The role of galectin-3 and galectin-3-binding protein in venous thrombosis.

Galectin-3-binding protein (gal3bp) and its receptor/ligand, galectin-3 (gal3), are secreted proteins that initiate signaling cascades in several diseases, and recent human proteomic data suggest they may play a role in venous thrombosis (VT). We hypothesized that gal3bp and gal3 may promote VT. Using a mouse stasis model of VT, we found that gal3bp and gal3 were localized on vein wall, red blood cells, platelets, and microparticles, whereas leukocytes expressed gal3 only. Gal3 was dramatically increased during early VT and gal3bp:gal3 colocalized in the leukocyte/endothelial cell interface, where leukocytes were partially attached to the vein wall. Thrombus size correlated with elevated gal3 and interleukin-6 (IL-6) vein wall levels. Recombinant gal3 promoted VT and increased vein wall IL-6 mRNA. Although recombinant gal3 restored the VT size in gal3(-/-) mice, it had no effect on IL6(-/-) mice, suggesting that gal3:gal3bp promotes VT through IL-6. Moreover, significantly fewer activated neutrophils were present in the gal3(-/-) vein walls. In a group of human patients, elevated circulating gal3bp correlated with acute VT. In conclusion, gal3bp:gal3 play a critical role in VT, likely via IL-6 and PMN-mediated thrombotic mechanisms, and may be a potential biomarker in human VT.

P-selectin inhibition therapeutically promotes thrombus resolution and prevents vein wall fibrosis better than enoxaparin and an inhibitor to von Willebrand factor.

OBJECTIVE: Aptamers are oligonucleotides targeting protein-protein interactions with pharmacokinetic profiles and activity reversal options. Although P-selectin and von Willebrand factor (vWF) have been implicated in the development of venous thrombosis (VT), no studies have directly compared aptamer efficacy with standard of care in VT. In this study, ARC5692, an anti-P-selectin aptamer, and ARC15105, an anti-vWF aptamer, were compared with low-molecular-weight heparin, enoxaparin, to test the efficacy of P-selectin or vWF inhibition in promoting thrombus resolution and preventing vein wall fibrosis, in a baboon model of VT. APPROACH AND RESULTS: Groups were as follows: treatment arm: animals received P-selectin or vWF aptamer inhibitors or enoxaparin (n=3 per group). Controls received no treatment (n=3). Prophylactic arm: animals received P-selectin inhibitor (n=4) or vWF inhibitor (n=3). Treatment arm: P-selectin-inhibitor demonstrated a significant improvement in vein recanalization by magnetic resonance venography (73% at day 21), and significantly decreased vein wall collagen, compared with all groups. Anti-P-selectin equaled enoxaparin in maintaining valve competency by ultrasound. All control animals had compromised valve competency post thrombosis. Prophylactic arm: animals receiving P-selectin and vWF inhibitors demonstrated improved vein recanalization by magnetic resonance venography versus controls (80% and 85%, respectively, at day 21). Anti-P-selectin protected iliac valve function better than anti-vWF, and both improved valve function versus controls. No adverse bleeding events were observed. CONCLUSIONS: The P-selectin inhibitor aptamer promoted iliac vein recanalization, preserved valve competency, and decreased vein wall fibrosis. The results of this work suggest that P-selectin inhibition maybe an ideal target in the treatment and prophylaxis of deep VT, warranting clinical trials.

Critical review of mouse models of venous thrombosis.

Deep vein thrombosis and pulmonary embolism are a significant health care concern, representing a major source of mortality and morbidity. In order to understand the pathophysiology of thrombogenesis and thrombus resolution, animal models are necessary. Mouse models of venous thrombosis contribute to our understanding of the initiation, propagation, and resolution of venous thrombus, as well as allow for the evaluation of new pharmaceutical approaches to prophylaxis and treatment of deep vein thrombosis. In this work we review the ferric chloride model, the inferior vena cava ligation model, the inferior vena cava stenosis models, and the electrolytic inferior vena cava model and compare their advantages and disadvantages.

Extracellular DNA traps promote thrombosis.

