Rodent Inferior Vena Cava Venoplasty Balloon Model.

Balloon venoplasty is a commonly used clinical technique to treat deep vein stenosis and occlusion as a consequence of trauma, congenital anatomic abnormalities, acute deep vein thrombosis (DVT), or stenting. Chronic deep venous obstruction is histopathologically characterized by thrombosis, fibrosis, or both. Currently, no direct treatment is available to target these pathological processes. Therefore, a reliable in vivo animal model to test novel interventions is necessary. The rodent survival inferior vena cava (IVC) venoplasty balloon model (VBM) allows the study of balloon venoplasty in non-thrombotic and post-thrombotic conditions across multiple time points. The local and systemic effect of coated and uncoated venoplasty balloons can be quantified via tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent assay (ELISA), zymography, vein wall and thrombus cellular analysis, whole blood and plasma assays, and histological analysis. The VBM is reproducible, replicates surgical human interventions, can identify local vein wall-thrombi protein changes, and allows multiple analyses from the same sample, decreasing the number of animals required per group.

Evaluation of Efficacy of 2 Extended-release Bupivacaine Products in a Porcine Model of Incisional Pain.

Extended-release (ER) local anesthetics are often incorporated in multi-modal analgesia or as an alternative when the effect of systemic analgesics may confound research. In this study, we compared the analgesic efficacy of 2 ER bupivacaine anesthetics with different ER mechanisms, a slow-release bupivacaine-meloxicam polymer (BMP) and a sucrose acetate isobutyrate bupivacaine (SABER-B) system. We used a full-thickness unilateral skin incision porcine model to evaluate the efficacy of these 2 ER bupivacaine analgesics. Eighteen male swine were randomized into 3 groups: control (saline; n = 6), bupivacaine:meloxicam (10 mg/kg, 0.3 mg/kg; n = 6), and SABER-B (10 mg/kg; n = 6). After surgery, pigs were assessed for changes in body weight, salivary cortisol level, and response to von Frey testing at 1, 3, 6, 24, 48, 72, 96, 120, and 168 h. Body weight and salivary cortisol levels were not significantly different between groups. Based on the von Frey testing, the pigs that received analgesics showed a significantly higher withdrawal threshold of nociceptive stimulus than those that received saline at 1, 3, 6, and 24 h after the surgery. At 48 h after surgery, the SABER-B group had a significantly higher withdrawal threshold than the saline group. The withdrawal threshold was not significantly different from the baseline measurement on intact skin at 3 and 6 h after surgery in the BMP group or 1 and 3 h for the SABERB group. The analgesic effects of BMP were greatest at 3 and 6 h after surgery and that of SABER-B as 1 and 3 h SABER-B provided an earlier onset of analgesia and longer analgesia duration than did BMP. This study demonstrates that ER bupivacaine can provide pigs with 24 to 48 h of analgesia for incisional pain.

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

BACKGROUND: This 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. METHODS: Using 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. RESULTS: Percent 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. CONCLUSIONS: Treatment 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.

Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA): Zone I Balloon Occlusion Time Affects Spinal Cord Injury in the Nonhuman Primate Model.

OBJECTIVES: Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA) has been used clinically to limit torso bleeding and restore central perfusion. The objective of this study was to determine the sequelae of prolonged REBOA in a nonhuman primate animal model. SUMMARY BACKGROUND DATA: Prolonged duration of REBOA is associated with adverse clinical outcomes. Threshold occlusion values tied to relative risk have yet to be determined. METHODS: Juvenile baboons were subjected to 40% to 55% total blood volume hemorrhage to achieve profound hypotension and shock. Zone I REBOA was performed for 60 minutes to assess acute injury and survival at 4 hours (group 1; n = 7). Post-REBOA 10-day survival and complications were then compared between 60 minutes (group 2; n = 8) and 30 minutes (group 3; n = 6) REBOA animals. RESULTS: Overall survival was 20/21 (95%). IL-6 and IL-8 were elevated at 1 and 4 hours in group 1 (P = 0.005; P = 0.001). Comparing 60-minute REBOA with 30-minute REBOA, there was (1) hypertension compared with normotension (P = 0.005), (2) increased base deficit (P = 0.003), (3) elevated Troponin I (P = 0.04), and histological evidence of kidney injury (P = 0.004). In addition, group 2 demonstrated paralysis with histopathologic changes of spinal cord ischemia (SCI) in 4/8 (50%), with no SCI in group 3 (P = 0.033). CONCLUSIONS: REBOA limits mortality in the primate model of severe hemorrhagic shock. However, unopposed balloon inflation in the distal thoracic aorta for 60 minutes results in high rates of spinal cord ischemia, an effect mitigated by limiting balloon inflation to 30 minutes.

