cava (IVC) of WT mice, venous thrombi formed and grew progressively until 5 days, and the thrombus weight decreased after that. Concomitantly, the gene expression of Il6 was detected in the thrombi. When Il6 KO mice were treated in the same manner, thrombus size was much larger than WT mice. The intrathrombotic IL-6 was produced by intrathrombotic infiltrated macrophages on double color immunofluorescence staining of thrombi. And the number of intrathrombotic IL-6-positive cells changed in proportion to the number of macrophages. Moreover, the blood flow of the IVC was more recovered in WT than in Il6 KO mice. And intrathrombotic Mmp2 , Mmp9 , and Plau mRNA expression was significantly reduced in Il6 KO mice than WT mice. Supportingly, the administration of anti-IL-6 antibody delayed the thrombus resolution in WT mice. Furthermore, IL-6 treatment enhanced gene expression of Plau , Mmp2 , and Mmp9 in WT-derived macrophages. These IL-6 effects were significantly suppressed by Stat3 inhibitor. Collectively, IL-6 can have a beneficial role in the thrombus resolution by upreglation of MMP-2, MMP-9 and alpha expression. IL-6 can be a good molecular target for the DVT treatment.

18F-fluorodeoxyglucose positron emission tomography/computed tomography enables the detection of recurrent same-site deep vein thrombosis by illuminating recently formed, neutrophil-rich thrombus.

Accurate detection of recurrent same-site deep vein thrombosis (DVT) is a challenging clinical problem. Because DVT formation and resolution are associated with a preponderance of inflammatory cells, we investigated whether noninvasive (18)F-fluorodeoxyglucose (FDG)-positron emission tomography (PET) imaging could identify inflamed, recently formed thrombi and thereby improve the diagnosis of recurrent DVT. We established a stasis-induced DVT model in murine jugular veins and also a novel model of recurrent stasis DVT in mice. C57BL/6 mice (n=35) underwent ligation of the jugular vein to induce stasis DVT. FDG-PET/computed tomography (CT) was performed at DVT time points of day 2, 4, 7, 14, or 2+16 (same-site recurrent DVT at day 2 overlying a primary DVT at day 16). Antibody-based neutrophil depletion was performed in a subset of mice before DVT formation and FDG-PET/CT. In a clinical study, 38 patients with lower extremity DVT or controls undergoing FDG-PET were analyzed. Stasis DVT demonstrated that the highest FDG signal occurred at day 2, followed by a time-dependent decrease (P<0.05). Histological analyses demonstrated that thrombus neutrophils (P<0.01), but not macrophages, correlated with thrombus PET signal intensity. Neutrophil depletion decreased FDG signals in day 2 DVT in comparison with controls (P=0.03). Recurrent DVT demonstrated significantly higher FDG uptake than organized day 14 DVT (P=0.03). The FDG DVT signal in patients also exhibited a time-dependent decrease (P<0.01). Noninvasive FDG-PET/CT identifies neutrophil-dependent thrombus inflammation in murine DVT, and demonstrates a time-dependent signal decrease in both murine and clinical DVT. FDG-PET/CT may offer a molecular imaging strategy to accurately diagnose recurrent DVT.

Abstract 16: FDG-PET/CT Enables the Detection of Recurrent, Same-Site Deep Vein Thrombosis by Illuminating Recently Formed Neutrophil-Rich Thrombus

Background: Specific detection of recurrent deep vein thrombosis (DVT) is a challenging clinical problem. As DVT formation and resolution are inflammatory processes, we experimentally investigated whether noninvasive 18F-fluorodeoxyglucose (FDG)-PET imaging could identify inflamed, recently formed thrombi and thereby improve the diagnosis of recurrent DVT. Methods: We established a stasis-induced DVT model in murine jugular veins and also a novel model of recurrent stasis DVT in mice. C57BL/6 mice (n = 30) underwent ligation of the jugular vein to induce stasis DVT. FDG-PET/CT was performed at DVT timepoints of day 2, 4, 7, 14, or 2+16 (same-site recurrent day 2 DVT overlying a 16 days old DVT). Antibody-based neutrophil depletion was performed in a subset of mice prior to DVT formation and FDG-PET/CT. In a clinical study, 38 patients with lower extremity DVT or controls undergoing FDG-PET were retrospectively analyzed. Results: Stasis DVT demonstrated that the highest FDG signal occurred day 2, followed by a time-dependent decrease (p&lt;0.05). Histological analyses demonstrated that thrombus neutrophils (p&lt;0.01), but not macrophages, correlated with thrombus PET signal intensity. Neutrophil depletion decreased FDG signals in day 2 DVT compared to that of control mice (p&lt;0.05). Recurrent DVT demonstrated significantly higher FDG uptake than organized day 14 DVT (p&lt;0.05). The FDG DVT signal in patients also exhibited a time-dependent decrease (p&lt;0.05). Recurrent day 2 DVT showed neutrophil-dominant infiltration pattern with few macrophages, similar to standalone day 2 DVT. Conclusions: Noninvasive FDG-PET/CT identifies neutrophil-dependent thrombus inflammation in murine DVT, and demonstrates a time-dependent signal in both murine and clinical DVT. FDG-PET/CT may offer a molecular imaging strategy to accurately diagnose recurrent DVT.