Neutrophil extracellular traps (NETs) are part of the innate immune response to infections. NETs are a meshwork of DNA fibers comprising histones and antimicrobial proteins. Microbes are immobilized in NETs and encounter a locally high and lethal concentration of effector proteins. Recent studies show that NETs are formed inside the vasculature in infections and noninfectious diseases. Here we report that NETs provide a heretofore unrecognized scaffold and stimulus for thrombus formation. NETs perfused with blood caused platelet adhesion, activation, and aggregation. DNase or the anticoagulant heparin dismantled the NET scaffold and prevented thrombus formation. Stimulation of platelets with purified histones was sufficient for aggregation. NETs recruited red blood cells, promoted fibrin deposition, and induced a red thrombus, such as that found in veins. Markers of extracellular DNA traps were detected in a thrombus and plasma of baboons subjected to deep vein thrombosis, an example of inflammation-enhanced thrombosis. Our observations indicate that NETs are a previously unrecognized link between inflammation and thrombosis and may further explain the epidemiological association of infection with thrombosis.

Impaired fibrinolytic system in ApoE gene-deleted mice with hyperlipidemia augments deep vein thrombosis.

BACKGROUND: Hyperlipidemia increases the level of blood plasminogen activator inhibitor-1 (PAI-1) that is responsible for regulating fibrinolysis by inhibiting both urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA). While this fibrinolytic pathway is well known, the role of PAI-1 in venous thrombosis (VT) under hyperlipidemic conditions has not been fully established. We sought to determine the effects of PAI-1 in an in vivo hyperlipidemic model of VT. METHODS: C57BL/6 wild-type (WT) mice, apolipoprotein E gene-deleted mice (ApoE-/-) having hyperlipidemia, and PAI-1 gene-deleted (PAI-1-/-) mice were used in this study. Inferior vena cava (IVC) ligation below the level of the renal veins was performed to create a stasis VT. Endpoints included measuring acute thrombosis (day 2) and chronic thrombosis (days 6 and 14). At euthanasia, blood samples were collected for plasmin and PAI-1 activity. In addition, the IVC and its thrombus were evaluated for thrombus weight (TW), u-PA activity, and differential leukocyte count while the vein wall only was analyzed for monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase (MMP) 2, and MMP-9. RESULTS: Compared to WT at day 2, ApoE-/-mice demonstrated a statistically significant 14% increase in TW (P < .05) and a significant 41% increase in circulating PAI-1 activity (P < .05), while showing a trend of decreased plasmin activity. In addition, TW in ApoE-/-mice was 45% higher than PAI-1-/-mice at day 2 (P < .05), 33% at day 6 (P < .01), and 41% at day 14 (P < .01). ApoE-/-mice exhibited undetectable levels of u-PA in both vein wall and thrombus, compared to WT, at all time points. Also, vein wall MMP-2 was significantly decreased by 64% at day 6 (P < .01) and 58% at day 14 (P < .05). MMP-9 was significantly decreased by 71% at day 2 (P < .01) and 48% at day 6 (P < .01), in ApoE-/-mice compared to WT mice. In addition, in ApoE-/-mice, MCP-1 was significantly decreased by 38% at day 2 (P < .01) and 67% at day 6 (P < .01) vs WT mice. As expected in ApoE mice, following a decrease in MCP-1, monocyte recruitment was significantly decreased at days 6 (P < .01) and 14 (P < .05). CONCLUSIONS: A significant increase of circulating PAI-1 levels in hyperlipidemic mice correlated with an early increase in TW due to impaired fibrinolysis. The undetectable levels of u-PA in ApoE-/-mice correlated to a decrease in vein wall MMP-2, MMP-9, MCP-1, and a decrease in monocyte recruitment diminishing thrombus resolution.

Statins, inflammation and deep vein thrombosis: a systematic review.

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism. The 2009 JUPITER trial showed a significant decrease in DVT in non-hyperlipidemic patients, with elevated C-reactive protein (CRP) levels, treated with rosuvastatin. The effects of statins on thrombosis are unclear, prompting this literature review. A literature search was performed (1950 to February 2011) with MEDLINE, EMBASE, and PUBMED databases including the following keywords: "statins", "hydroxymethylglutaryl-CoA reductase inhibitors", "VTE", "PE", "DVT", and either "anti-coagulation" or "inflammation". Editorials, reviews, case reports, meta-analysis and duplicates were excluded. Inflammatory biomarkers of DVT, include interleukin (IL)-6, CRP, IL-8, and monocyte chemotactic protein 1 (MCP-1). Statin therapy reduces IL-6 expression of CRP and MCP-1, usually elevated in VTE. Reduction of IL-6 induced MCP-1 has been linked to vein wall fibrosis, promoting post thrombotic syndrome (PTS) and recurrent DVT in patients. Also, our review suggests that the anti-thrombotic effects are likely exhibited through the anti-inflammatory properties of statins. This work supports that statin therapy has the ability to decrease the incidence and recurrence of VTE and the potential to decrease PTS. This is mainly due to the anti-inflammatory effects of statins and may explain why normolipidemic patients, with elevated CRP, appear to have the greatest reduction in VTE. Given their low risk of bleeding, statins have the potential to serve as a safe adjunctive pharmacological therapy to current treatments in select patients with VTE, however further investigations into this concept are needed and essential.