Use of GMI-1271, an E-selectin antagonist, in healthy subjects and in 2 patients with calf vein thrombosis.

BACKGROUND: There is an unmet need for antithrombotic treatments for venous thromboembolic disease that do not increase bleeding risk. Selectins are cell adhesion molecules that augment thrombosis by activating immune cells to initiate the coagulation cascade. GMI-1271, a potent small-molecule E-selectin antagonist, has been shown in mouse models to decrease thrombus burden with a low risk of bleeding. METHODS: A first-in-human study of GMI-1271 was conducted to assess its safety, tolerability, and pharmacokinetic (PK) profile. As a secondary end point, biomarkers of coagulation, cell adhesion, and leukocyte/platelet activation were evaluated. Aims 1 and 2 were performed in healthy volunteers and evaluated single and multiple doses of the study drug, respectively. Aim 3 included 2 patients with isolated calf-level deep vein thrombosis (DVT). RESULTS: GMI-1271 showed consistent PK parameters for doses ranging from 2 to 40 mg/kg. Plasma levels increased in a linear manner with respect to dose, while clearance, volume of distribution, and half-life were not dose dependent. No accumulation was seen with multiple consecutive doses. No serious adverse events (grade 3 or 4) were reported. Biomarker analysis demonstrated a trend in reduction of soluble E-selectin (sEsel) levels with GMI-1271 exposure, while exposure did not impact laboratory testing of coagulation. Two patients with calf vein DVT were treated with GMI-1271 and demonstrated rapid improvement of symptoms after 48 hours, with repeat ultrasound showing signs of clot resolution. CONCLUSIONS: We demonstrate that GMI-1271 is safe in healthy volunteers and provide proof of concept that an E-selectin antagonist is a potential therapeutic approach to treat venous thrombosis.

Rapid, Reproducible, Quantifiable NMR Metabolomics: Methanol and Methanol: Chloroform Precipitation for Removal of Macromolecules in Serum and Whole Blood.

BACKGROUND: Though blood is an excellent biofluid for metabolomics, proteins and lipids present in blood can interfere with 1d-¹H NMR spectra and disrupt quantification of metabolites. Here, we present effective macromolecule removal strategies for serum and whole blood (WB) samples. METHODS: A variety of macromolecule removal strategies were compared in both WB and serum, along with tests of ultrafiltration alone and in combination with precipitation methods. RESULTS: In healthy human serum, methanol:chloroform:water extraction with ultrafiltration was compared to methanol precipitation with and without ultrafiltration. Methods were tested in healthy pooled human serum, and in serum from patients with sepsis. Effects of long-term storage at -80 °C were tested to explore the impact of macromolecule removal strategy on serum from different conditions. In WB a variety of extraction strategies were tested in two types of WB (from pigs and baboons) to examine the impact of macromolecule removal strategies on different samples. CONCLUSIONS: In healthy human serum methanol precipitation of serum with ultrafiltration was superior, but was similar in recovery and variance to methanol:chloroform:water extraction with ultrafiltration in pooled serum from patients with sepsis. In WB, high quality, quantifiable spectra were obtained with the use of a methanol: chloroform precipitation.

Safety Considerations When Working with Humanized Animals.

Research using laboratory animals has been revolutionized by the creation of humanized animal models, which are immunodeficient animals engrafted with human cells, tissues, or organs. These animal models provide the research community a unique and promising opportunity to mimic a wide variety of disease conditions in humans, from infectious disease to cancer. A vast majority of these models are humanized mice like those injected with human CD34+ hematopoietic stem cells and patient-derived xenografts. With this technology comes the need for the animal research enterprise to understand the inherent and potential risks, such as exposure to bloodborne pathogens, associated with the model development and research applications. Here, we review existing humanized animal models and provide recommendations for their safe use based on regulatory framework and literature. A risk assessment program-from handling the human material to its administration to animals and animal housing-is a necessary initial step in mitigating risks associated with the use of humanized animals in research. Ultimately, establishing institutional policies and guidelines to ensure personnel safety is a legal and ethical responsibility of the research institution as part of the occupational health and safety program and overall animal care and use program.

