Publications by authors named "Daniel D Myers"

58 Publications

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

Res Pract Thromb Haemost 2020 Feb 11;4(2):193-204. Epub 2020 Feb 11.

Department of Internal Medicine Division of Hematology/Oncology University of Michigan Ann Arbor MI USA.

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.
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http://dx.doi.org/10.1002/rth2.12279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040550PMC
February 2020

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

Ann Surg 2019 Jun 7. Epub 2019 Jun 7.

Department of Surgery, Uniformed Services University, Bethesda, MD.

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.
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http://dx.doi.org/10.1097/SLA.0000000000003408DOI Listing
June 2019

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

Metabolites 2018 Dec 14;8(4). Epub 2018 Dec 14.

NMR Metabolomics Laboratory, Department of Clinical Pharmacy, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA.

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.
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http://dx.doi.org/10.3390/metabo8040093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316042PMC
December 2018

Safety Considerations When Working with Humanized Animals.

ILAR J 2018 12;59(2):150-160

Unit for Laboratory Animal Medicine, University of Michigan Medical School in Ann Arbor, Michigan.

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.
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http://dx.doi.org/10.1093/ilar/ily012DOI Listing
December 2018

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

Methodist Debakey Cardiovasc J 2018 Jul-Sep;14(3):173-181

UNIVERSITY OF MICHIGAN, ANN ARBOR, MICHIGAN.

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.
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http://dx.doi.org/10.14797/mdcj-14-3-173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217569PMC
January 2019

Analgesic Efficacy and Hematologic Effects of Robenacoxib in Mice.

J Am Assoc Lab Anim Sci 2018 05;57(3):258-267

Unit for Laboratory Animal Medicine, Department of Surgery, Vascular Surgery, University of Michigan Medical School, Ann Arbor, Michigan.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5966233PMC
May 2018

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

Thromb Haemost 2017 06 16;117(6):1171-1181. Epub 2017 Mar 16.

Daniel D. Myers, Jr., DVM, MPH, DACLAM, University of Michigan, North Campus Research Complex, Building 26, Room 263N, 2800 Plymouth Road, Ann Arbor, MI 48109-2800, USA, Tel.: +1 734 763 0940, E-mail:

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.
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http://dx.doi.org/10.1160/TH16-04-0323DOI Listing
June 2017

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

Arterioscler Thromb Vasc Biol 2017 05 23;37(5):942-948. Epub 2017 Feb 23.

From the Unit for Laboratory Animal Medicine (P.A.L., D.D.M.) and Conrad Jobst Vascular Research Laboratories (P.A.L., D.M.C., J.A.D., T.O.J., A.E.H., A.R.M., B.T.G., T.W.W., D.D.M.), University of Michigan, Ann Arbor; and Global Clinical Research, Research & Development (R.M.K., E.R.) and Exploratory Clinical and Translational Research (C.E.F., Y.S.), Bristol-Myers Squibb Company, Princeton, NJ.

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; <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.
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http://dx.doi.org/10.1161/ATVBAHA.116.308649DOI Listing
May 2017

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

J Vasc Surg Venous Lymphat Disord 2016 Apr 24;4(2):221-30. Epub 2015 Nov 24.

Jobst Vascular Research Laboratories, Section of Vascular Surgery, Department of Surgery, University of Michigan Health System, Ann Arbor, Mich; Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, Mich. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jvsv.2015.09.010DOI Listing
April 2016

Practice patterns of adjunctive therapy for venous leg ulcers.

Phlebology 2017 Feb 9;32(1):19-26. Epub 2016 Jul 9.

6 Vascular Surgery, University of Maryland School of Medicine, Baltimore, MD, USA.

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.
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http://dx.doi.org/10.1177/0268355515625526DOI Listing
February 2017

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

J Clin Invest 2016 Feb;126(2):483-94

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-αIIbβ3 mAb fused to a thrombin-activatable, low-molecular-weight pro-uPA (PLT/uPA-T). PLT/uPA-T recognizes human αIIbβ3 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.
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http://dx.doi.org/10.1172/JCI81470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731161PMC
February 2016

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

Arterioscler Thromb Vasc Biol 2015 Apr 5;35(4):829-37. Epub 2015 Feb 5.

From the Section of Vascular Surgery, Department of Surgery, Conrad Jobst Vascular Research Laboratories (J.A.D., S.K.W., C.M.A., A.E.H., N.K.D., J.E.G., K.J.R., P.K.H., T.W.W., D.D.M.), Unit for Laboratory Animal Medicine (C.M.A., P.A.L., D.D.M.), and Department of Radiology (S.E.L.), University of Michigan, Ann Arbor; and Research and Development, Archemix Corporation, Cambridge, MA (R.G.S.).