Interleukin-6: a potential target for post-thrombotic syndrome.

BACKGROUND: Deep vein thrombosis (DVT) and its associated sequelae, post-thrombotic syndrome (PTS), are significant health care problems in the United States. It is estimated that a maximum of 60% of patients diagnosed with DVT develop PTS, which is characterized by extensive perivenous and mural fibrosis. Interleukin-6 (IL-6) has been linked to fibrosis, and high circulating plasma levels have been found to increase the risk of developing DVT. The aim of this study was to elucidate the role of IL-6 in the progression of vein wall fibrosis by using a mouse model of DVT. METHODS AND RESULTS: C57BL/6 mice (n = 136) were treated with either anti-IL-6 monoclonal antibody or control rat-immunoglobulin G. Thrombus was induced by using an inferior vena cava ligation model. The inferior vena cava and thrombus were harvested at days 2, 6, or 14 for thrombus weight, gene expression of IL-6 and/or C-C motif chemokine ligand 2 (CCL2), inflammatory cell recruitment, and morphometric analysis of vein wall fibrosis. Mice treated with anti-IL-6 had smaller thrombus weights at day 2, decreased vein wall gene expression and protein concentration of CCL2 at day 2, and impaired vein wall influx of monocytes from days 2 to 6, as compared with controls. Intimal thickness was reduced by 44% (p < 0.05) and vein wall collagen deposition was decreased by 30% at day 14 in the anti-IL-6 group (p < 0.05). CONCLUSIONS: Neutralizing IL-6 throughout venous thrombogenesis decreased the production of CCL2, reduced monocyte recruitment, and decreased vein wall intimal thickness and fibrosis. These results suggest that IL-6 may serve as a therapeutic target to prevent the fibrotic complications seen in PTS.

Leukocyte- and platelet-derived microparticles correlate with thrombus weight and tissue factor activity in an experimental mouse model of venous thrombosis.

Microparticles (MP) are lipid vesicles from platelets, leukocytes and endothelial cells that are involved in early thrombogenesis. We evaluated a detailed time-course analysis of MPs on thrombogenesis and the associated tissue factor (TF) activity in wild-type, in gene-deleted for E- and P-selectins and with high levels of P-selectin expression after the initiation of venous thrombosis in mice. Inferior vena cava (IVC) ligation was performed on C57BL/6 mice (n = 191, 59 = wild-type [WT], 55 = gene-deleted for E- and P - selectins [knock-outs, EPKO] and 77 = elevated levels of soluble P-selectin, named Delta Cytoplasmic Tail (DeltaCT). Animals were euthanised at various time points to assess MP production, origin and thrombus weight. MPs were re-injected into separate mice at concentrations of 80,000 and 160,000 units, as well as from different ages. In addition, MPs from thrombosed animals were pooled and TF activity quantitated using a chromogenic assay. Thrombus weight correlated negatively with MPs derived from leukocytes, and positively with MPs derived from platelets for WT animals (p < 0.05), while MPs from platelets presented a positive correlation to thrombus weight in the WT and EPKO groups (p < 0.01). Total MPs correlated negatively with thrombus weight in the DeltaCT group (p < 0.05). MP re-injections led to greater thrombus weight, while older MP reinjections tended to form larger thrombus than younger. Finally, TF bearing MPs showed a significant correlation to MP concentrations (R = 0.99). In conclusion, MPs appear to be an important element in venous thrombogenesis.

Tissue inhibitor of metalloproteinase-1 is an early marker of acute endothelial dysfunction in a rodent model of venous oxidative injury.