Venous Thrombosis and Post-Thrombotic Syndrome: From Novel Biomarkers to Biology.

Deep vein thrombosis (DVT) is a common disease that carries serious ramifications for patients, including pulmonary embolism and post-thrombotic syndrome (PTS). Although standard treatment for DVT is anticoagulation, this carries an added risk of bleeding and increased medication monitoring. Identifying those at risk for DVT and PTS can be difficult, and current research with murine models is helping to illuminate the biologic changes associated with these two disorders. Potential novel biomarkers for improving the diagnosis of DVT and PTS include ICAM-1, P-selectin, and cell-free DNA. Inhibition of factor XI, P- and E-selectin, and neutrophil extracellular traps holds promise for novel clinical treatment of DVT. Experimental research on PTS suggests potential cellular and mediator therapy targets of TLR9, MMP-2 and-9, PAI-1, and IL-6. Although many important concepts and mechanisms have been elucidated through research on DVT and PTS, more work must be done to translate experimental findings to the clinical arena. This review examines the currently used murine models of DVT, biomarkers involved in the pathophysiology and diagnosis of DVT and PTS, and potential pharmacologic targets for PTS treatment.

Analgesic Efficacy and Hematologic Effects of Robenacoxib in Mice.

NSAID analgesics may confound models that require inflammation to mimic disease development in humans. This effect presents a challenge for veterinary staff and investigators, because surgery is often necessary to create mouse models of disease and NSAID are first-line analgesics used to treat postoperative pain. We evaluated robenacoxib, a NSAID highly selective for cyclooxygenase 2, in a carrageenan paw edema (CPE) assay and surgical model of venous thrombosis (VT). We generated a mouse-specific dose-response curve by using the CPE assay for robenacoxib doses of 3.2, 10, 32 and 100 mg/kg SC. Electronic von Frey assay, calipers, and novel software for measuring open-field activity revealed that all robenacoxib doses provided, identified effective analgesia at 3 and 6 h, compared with saline. In addition, the 100-mg/kg dose had measurable antiinflammatory effects but yielded adverse clinical side effects. Because the 32-mg/kg dose was the highest analgesic dose that did not decrease paw swelling, we evaluated it further by using the same nociceptive and behavioral assays in addition to a novel nest-consolidation test, and assessment of blood clotting and hematologic parameters in the surgical VT model. A single preemptive dose of either 32 mg/kg SC robenacoxib or 5 mg/kg SC carprofen protected against secondary hyperalgesia at 24 and 48 h. Neither drug altered clot formation or hematology values in the VT model. The open-field activity software and our novel nest consolidation test both identified significant postoperative discomfort but did not differentiate between saline and analgesia groups. In light of these data, a single preemptive subcutaneous dose of 32 mg/kg of robenacoxib or 5 mg/kg of carprofen did not impede this VT mode but also failed to provide sufficient postoperative analgesia.

E-selectin inhibition with GMI-1271 decreases venous thrombosis without profoundly affecting tail vein bleeding in a mouse model.