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.
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http://dx.doi.org/10.1161/ATVBAHA.114.304457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489932PMC
April 2015

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

Blood 2015 Mar 26;125(11):1813-21. Epub 2014 Nov 26.

Department of Surgery, Section of Vascular Surgery, Conrad Jobst Vascular Research Laboratories, and Unit for Laboratory Animal Research, University of Michigan, Ann Arbor, MI.

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.
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http://dx.doi.org/10.1182/blood-2014-04-569939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357586PMC
March 2015

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

J Am Assoc Lab Anim Sci 2014 Sep;53(5):485-93

Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, Michigan, USA.

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 (IL1β, 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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4181690PMC
September 2014

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

J Vasc Surg Venous Lymphat Disord 2013 Apr;1(2):117-1125

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.
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http://dx.doi.org/10.1016/j.jvsv.2012.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3752921PMC
April 2013

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

Comp Med 2013 Jun;63(3):244-51

Merck, Rahway, New Jersey, USA.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3690430PMC
June 2013

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

Thromb Haemost 2013 Jun 28;109(6):1158-69. Epub 2013 Mar 28.

Department of Surgery, Section of Vascular Surgery, Conrad Jobst Vascular Research Laboratories, University of Michigan, North Campus Research Complex (NCRC), 2800 Plymouth Road, B26, R251N, Ann Arbor, MI 48105-0654, USA.

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.
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http://dx.doi.org/10.1160/TH12-09-0711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822196PMC
June 2013

Current perspectives on venous disease.

Authors:
Daniel D Myers

Expert Rev Cardiovasc Ther 2012 Jul;10(7):839-41

Section of Vascular Surgery/ULAM Conrad Jobst Vascular Research Laboratories, North Campus Research Complex, Bldg 26, Room 263N, 2800 Plymouth Road, Ann Arbor, MI 48109-2800, USA.

24th Annual Meeting of the American Venous Forum Orlando, FL, USA, 8-11 February 2012: The American Venous Forum unites authorities on all facets of venous disease, the pathophysiology of venous disease and its treatment. The goal of this meeting was to educate attendees about current and novel clinical strategies to effectively manage venous disease.
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http://dx.doi.org/10.1586/erc.12.73DOI Listing
July 2012

Nonhuman primate models of thrombosis.

Authors:
Daniel D Myers

Thromb Res 2012 May 10;129 Suppl 2:S65-9. Epub 2012 Mar 10.

Department of Surgery, Section of Vascular Surgery, Conrad Jobst Vascular Research Laboratories, University of Michigan, Ann Arbor, MI 48109-0654, USA.

Arterial thrombosis and its associated clinical diseases make it one of the leading causes of death today. The majority of myocardial infarctions are caused by arterial thrombosis with acute myocardial infarction being the number one killer of individuals at a premature age. Venous thromboembolism (VTE) has a national morbidity exceeding 900,000 persons, with approximately 300,000 fatalities annually. Unfortunately, the incidence of this disease has not significantly changed over the past 25 years, possibly due to the increasing identification of new cases. The use of nonhuman primate models of vascular disease is essential to our understanding of the mechanisms that promote thrombogenesis in humans. These large animal models have close phylogenetic, anatomic, and physiologic similarities to humans, which make them necessary for many translational vascular research applications. Thus, translational animal models can help accelerate the development of new pharmacologic and medical devices targeted at limiting vascular inflammation and thrombosis. This review provides a brief overview of several nonhuman primate models used for the development of novel interventions directed at thrombotic diseases.
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http://dx.doi.org/10.1016/j.thromres.2012.02.037DOI Listing
May 2012

Critical review of mouse models of venous thrombosis.

Arterioscler Thromb Vasc Biol 2012 Mar;32(3):556-62

Department of Surgery, Conrad Jobst Vascular Research Laboratories, University of Michigan, A570 MSRB II, Dock #6, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0654, USA.

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.
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http://dx.doi.org/10.1161/ATVBAHA.111.244608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292052PMC
March 2012

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

J Thromb Thrombolysis 2012 May;33(4):371-82

Department of Surgery, Section of Vascular Surgery, Conrad Jobst Vascular Research Laboratories, School of Medicine, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA.

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.
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http://dx.doi.org/10.1007/s11239-012-0687-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338886PMC
May 2012

Inflammation and thrombosis: new insights.

Front Biosci (Schol Ed) 2012 Jan 1;4:620-38. Epub 2012 Jan 1.