Although there are extensive research data regarding arterial endothelial dysfunction, the effects of venous endothelial dysfunction are not well characterized. Matrix metalloproteinases (MMPs) have a defined role in vascular remodeling. MMPs are endopeptidases that are capable of degrading extracellular matrix proteins. We hypothesize that tissue inhibitor of metalloproteinase-1 (TIMP-1) can serve as an indicator of acute venous endothelial dysfunction in a rat model of oxidative injury. The experimental groups evaluated were as follows: rats not undergoing oxidative injury (controls), rats that received rose bengal but no laser (shams), and rats that received both rose bengal and laser illumination, resulting in an oxidative injury. Animals were evaluated at baseline (control, shams) and at 1 hr and 1 day post-oxidative injury. mRNA expression was determined by gene array technology and real-time polymerase chain reaction, plasma and vein wall TIMP-1 protein concentrations were determined by enzyme-linked immunosorbent assay, and vein wall morphometrics (cells/five high-power fields) were performed. B-cell lymphoma 2-like gene expression was upregulated at both 1 hr and 1 day post-injury. TIMP-1 protein and mRNA expression were significantly increased post-oxidative injury. One hour postinjury, vein wall polymorphonuclear leukocytes were present in significant numbers. Our results support the hypothesis that increased expression of TIMP-1 in venous endothelium and plasma may serve as an early indicator of endothelial dysfunction.

Dose-dependent thrombus resolution due to oral plaminogen activator inhibitor (PAI)-1 inhibition with tiplaxtinin in a rat stenosis model of venous thrombosis.

This study aimed to evaluate a small-molecule PAI-1 inhibitor (PAI-039; tiplaxtinin) in a rodent stenosis model of venous thrombosis in a two-phase experiment. Phase 1 determined the efficacy of tiplaxtinin against Lovenox (LOV), while phase 2 determined the dose-dependent efficacy. For both phases, drug treatment began 24 hours after surgically induced venous thrombosis and continued for four days. Phase 1 animals (n = 24) receiving low-dose (LD; 1 mg/kg oral gavage) PAI-1 inhibitor demonstrated a 52% decrease in thrombus weight (TW) versus controls (p < 0.05) with significant reductions in active plasma PAI-1, while the high-dose (HD; 10 mg/kg oral gavage) group demonstrated a 23% reduction in TW versus controls. Animals treated subcutaneously with LOV (3 mg/kg) showed a 39% decrease in TW versus controls (p < 0.05). Coagulation tests (aPTT and TCT) were significantly different in LOV compared to PAI-1 inhibitor groups. PAI-039 treatment was also associated with significantly increased return of inferior vena cava blood flow four days post-thrombosis versus controls (p < 0.05). In phase 2 (n = 30), TW was reduced from the 0.5 mg/kg to 5 mg/kg experimental groups, with the 10 mg/kg group demonstrating a paradoxical increase. The 5 mg/kg group showed statistically significant decreases in TW versus controls after four treatment days (p < 0.05). This is the first study to demonstrate dose related effects of PAI-039 on increasing thrombus resolution and inferior vena cava blood flow without adverse effects on anti-coagulation in a rat stenosis model of venous thrombosis.

Prophylactic P-selectin inhibition with PSI-421 promotes resolution of venous thrombosis without anticoagulation.

P-selectin inhibition has been evaluated as a therapeutic for prevention and treatment of venous thrombosis. In this study, a novel oral small-molecule inhibitor of P-selectin, PSI-421, was evaluated in a baboon model of stasis induced deep vein thrombosis (DVT). Experimental groups included i) primates receiving a single oral dose of 1 mg/kg PSI-421 two days prior and continued six days after thrombosis (n = 3); ii) primates receiving a single daily subcutaneous dose of 0.57 mg/kg enoxaparin sodium two days prior and continued six days post thrombosis (n = 3); and iii) primates receiving no treatment (n = 3). PSI-421 treated primates had greater percent vein reopening and less vein wall inflammation than the enoxaparin and controls at day 6. Microparticle tissue factor activity (MPTFA) was significantly lower in the animals receiving PSI-421 immediately after thrombosis (T+6 hours day 0) suggesting lower potential for thrombogenesis in these animals. PSI-421 also reduced soluble P-selectin levels versus controls at T+6 hours day 0, day 2 and 6. Experimental animals in any group showed no adverse effects on coagulation. This study is the first to demonstrate a reduction in MPTFA associated with vein reopening and reduced vein inflammation due to oral P-selectin inhibition in a baboon model of DVT.

Resolution of venous thrombosis using a novel oral small-molecule inhibitor of P-selectin (PSI-697) without anticoagulation.