Selectins, such as E-selectin (CD62E), function in venous thrombosis by binding and activating immune cells to initiate the coagulation cascade. GMI-1271 is a small molecule antagonist that inhibits E-selectin activity. Here we determine whether inhibition of E-selectin is sufficient to decrease acute venous thrombosis and associated inflammatory events in both prophylactic and treatment protocols without significantly affecting haemostasis. Male C57BL/6 mice underwent surgery for experimental thrombosis induction and were harvested at peak thrombus formation in our animal model, two days post induction. Groups included non-thrombosed true controls, shams, controls, and prophylactic or treatment groups of GMI-1271 (10 mg/kg intraperitoneal BID (twice a day) and low-molecular-weight heparin (LMWH, Lovenox 6 mg/kg subcutaneously (SC), once a day (SID). Compared with control animals, prophylaxis or treatment with LMWH and GMI-1271 in a dose-dependent manner significantly decreased thrombosis. GMI-1271 significantly lowered tail bleeding times when compared to LMWH. GMI-1271 and LMWH prophylactically administered significantly decreased vein wall neutrophil cell extravasation. However, all treatment and prophylactic therapies significantly decreased vein wall monocyte extravasation versus controls. GMI-1271 prophylactic therapy significantly decreased intra-thrombus cell counts versus control animals and other treatment groups. Immunohistochemistry confirmed that both treatments with GMI-1271 and LMWH significantly decreased activated leukocyte migration. GMI-1271 therapy significantly decreased thrombus weight and resulted in significantly lower bleeding times than LMWH. GMI-1271 treated mice showed decreased local and systemic inflammatory effects while modulating neutrophil activation, suggesting that GMI-1271 is a viable therapeutic candidate for venous thrombosis prophylaxis and treatment.

Apixaban Versus Warfarin for Mechanical Heart Valve Thromboprophylaxis in a Swine Aortic Heterotopic Valve Model.

OBJECTIVE: Warfarin is the current standard for oral anticoagulation therapy in patients with mechanical heart valves, yet optimal therapy to maximize anticoagulation and minimize bleeding complications requires routine coagulation monitoring, possible dietary restrictions, and drug interaction monitoring. As alternatives to warfarin, oral direct acting factor Xa inhibitors are currently approved for the prophylaxis and treatment of venous thromboembolism and reduction of stroke and systemic embolization. However, no in vivo preclinical or clinical studies have been performed directly comparing oral factor Xa inhibitors such as apixaban to warfarin, the current standard of therapy. APPROACH AND RESULTS: A well-documented heterotopic aortic valve porcine model was used to test the hypothesis that apixaban has comparable efficacy to warfarin for thromboprophylaxis of mechanical heart valves. Sixteen swine were implanted with a bileaflet mechanical aortic valve that bypassed the ligated descending thoracic aorta. Animals were randomized to 4 groups: control (no anticoagulation; n=4), apixaban oral 1 mg/kg twice a day (n=5), warfarin oral 0.04 to 0.08 mg/kg daily (international normalized ratio 2-3; n=3), and apixaban infusion (n=4). Postmortem valve thrombus was measured 30 days post-surgery for control-oral groups and 14 days post-surgery for the apixaban infusion group. Control thrombus weight (mean) was significantly different (1422.9 mg) compared with apixaban oral (357.5 mg), warfarin (247.1 mg), and apixiban 14-day infusion (61.1 mg; P<0.05). CONCLUSIONS: Apixaban is a promising candidate and may be a useful alternative to warfarin for thromboprophylaxis of mechanical heart valves. Unlike warfarin, no adverse bleeding events were observed in any apixaban groups.

Practice patterns of adjunctive therapy for venous leg ulcers.

Objectives Venous leg ulcers (VLU) are the most severe clinical sequelae of venous reflux and post thrombotic syndrome. There is a consensus that ablation of refluxing vein segments and treatment of significant venous obstruction can heal VLUs. However, there is wide disparity in the use and choice of adjunctive therapies for VLUs. The purpose of this study was to assess these practice patterns among members of the American Venous Forum. Methods The AVF Research Committee conducted an online survey of its own members, which consisted of 16 questions designed to determine the specialty of physicians, location of treatment, treatment practices and reimbursement for treatment of VLUs Results The survey was distributed to 667 practitioners and a response rate of 18.6% was achieved. A majority of respondents (49.5%) were vascular specialists and the remaining were podiatrists, dermatologists, primary care doctors and others. It was found that 85.5% were from within the USA, while physicians from 14 other countries also responded. Most of the physicians (45%) provided adjunctive therapy at a private office setting and 58% treated less than 5 VLU patients per week. All respondents used some form of compression therapy as the primary mode of treatment for VLU. Multilayer compression therapy was the most common form of adjunctive therapy used (58.8%) and over 90% of physicians started additional modalities (biologics, negative pressure, hyperbaric oxygen and others) when VLUs failed compression therapy, with a majority (65%) waiting less than three months to start them. Medicare was the most common source of reimbursement (52.4%). Conclusions Physicians from multiple specialties treat VLU. While most physicians use compression therapy, there is wide variation in the selection and point of initiation for additional therapies once compression fails. There is a need for high-quality data to help establish guidelines for adjunctive treatment of VLUs and to disseminate them to physicians across multiple specialties to ensure standardized high-quality treatment of patients with VLUs.