Unit for Laboratory Animal Medicine, University of Michigan Medical Center, Ann Arbor, MI, USA.

Vessel wall endothelial damage initiates a local inflammatory response, which promotes a prothrombotic state driven by tissue factor, adhesion molecules, and pro-inflammatory cytokines. Understanding how natural inflammatory mechanisms promote a procoagulant state, may lead to the development of new pharmacological interventions targeted at thrombosis.
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http://dx.doi.org/10.2741/s289DOI Listing
January 2012

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

Thromb Res 2012 Oct 20;130(4):640-5. Epub 2011 Dec 20.

Department of Surgery, Section of Vascular Surgery, University of Michigan, Ann Arbor, Michigan, USA; Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, Michigan, USA.

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.
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http://dx.doi.org/10.1016/j.thromres.2011.11.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3336042PMC
October 2012

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

J Vasc Surg 2012 Mar 25;55(3):815-22. Epub 2011 Nov 25.

Department of Surgery, Section of Vascular Surgery, Conrad Jobst Vascular Research Laboratories, University of Michigan, Ann Arbor, MI 48109, USA.

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.
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http://dx.doi.org/10.1016/j.jvs.2011.08.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3289767PMC
March 2012

Electrolytic inferior vena cava model (EIM) of venous thrombosis.

J Vis Exp 2011 Jul 12(53):e2737. Epub 2011 Jul 12.

Conrad Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan, USA.

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential therapeutic compounds. New compounds to be utilized in the treatment and prevention of DVT are currently being developed. The delivery of potential therapeutic antagonist compounds to an affected thrombosed vein has been problematic. In the context of therapeutic applications, a model that uses partial stasis and consistently generates thrombi within a major vein has been recently established. The Electrolytic Inferior vena cava Model (EIM) is mouse model of DVT that permits thrombus formation in the presence of continuous blood flow. This model allows therapeutic agents to be in contact with the thrombus in a dynamic fashion, and is more sensitive than other models of DVT. In addition, this thrombosis model closely simulates clinical situations of thrombus formation and is ideal to study venous endothelial cell activation, leukocyte migration, venous thrombogenesis, and to test therapeutic applications. The EIM model is technically simple, easily reproducible, creates consistent thrombi sizes and allows for a large sample (i.e. thrombus and vein wall) which is required for analytical purposes.
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http://dx.doi.org/10.3791/2737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196180PMC
July 2011

Mouse complete stasis model of inferior vena cava thrombosis.

J Vis Exp 2011 Jun 15(52). Epub 2011 Jun 15.

Conrad Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan, USA.

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern (1-2). After heart disease and stroke, VTE is the third most common vascular disease (3). In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually (3). A reliable in vivo animal model to study the mechanisms of this disease is necessary. The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays (4-8). The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.
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http://dx.doi.org/10.3791/2738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3346045PMC
June 2011

Evaluation of soluble P-selectin as a marker for the diagnosis of deep venous thrombosis.

Clin Appl Thromb Hemost 2011 Aug 17;17(4):425-31. Epub 2011 May 17.

Conrad Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan Medical Center, Ann Arbor, MI, USA.

Objective: The combination of D-dimer and Wells score can exclude, but not confirm, the diagnosis of deep venous thrombosis (DVT). Since thrombosis and inflammation are interrelated, we evaluated the combination of soluble P-selectin (sPsel) with other inflammatory biomarkers for the diagnosis of DVT.

Methods: Sixty-two positive and one hundred and sixteen patients with negative DVT, by duplex scan, were prospectively evaluated for sPsel, D-dimer, C-reactive protein (CRP), microparticles (MPs; total, leukocyte, and platelet-derived and tissue factor positive microparticles), and clinical Wells score.

Results: Biomarkers and clinical scores that differentiated DVT positives from negatives were sPsel (87.3 vs 53.4 ng/mL, P < .0001), D-dimer (5.8 vs 2.1 mg/ L, P < .0001), CRP (2.1 vs 0.8 μg/mL, P < .0005), and Wells score (3.2 vs 2.0, P < .0001). For MP analysis, platelet-derived MPs were found to differentiate DVT from negatives. Using multivariable logistic regression, a combination of sPsel and Wells score could establish the diagnosis of DVT (cut point ≥ 90 ng/mL + Wells ≥ 2), with a specificity of 96% and positive predictive value (PPV) of 100%, and could exclude DVT diagnosis (cut point ≤ 60 ng/mL and Wells <2) with a sensitivity of 99%, a specificity of 33%, and a negative predictive value (NPV) of 96%.