P-selectin inhibition has been shown to decrease thrombogenesis in multiple animal species. In this study, we show that a novel oral small-molecule inhibitor of P-selectin, PSI-697, promotes thrombus resolution and decreases inflammation in a baboon model of venous thrombosis. Experimental groups consisted of the following: 1) primates receiving a single oral dose of PSI-697 (30 mg/kg) daily starting three days pre-iliac vein balloon occlusion, and continued for six days; 2) primates receiving a single treatment dose of a low-molecular-weight-heparin (LMWH) (1.5 mg/kg) daily starting one day pre-iliac balloon occlusion, and continued for six days; and 3) primates receiving a single oral dose of a vehicle control daily starting three days pre-iliac vein balloon occlusion, and continued for six days. Animals receiving PSI-697, although thrombosed after balloon deflation, demonstrated greater than 80% vein lumen opening over time, with no opening (0%) for vehicle control (p < 0.01). LMWH opening evident after balloon deflation slightly deteriorated over time compared to PSI-697. PSI-697 therapy also significantly decreased vein wall inflammation determined by magnetic resonance venography (MRV). Importantly, this beneficial opening occurred without measured anticoagulation. Animals receiving PSI-697 demonstrated significantly increased plasma D-dimer levels versus LMWH and control animals six hours post thrombus induction (p < 0.01). This study is the first to demonstrate the effectiveness of oral P-selectin inhibition to modify venous thrombogenesis, increase vein lumen opening, and decrease inflammation in a large animal model.

Correspondence of ultrasound elasticity imaging to direct mechanical measurement in aging DVT in rats.

Previous ultrasound elasticity imaging experiments supported a generally accepted concept that the hardness of deep venous thrombi increases with thrombus aging. Results also showed that this noninvasive imaging technique can accurately predict thrombus age through strain estimates, in a well-controlled animal study. In the present study, as an alternative means to characterize elastic properties of thrombi, we used a direct mechanical measurement system to estimate Young's modulus of ex vivo thrombi. Unlike conventional indentation tests, the device uses a specific compression geometry for cylindrical tissue specimens. We also proposed an approximation scheme to retrieve Young's modulus from force-displacement measurements made using the device. Finite element simulations and calibrations on tissue-mimicking phantoms validated the system. Then, using two groups of rats with surgically-induced thrombi, we further investigated the correlation between Young's modulus measured ex vivo and elasticity images reconstructed in vivo. This comparison was accomplished by converting the intrathrombus strains measured in the in vivo studies into Young's modulus estimates using a model-based approach. Good agreement between time-dependent Young's modulus estimates observed in vivo and direct measurements of Young's modulus using the mechanical device helps to confirm the ability of elasticity imaging to age deep venous thrombi for efficient treatment.

Gender differences in deep venous thrombosis in a rat model: a preliminary study.

The purpose of this study was to determine whether gender differences have an effect on inflammation and thrombosis in a rat model of venous thrombosis. A thrombus was created in mature female (n = 12) and male (n = 12) Sprague Dawley rats (Rattus norvegicus) by ligating the inferior vena cava (IVC). The IVC containing the thrombus was harvested at 1 and 3 days postligation, weighed, measured, and submitted for immunohistochemical analysis. In addition, hematology was performed at selected time points. There were no statistically significant differences in thrombus mass (mean +/- 1 standard deviation) between female and male rats at 1 (683 +/- 47.7 x 10(-4) versus 660 +/- 112.0 x 10(-4) g/cm) or 3 (683 +/- 83.3 x 10(-4) versus 580 +/- 86.0 x 10(-4) g/cm) days post-ligation. Females had significantly more platelets than did males on day 1 (741 +/- 37.2 versus 523 +/- 55.1 K/microL, P < 0.01). Day 3 males showed significant increases in vein wall neutrophils (18.0 +/- 2.30 versus 11.2 +/- 1.38, P < 0.05), ED-1-positive monocytes (54.4 +/- 16.0 versus 18.7 +/- 5.63, P < 0.05), and circulating white blood cells (15.4 +/- 0.947 x 10(3) versus 10.9 +/- 0.714 x 10(3)/microL, P < 0.01) at post-thrombosis when compared with females. We conclude that although female rats had greater thrombus mass, the male rats demonstrated more inflammatory cells in circulation and in their vein walls. This finding suggests that inflammation plays a role in thrombus resolution.