1D-¹H-nuclear magnetic resonance metabolomics reveals age-related changes in metabolites associated with experimental venous thrombosis.

OBJECTIVE: Age is a significant risk factor for the development of venous thrombosis (VT), but the mechanism(s) that underlie this risk remain(s) undefined and poorly understood. Aging is known to adversely influence inflammation and affect metabolism. Untargeted metabolomics permits an agnostic assessment of the physiological landscape and lends insight into the mechanistic underpinnings of clinical phenotypes. The objective of this exploratory study was to test the feasibility of a metabolomics approach for identifying potential metabolic mechanisms of age-related VT. METHODS: We subjected whole blood samples collected from young and old nonthrombosed controls and VT mice 2 days after thrombus induction using the electrolytic inferior vena cava, to a methanol:chloroform extraction and assayed the resulting aqueous fractions using 1D-(1)H- nuclear magnetic resonance. Normalized mouse metabolite data were compared across groups using analysis of variance (ANOVA) with Holm-Sidak post-testing. In addition, associations between metabolite concentrations and parameters of thrombosis such as thrombus and vein wall weights, and markers of inflammation, vein wall P- and E-selectin levels, were assessed using linear regression. The relatedness of the found significant metabolites was visually assessed using a bioinformatics tool, Metscape, which generates compound-reaction-enzyme-gene networks to aid in the interpretation of metabolomics data. RESULTS: Old mice with VT had a greater mean vein wall weight compared with young mice with VT (P < .05). Clot weight differences between old and young mice followed the same trend as vein wall weight (0.011 ± 0.04 g vs 0.008 ± 0.003 g; P = not significant). Glutamine (ANOVA, P < .01), proline (ANOVA, P < .01), and phenylalanine (ANOVA, P < .05) levels were increased in old VT mice compared with age-matched controls and young VT mice. Betaine and/or trimethylamine N-oxide levels were increased in aged mice compared with young animals. Vein wall weight was strongly associated with glutamine (P < .05), and phenylalanine (P < .01) concentrations and there was a trend toward an association with proline (P = .09) concentration. Vein wall P-selectin, but not E-selectin levels, were increased in old VT mice and were associated with the three found metabolites of age-related VT. Collectively, with the addition of glutamate, these metabolites form a single compound-reaction-enzyme-gene network that was generated by Metscape. CONCLUSIONS: We used 1D-(1)H-nuclear magnetic resonance-metabolite profiling to identify, for the first time, in an experimental model, three potential metabolites, glutamine, phenylalanine, and proline, associated with age-related VT. These metabolites are metabolically related and their levels are associated with vein wall weight and P-selectin concentrations. In aggregate, these findings provide a "roadmap" of pathways that could be interrogated in future studies, which could include provocation of the glutamine, phenylalanine, and proline pathways in the vein wall. This study introduces metabolomics as a new approach to furthering knowledge about the mechanisms of age-related VT.

A chimeric platelet-targeted urokinase prodrug selectively blocks new thrombus formation.

The use of fibrinolytic agents to prevent new thrombus formation is limited by an increased risk of bleeding due to lysis of hemostatic clots that prevent hemorrhage in damaged blood vessels. We sought to develop an agent that provides thromboprophylaxis without carrying a significant risk of causing systemic fibrinolysis or disrupting hemostatic clots. We previously showed that platelet (PLT) α granule-delivered urokinase plasminogen activator (uPA) is highly effective in preventing thrombosis, while being associated with little systemic fibrinolysis or bleeding. Here, we generated a chimeric prodrug composed of a single-chain version of the variable region of an anti-αIIbß3 mAb fused to a thrombin-activatable, low-molecular-weight pro-uPA (PLT/uPA-T). PLT/uPA-T recognizes human αIIbß3 on both quiescent and activated platelets and is enzymatically activated specifically by thrombin. We found that this prodrug binds tightly to human platelets even after gel filtration, has a prolonged half-life in mice transgenic for human αIIb compared with that of uPA-T, and prevents clot formation in a microfluidic system. Importantly, in two murine injury models, PLT/uPA-T did not lyse preexisting clots, even when administration was delayed by as little as 10 minutes, while it concurrently prevented the development of nascent thrombi. Thus, PLT/uPA-T represents the prototype of a platelet-targeted thromboprophylactic agent that selectively targets nascent over preexisting thrombi.