Conclusion: This study establishes a biomarker and clinical profile combination that can both confirm and exclude the diagnosis of DVT.
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http://dx.doi.org/10.1177/1076029611405032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306250PMC
August 2011

Male mice have increased thrombotic potential: sex differences in a mouse model of venous thrombosis.

Thromb Res 2011 May 5;127(5):478-86. Epub 2011 Feb 5.

Department of Surgery, Section of Vascular Surgery, University of Michigan, Ann Arbor, MI 48109-0654, USA.

Introduction: Our objectives were to characterize sex differences during venous thrombosis, using the electrolytic inferior vena cava model of the disease.

Materials And Methods: Male and female C57BL/6 mice (6-8 weeks) underwent inferior vena cava thrombosis. Time points included 6 hours, day 2, day 6, and day 14 post surgery, along with surgically naïve true controls and surgical shams. Analyses included thrombus weight, vein wall morphometrics, vein wall protein and gene expression for P-selectin, interleukin-1β, and tumor necrosis factor-α; hematology, soluble P-selectin, and plasma microparticle tissue factor activity assays.

Results: Male venous thrombi were significantly larger than females at days 2 (13.1 ± 1.0 vs. 6.8 ± 0.5 × 10(-3) grams, p < 0.01), 6 (10.4 ± 0.8 vs. 5.4 ± 0.5 × 10(-3) grams, p < 0.01) and 14 (6.3 ± 0.5 vs. 4.1 ± 0.3 × 10(-3) grams, p < 0.01). Both male and female mice exhibited significantly increased vein wall P-selectin at 6 hours, vs. true controls (p < 0.05). Males had increased vein wall interleukin-1β, versus females, at 6 hours (180.926 ± 24.596 vs. 60.417 ± 10.478 pg/mL, p < 0.05) and day 6 (76.966 ± 13.081 vs. 33.834 ± 4.198 pg/mL, p < 0.01). Males showed decreased tumor necrosis factor-α expression (-66 %) at 6 hours. Females had increased tumor necrosis factor-α expression at 6 hours (+541%) and day 6 (+539%). Both sexes demonstrated decreased peripheral platelets at 6 hours (p < 0.05), coinciding with thrombogenesis. Plasma P-selectin increased in both sexes, versus controls, through day 6 (p < 0.05).

Conclusions: Males had significantly larger venous thrombi than females. Sex differences in vascular anatomy and response to inflammation may influence thrombus formation in our mouse thrombosis model.
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http://dx.doi.org/10.1016/j.thromres.2011.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3081977PMC
May 2011

Noninvasive treatment of deep venous thrombosis using pulsed ultrasound cavitation therapy (histotripsy) in a porcine model.

J Vasc Interv Radiol 2011 Mar 30;22(3):369-77. Epub 2010 Dec 30.

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.

Purpose: This study evaluated histotripsy as a noninvasive, image-guided method of thrombolysis in a porcine model of deep vein thrombosis. Histotripsy therapy uses short, high-intensity, focused ultrasound pulses to cause mechanical breakdown of targeted soft tissue by acoustic cavitation, which is guided by real-time ultrasound imaging. This is an in vivo feasibility study of histotripsy thrombolysis.

Methods And Materials: Acute thrombi were formed in the femoral vein of juvenile pigs weighing 30-40 kg by balloon occlusion with two catheters and thrombin infusion. A 10-cm-diameter 1-MHz focused transducer was used for therapy. An 8-MHz ultrasound imager was used to align the clot with the therapy focus. Therapy consisted of five cycle pulses delivered at a rate of 1 kHz and peak negative pressure between 14 and 19 MPa. The focus was scanned along the long axis of the vessel to treat the entire visible clot during ultrasound exposure. The targeted region identified by a hyperechoic cavitation bubble cloud was visualized via ultrasound during treatment.

Results: Thrombus breakdown was apparent as a decrease in echogenicity within the vessel in 10 of 12 cases and in 7 cases improved flow through the vein as measured by color Doppler. Vessel histology found denudation of vascular endothelium and small pockets of hemorrhage in the vessel adventitia and underlying muscle and fatty tissue, but perforation of the vessel wall was never observed.

Conclusions: The results indicate histotripsy has potential for development as a noninvasive treatment for deep vein thrombosis.
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http://dx.doi.org/10.1016/j.jvir.2010.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3053086PMC
March 2011

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

Ann Vasc Surg 2011 Feb 4;25(2):229-39. Epub 2010 Dec 4.

Section of Vascular Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI, USA.

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.
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http://dx.doi.org/10.1016/j.avsg.2010.09.003DOI Listing
February 2011