New and effective treatment of experimentally induced venous thrombosis with anti-inflammatory rPSGL-Ig.

BACKGROUND: P-selectin antagonism decreases thrombosis and inflammation in animal models of venous thrombosis (VT) prophylaxis. This study defines results using a P-selectin receptor antagonist for VT treatment. METHODS: Eight juvenile baboons underwent 6 h of iliofemoral venous stasis to produce an occlusive VT. Two days later, animals were treated for 14 days with rPSGL-Ig, 4 mg/kg (n3), LMWH (n2) or saline (n3) and treatment continued weekly (rPSGL-Ig) or daily (LMWH, saline). The animals were examined and sacrificed 14 days after treatment initiation (n4) or on day 90 (n4). RESULTS: Percent spontaneous vein reopening revealed a significant increase (p <0.05) in the proximal iliac vein in rPSGL-Ig and LMWH animals compared to controls (62%, 70% vs 8%), without differences in inflammation. No anticoagulation, thrombocytopenia, or wound complications were found in rPSGL-Ig animals. At 90 days, recanalization with iliac vein valve competence was found in treated animals. CONCLUSIONS: rPSGL-Ig successfully treated established VT without anticoagulation.

P-selectin inhibition decreases post-thrombotic vein wall fibrosis in a rat model.

BACKGROUND: Post-deep vein thrombosis (DVT) venous insufficiency is a vexing problem despite effective anticoagulation, and is characterized by vein wall fibrosis. This study tested the hypothesis that P-selectin inhibition would decrease post-thrombotic vein wall fibrosis and associated profibrotic mediators. METHODS: A rat stasis model of DVT was used to produce a 2-day-old DVT. Rats then received either intravenous saline (control), rPSGL-Ig (4 mg/kg) once, or daily subcutaneous low molecular weight heparin (LMWH) (0.5 mg/kg). Inferior vena cava wall was harvested 7 days after treatment and processed for thrombus size; leukocyte content; profibrotic mediators by enzyme-linked immunosorbent assay; collagen I and III mRNA expression by semiquantitative real-time polymerase chain reaction; and for collagen protein. RESULTS: Thrombus mass and leukocyte counts were similar between the groups. Treatment with rPSGL-Ig and LMWH resulted in less vein wall collagen (P <.05). rPSGL-Ig treatment (and a similar trend for LMWH) was associated with decreased profibrotic mediators, including less IL-13, MCP-1, bFGH, and transforming growth factor-beta (P <.05). Collagen III gene expression, but not collagen I gene expression, was increased with LMWH treatment (P <.05). CONCLUSIONS: P-selectin inhibition with rPSGL-Ig or LMWH decreases post-DVT vein wall fibrosis, and is associated with decreased vein wall profibrotic mediators. This effect is independent of thrombus mass and vein wall leukocytes.

Staging deep venous thrombosis using ultrasound elasticity imaging: animal model.

Deep venous thrombi undergo progressive hardening with age. However, the evolution rate remains poorly characterized by both invasive and noninvasive techniques. In a previous study (Emelianov et al. 2002), we demonstrated the potential of ultrasound elasticity imaging to noninvasively detect and age thrombus using a rat-based model. Knowing that thrombi harden over time is useful, but the value of the technique relies on whether the age of a thrombus can be predicted from strain estimates, and how accurate these predictions are. The objective of the present study is to answer these two questions. In the previous study, thrombus elasticity changes were monitored only on day 3, 6 and 9 after surgically induced formation of thrombosis in rat inferior vena cavas. In this study, ultrasound elasticity imaging was performed on two independent groups of rats (16 in total) starting from day 3 through day 10 with more temporal samples through the thrombus maturation process. For each rat, thrombus hardness was quantified at each scan interval by measures of normalized strains and reconstructed relative Young's moduli. In both groups, strain magnitudes exhibit progressive decrease as clots age. The relationship between the normalized strain and the clot age was developed from the first group and evaluated by the second group. Statistical analysis showed that the age estimation accuracy is within 0.8 day. If further research can successfully transfer the animal clot-hardening model to human patients, we believe that elasticity imaging will become a key component of venous compression ultrasound for effective diagnosis and treatment of deep venous thrombosis.

The electrolytic inferior vena cava model (EIM) to study thrombogenesis and thrombus resolution with continuous blood flow in the mouse.