Contrast ultrasound for the quantification of deep vein thrombosis in living mice: effects of enoxaparin and P2Y12 receptor inhibition

We examined the applicability of contrast-enhanced ultrasound (CEUS) for imaging of murine deep vein thrombosis (DVT) and measured the effects of enoxaparin, ticagrelor and P2Y(12) receptor deficiency in vivo. Deep vein thrombosis was induced by exposure to ferric chloride or ligation of the infrarenal vena cava of C57BL/6 mice after pretreatment with enoxaparin, ticagrelor or vehicle and in P2Y(12-/-) mice. Initial thrombus growth was visualized by intravital microscopy. Thrombi were weighed and examined by immunohistochemistry. CEUS was performed with a standard ultrasound system (Vivid 7, GE Healthcare) in the open abdominal cavity after injection of stabilized sulphur hexafluoride microbubbles. Incubation with ferric chloride resulted in non-occluding platelet-containing thrombus growth within 15-25 min. Sham-operated mice, enoxaparin- and ticagrelor-pretreated wild-type and P2Y(12-/-) mice developed only small thrombi. After injection of the contrast agent, growing thrombi were delineated clearly as negative contrast on CEUS. Thrombus size on CEUS after 25 min was significantly smaller in enoxaparin- (0.3 ± 0.1 mm(2)) and ticagrelor-treated (0.5 ± 0.1 mm(2)) wild-type and in P2Y(12-/-) mice (0.4 ± 0.1 mm(2)) as compared with vehicle-treated wild-type mice (2.0 ± 0.3 mm(2)) in the maximal sagittal plane (P < 0.001, n = 5-10). CEUS-derived thrombus size correlated linearly with thrombus weight and also reflected the extent of ligation-induced DVT. Contrast-enhanced ultrasound allowed the real-time quantification of DVT in living mice. Genetic and pharmacologic antithrombotic interventions were well reflected by CEUS and suggested an important role of the platelet P2Y(12) receptor in early DVT formation.

Immunohistochemical detection of intrathrombotic macrophage-derived cytokines and its application to thrombus age estimation in murine deep vein thrombosis model

We immunohistochemically examined the expression of IFNγ, TNFα, and TNF receptor p55 (TNF-Rp55) in a stasis-induced venous thrombus murine model. IFNγ(+), TNFα(+), and TNF-Rp55(+) cells could be first detected 7, 3, and 3 days after inferior vena cava (IVC) ligation. Thereafter, the numbers of these positive cells increased with time after IVC ligation. Double-color immunofluorescence analyses demonstrated that each molecule was expressed by intrathrombotic macrophages. In all samples with postligation intervals of 7 days or less, IFNγ/macrophage ratio (IFNγ/Mϕ), TNFα/macrophage ratio (TNFα/Mϕ), and TNF-Rp55/macrophage ratio (TNF-Rp55/Mϕ) were <0.2, <0.2, and <0.4, respectively. In contrast, IFNγ/Mϕ and TNFα/Mϕ were greater than 0.2 and 0.3 at ≥10 days after the IVC ligation, respectively. These observations suggested that IFNγ/Mϕ ratios of >0.2 and TNFα/Mϕ ratios of >0.3 indicated thrombus age of ≥10 days. Moreover, TNF-Rp55/Mϕ ratios of >0.7 strongly suggested thrombus age of >14 days. The present study demonstrated that the immunohistochemical detection of IFNγ, TNFα, and TNF-Rp55 was suitable to estimate the age of venous thrombi. However, the variation in TNF-Rp55/Mϕ ratios is wider than those of the other two markers. Thus, IFNγ and TNFα could be more practical markers.