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.

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.

Effects of analgesic use on inflammation and hematology in a murine model of venous thrombosis.

Venous thrombosis (VT) is a significant cause of morbidity and mortality in humans. Surgical animal models are crucial in studies investigating the pathogenesis of this disease and evaluating VT therapies. Because inflammation is critical to both the development and resolution of VT, analgesic medications have the potential to adversely affect multiple parameters of interest in VT research. The objective of this study was to determine how several common analgesics affect key variables in a murine ligation model of deep vein thrombosis. Male C57BL/6 mice were randomly assigned to receive either local (bupivacaine) or systemic parenteral analgesia (buprenorphine, tramadol, or carprofen) or 0.9% NaCl (control). All mice underwent laparotomy and ligation of the inferior vena cava, and treatment was continued until euthanasia at 6 or 48 h after surgery. Analysis of harvested tissues and blood included: hematology, thrombus weight, serum and vein-wall cytokines (IL1ß, IL6, IL10, TNFα), soluble P-selectin, and vein-wall leukocyte infiltration. Compared with 0.9% NaCl, all of the analgesics affected multiple parameters important to VT research. Carprofen and tramadol affected the most parameters and should not be used in murine models of VT. Although they affected fewer parameters, a single dose of bupivacaine increased thrombus weight at 6 h, and buprenorphine was associated with reduced vein wall macrophages at 48 h. Although we cannot recommend the use of any of the evaluated analgesic dosages in this mouse model of VT, buprenorphine merits additional investigation to ensure the highest level of laboratory animal care and welfare.

Soluble P-selectin for the diagnosis of lower extremity deep venous thrombosis.

OBJECTIVE: Although duplex ultrasound is the standard for the diagnosis of lower extremity deep venous thrombosis (LE-DVT), imaging is not always available. The use of D-dimer can exclude (high-sensitivity), but not rule in (low-specificity) LE-DVT. Previously, we demonstrated that soluble P-selectin (sP-sel) in combination with the Wells score, establishes the diagnosis of LE-DVT with a specificity of 96% and a positive predictive value of 100%. In order to validate our previous results, we applied the model to a separate but similar patient cohort. Additionally, we analyzed the role of biomarkers for diagnosing upper extremity DVT (UE-DVT). METHODS: Between April 2009 and March 2012, all patients presenting for a duplex ultrasound exam with concern of DVT were screened. Demographics, clinical data, D-dimer, sP-sel, C-reactive protein, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, and von Willebrand factor levels were prospectively collected in 279 patients (234 LE-DVT, 45 UE-DVT). Continuous and categorical variables among patients with DVT were compared with patients without DVT. The diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were then calculated using our previously derived cut points to rule in or exclude DVT. RESULTS: Among 234 patients evaluated for LE-DVT, 112 (48%) patients had a confirmed LE-DVT with significant differences in all biomarkers. When Wells score ≥2, sP-sel could rule in LE-DVT with a specificity of 97.5% and a positive predictive value of 91%, which was more accurate than Wells score ≥2 and D-dimer (specificity, 65%; positive predictive value, 69%). When Wells score was <2, D-dimer was superior to sP-sel for excluding the diagnosis of LE-DVT (sensitivity, 98%; negative predictive value, 95% vs sensitivity, 91%; negative predictive value, 79%). The use of additional biomarkers did not increase accuracy. Had imaging not been available, we could have correctly ruled in or ruled out LE-DVT in 29% (67/234) of patients. The use of sP-sel in UE-DVT was nondiagnostic. CONCLUSIONS: We demonstrate that when Wells score ≥2, sP-sel is an excellent biomarker to rule in LE-DVT. Different from our previous study, D-dimer and a Wells score <2 was most sensitive at excluding a diagnosis of LE-DVT. Combined, Wells score, sP-sel, and D-dimer can both rule in and exclude LE-DVT in approximately one-third of patients.

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.

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.