Previously, we presented the electrolytic inferior vena cava (IVC) model (EIM) during acute venous thrombosis (VT). Here, we present our evaluation of the EIM for chronic VT time points in order to determine whether this model allows for the study of thrombus resolution. C57BL/6 mice (n=191) were utilised. In this model a copper-wire, inserted into a 25-gauge needle, is placed in the distal IVC and another subcutaneously. An electrical current (250 µAmp/15 minutes) activates the endothelial cells, inducing thrombogenesis. Ultrasound, thrombus weight (TW), vein wall leukocyte counts, vein wall thickness/fibrosis scoring, thrombus area and soluble P-selectin (sP-sel) were performed at baseline, days 1, 2, 4, 6, 9, 11 and 14, post EIM. A correlation between TW and sP-sel was also determined. A thrombus formed in each mouse undergoing EIM. Blood flow was documented by ultrasound at all time points. IVC thrombus size increased up to day 2 and then decreased over time, as shown by ultrasound, TW, and sP-sel levels. TW and sP-sel showed a strong positive correlation (r=0.48, p<0.0002). Vein wall neutrophils were the most common cell type present in acute VT (up to day 2) with monocytes becoming the most prevalent in chronic VT (from day 6 to day 14). Thrombus resolution was demonstrated by ultrasound, TW and thrombus area. In conclusion, the EIM produces a non-occlusive and consistent IVC thrombus, in the presence of constant blood flow, allowing for the study of VT at both acute and chronic time points. Thrombus resolution was demonstrated by all modalities utilised in this study.

Prothrombotic effects of thrombolytic therapy in a rat (Rattus norvegicus) model of venous thrombolysis.

The use of thrombolytic agents has greatly improved patient outcomes, but the prothrombotic response to these drugs in vivo is unknown. Approximately 24 h after we induced thrombosis in male Sprague-Dawley rats, we placed an infusion line in the inferior vena cava and administered either saline or a thrombolytic agent (tissue plasminogen activator [tPA] or plasmin) for 30 min. Blood was drawn immediately after infusion; rats were euthanized 24 h after infusion for collection of blood and tissue (inferior vena cava and thrombus). Thrombus size was decreased in the tPA-treated rats but not in those that received saline or plasmin; this change correlated with the significant rise in D-dimer levels noted immediately after infusion in the tPA-treated rats. Plasma soluble P-selectin, a prothrombotic marker, was elevated at 24 h in the plasmin group compared with the other treatment groups. There were no significant differences in plasma C3a, C5a, or C5b9 levels or in thrombus C3 levels between groups. According to ultrastructural analysis, thrombus structure and vein wall effects did not differ between groups. Local tPA did not induce a prothrombotic state during acute DVT or after thrombolytic therapy in a rodent model of venous thrombolysis. Conversely, levels of the prothrombotic marker plasma soluble P-selectin increased when plasmin was administered.

Myeloid cell tissue factor does not contribute to venous thrombogenesis in an electrolytic injury model.

INTRODUCTION: Tissue factor (TF) is a potent initiator of the extrinsic coagulation cascade. The role and source of TF in venous thrombotic disease is not clearly defined. Our study objective was to identify the contribution of myeloid cell TF to venous thrombogenesis in mice. MATERIALS AND METHODS: The mouse electrolytic inferior vena cava model was used to induce thrombosis. The following groups of mice were used (1) TF(flox/flox)LysMCre(+) mice that have reduced TF expression in myeloid cells, (2) TF(flox/flox)LysMCre(-) littermate controls, (3) Wild type mice given a monoclonal anti-mouse TF antibody (1H1) to inhibit TF activity, and (4) Wild type mice given rat IgG. Evaluations at baseline, day 2, and day 6 post thrombosis included thrombus weight, vein wall inflammatory cell migration, vein wall TF mRNA, and plasma D-dimer levels. RESULTS: Inhibition of TF significantly decreased thrombus weight 2days post venous thrombosis. In contrast, TF(flox/flox)LysMCre(+) had no change in thrombus weight when compared to littermate controls. The absence of myeloid cell TF did not affect infiltration of neutrophils or monocytes into the vein wall. TF mRNA expression in the vein wall decreased at 2days but then returned to baseline levels by 6days post thrombosis. D-dimer levels peaked at 2days post thrombosis in mice with or without myeloid cell TF. CONCLUSIONS: TF is important in the formation of venous thrombi in the macrovasculature. However, TF expression by myeloid cells does not significantly contribute to venous thrombogenesis in this model.