Abstract 348: Fibrin Targeted in vivo Imaging of Tissue Plasminogen Activator-induced Thrombolysis of Mouse Deep Vein Thrombosis

Introduction Tissue plasminogen activator (tPA) is increasingly used to treat deep vein thrombosis (DVT) to prevent the post-thrombotic syndrome. However, while aging venous thrombi are likely to become resistant to fibrinolysis, the age-dependent response to fibrinolysis has not been systematically assessed. Here we used confocal intravital microscopy (IVM) to dynamically study fibrinolysis of murine thigh DVT at high-resolution using thrombus angiography and a fibrin-specific sensor, FTP11-CyAm7 (FTP11). We hypothesized that fibrinolysis could be tracked and quantified in vivo using serial IVM and molecular imaging of fibrin. Methods C57Bl/6 mice underwent topical 7.5% ferric chloride injury to induce femoral DVT. At day 1 (n=6) or day 4 (n=5) post DVT, IVM was performed during tPA infusion. Mice were i.v. injected with a bolus of tPA, heparin and plasminogen, followed by a continuous 1 hour infusion of tPA+heparin. FTP11, injected 45 min prior to lysis, enabled molecular imaging of fibrin. DVT area and length were quantified using FITC-dextran venography before and after lysis. IVM image stacks were analyzed by NIH ImageJ. After sacrifice, fluorescence microscopy (FM) and Carstair’s staining were performed. Results Compared to day 4, day 1 DVT fibrinolysis showed a greater net decrease in thrombus area (32±11 vs. 6±4%, p=0.004) and FTP11 signal (54±17 vs. 11±8%, p=0.004) after 60 min. Of note, day 1 more lyseable DVT had significantly higher baseline FTP11 signal (target-to-background ratio 1.91±0.86 vs. 1.10±0.06, p=0.004), consistent with greater thrombus permeability at day 1 vs. day 4. The net lysis correlated well with both the baseline FTP11 thrombus signal (r=0.62, p=0.048) and change in FTP11 signal (r=0.79, p=0.006). IVM angiography revealed a trend of increased lysis in the distal thrombus edge, compared to the middle or proximal edge (day 1: 44±11% vs. 26±16% and 29±26%, p&gt;0.05). FTP11 signal colocalized with fibrin-rich thrombi on FM and Carstair’s staining. Conclusion Chemically induced murine DVT lyse more effectively at day 1 than day 4, as tracked by serial IVM molecular imaging. The FTP11-CyAm7 fibrin signal differentiates DVT of different ages and informs the magnitude of triggered fibrinolysis.

Abstract 19333: Ectonucleoside Triphosphate Diphosphohydrolase-1 (ENTDP-1/CD39) Regulates the Inflammatory Response in a Murine Model of Venous Thrombosis

Background: Deep vein thrombosis (DVT) is a major health concern in the United States, carrying significant morbidity, mortality and socioeconomic cost. CD39 (ectonucleoside triphosphate diphosphohydrolase 1) is a circulating and membrane-spanning protein on the surface of endothelial and some leukocyte populations that is responsible for the phospohydrolysis of ATP to ADP and ADP to AMP. We hypothesized that CD39 plays a protective role in modulating inflammatory responses in a murine model of DVT. Methods: C57BL/6 (WT) and CD39-/- mice underwent inferior vena cava (IVC) ligation. IVCs were harvested at 48h and 6 days post IVC ligation. Vena cava and thrombi were characterized. Leukocytes infiltrating the vein wall were quantified per high power field (hpf) by H&amp;E. Additionally, gene expression of macrophage markers was evaluated using qRT-PCR. Results: Thrombosed IVC weights were higher six days post IVC ligation in CD39-/- mice compared to IVC-ligated WT mice (35.6 mg ± 4.0 vs. 19.5 ± 2.6, n=3 per group, p&lt;0.05, respectively). Additionally, CD39-/- mice had a trend toward increased leukocyte infiltration into the vessel wall at all timepoints with an 80% increase in total leukocytes at 48h (225 ± 26 cells/hpf, n=3 vs. 124 ± 29 cells/hpf n=4, p=0.061, respectively), driven by neutrophilic (PMN) predominance (181 ± 15 cells/hpf, n=3 vs. 103 ± 23 cells/hpf, n=4, p 0.046, respectively) followed by a later trend toward increased macrophage and lymphocyte infiltration in CD39-/- mice. Consistent with these findings, CD39 gene-deletion was associated with a &gt;40-fold increase in mRNA expression of CCR2 (52.35 ± 0.73 vs. 1.17 ± 0.10, n=2 per group, p&lt;0.0002, respectively), a marker of pro-inflammatory macrophages, in the vessel wall compared to WT 6 days post IVC ligation. CD39-/- mice also had 15-fold increased expression (9.62 ± 0.43 vs. 0.62 ± 0.56, n=2 per group, p=0.006, respectively) of CD301b versus WT mice 6 days post IVC ligation. Conclusion: CD39 appears to be a critical regulator of the early and late inflammatory responses in murine DVT with increased thrombus burden, leukocyte infiltration and inflammation-related gene expression in CD39 gene-deleted mice. CD39 may serve a previously unexplored, protective role in the pathophysiology of DVT.

Abstract 28: Serial in Vivo Imaging of Thrombus Inflammation Predicts Venous Thrombus Resolution

Introduction: Inflammation following deep vein thrombosis (DVT) critically modulates thrombus resolution. However it is unknown whether the degree of inflammation in vivo predicts the magnitude of thrombus resolution. Here, we utilize serial multichannel intravital fluorescence microscopy (IVFM) to spatially map and quantify macrophage infiltration in thrombus, and then to determine the degree of thrombus resolution in vivo . Methods: C57Bl/6 mice (n=5) underwent topical ferric chloride injury to induce femoral venous thrombosis. On day 3, mice were i.v. injected with macrophage-targeted nanoparticles (CLIO-AF555, ex/em 555/565nm). On day 4, survival IVFM of thrombus macrophages was performed. In addition, venography was concomitantly performed (pre-injection of FITC-dextran 45 minutes before IVFM, MW 2000 kD, 490/520nm) to quantify DVT area and length in vivo at day 4 and 6. IVFM image analysis was performed using ImageJ. Mid-luminal z-stacks (40 μm thickness) were analyzed. Thrombus ROIs were outlined using FITC-dextran generated angiograms, and used calculating macrophage target-to-background ratios (TBR) and thrombus area and length. Contralateral sham femoral veins were imaged as controls. Imaging was followed by histopathology and fluorescence microscopy of the cryosectioned tissue. Results: At day 4, the baseline femoral DVT area and length were 0.22±0.08mm 2 and 1.29±0.16mm (mean±sd). The day 4 macrophage activity was noted to be heterogeneous with peripheral infiltration of the thrombus, and occasionally with some deeper penetration into the thrombus interior. At day 6, the thrombus area and length were reduced compared to day 4 (p&lt;0.005 for both). The day 4 thrombus macrophage TBR significantly predicted the degree of thrombus reduction (correlation of initial macrophage TBR and Δthrombus area, r=0.97, p=0.001). Conclusion: This serial in vivo assessment of murine DVT resolution demonstrates that degree of macrophage infiltration at day 4 predicts the magnitude of thrombus resolution at day 6. Molecular imaging of DVT inflammation in vivo could provide new insights into DVT resolution and the development of the post-thrombotic syndrome, and also in evaluating inflammatory-modulating therapies for improved DVT resolution.

Generation and Application of Active Recombinant Mouse Tissue Factor and Its Function-blocking Monoclonal Antibody*

Tissue factor (TF) is a cell surface glycoprotein playing an important role in the initiation of the blood coagulation cascade. The functions of TF in other physiological or pathological activities, such as tumor cell migration, are also acknowledged gradually in recent years. A recombinant protein of mouse tissue factor (mTF) extracellular part fused with His tag was constructed, and it was expressed and purified successfully in high-level soluble form. This recombinant mTF can effectively initiate plasma clotting in vitro and enhance blood coagulation in vivo, which prove its biological activity. Using this recombinant mTF as immunogen, a murine monoclonal function-blocking antibody to mTF was also generated, which can inhibit mTF-initiated plasma clotting in vitro. And by treating with this function-blocking antibody, the thrombus formation in a murine deep vein thrombosis model was attenuated successfully, which suggests the important role of tissue factor in deep vein thrombosis. In all, with the active mTF recombinant protein and the mTF function-blocking antibody, the functional investigations of TF in murine models of various research areas become more convenient and feasible.