Inferior vena cava branch variations in C57BL/6 mice have an impact on thrombus size in an IVC ligation (stasis) model.

BACKGROUND: Animal models of venous thrombosis (VT) are critical tools for those investigating the VT mechanism. Recently, inferior vena cava (IVC) branches have been subject to debate, causing controversy in the field. OBJECTIVES: To understand how the variability of IVC branches, in commonly used C57BL/6 mice, have an impact on thrombus formation in the IVC ligation model. METHODS: C57BL/6 male mice (n = 46), 20-25 g, were subjected to the IVC ligation model with various interruptions of the IVC branches. Control animals (n = 50) had all branches interrupted. Two days after IVC ligation, thrombus weight (TW), as a parameter of thrombus size, was assessed. RESULTS: We found four different anatomical patterns. Side branches were more prevalent on the mouse's right side (34%) compared with the left (20%). In mice where side branches were absent (21%), back branches appeared larger. Also, 25% of mice had both side branches. Controls that had all IVC branches interrupted had the most consistent and largest TW (32.6 mg to 34.7 mg) while groups that had no back branches interrupted had the smallest TW (3.6-9.7 mg), a 4 to 9-fold decrease. All groups with open back branches had significantly smaller TW (P < 0.05) than controls. CONCLUSIONS: Variations in TW were observed based on different branch interruption patterns, compared with the fully ligated controls. Having two back branches was the most consistent anatomy and open back branches had the largest negative impact on thrombus size. This work confirms that the IVC branches significantly affect thrombus burden in C57BL/6 mice and further studies should be conducted in order to standardize this and other animal models of VT.

Plasminogen activator-1 overexpression decreases experimental postthrombotic vein wall fibrosis by a non-vitronectin-dependent mechanism.

BACKGROUND: Factors associated with postthrombotic syndrome are known clinically, but the underlying cellular processes at the vein wall are not well delineated. Prior work suggests that vein wall damage does not correlate with thrombus resolution but rather with plasminogen activator-1 (PAI-1) and matrix metalloproteinase (MMP) activity. OBJECTIVE: We hypothesized that PAI-1 would confer post venous thrombosis (VT) vein wall protection via a vitronectin (Vn)-dependent mechanism. METHODS: A stasis model of VT was used with harvest over 2 weeks, in wild-type, Vn(-/-) , and PAI-1-overexpressing mice (PAI-1 Tg). RESULTS: PAI-1 Tg mice had larger VT at 6 and 14 days, compared to controls, but Vn(-/-) mice had no alteration of VT resolution. Gene deletion of Vn resulted in an increase in, rather than the expected decrease in, circulating PAI-1 activity. While both Vn(-/-) and PAI-1 Tg had attenuated intimal fibrosis, PAI-1 Tg had significantly less vein wall collagen and a compensatory increase in collagen III gene expression. Both Vn(-/-) and PAI-1 Tg vein wall had less monocyte chemotactic factor-1 and fewer macrophages (F4/80), with significantly less MMP-2 activity and decreased TIMP-1 antigen. Ex vivo assessment of transforming growth factor ß-mediated fibrotic response showed that PAI-1 Tg vein walls had increased profibrotic gene expression (collagens I and III, MMP-2, and α-smooth muscle actin) compared with controls, opposite of the in vivo response. CONCLUSIONS: The absence of Vn increases circulating PAI-1, which positively modulates vein wall fibrosis in a dose-dependent manner. Translationally, PAI-1 elevation may decrease vein wall damage after deep vein thrombosis, perhaps by decreasing macrophage-mediated activities.

Plasma DNA is Elevated in Patients with Deep Vein Thrombosis.

OBJECTIVE: To investigate if plasma DNA is elevated in patients with deep vein thrombosis (DVT) and to determine whether there is a correlation with other biomarkers of DVT. BACKGROUND: Leukocytes release DNA to form extracellular traps (ETs), which have recently been linked to experimental DVT. In baboons and mice, extracellular DNA co-localized with von Willebrand factor (VWF) in the thrombus and DNA appeared in circulation at the time of thrombus formation. ETs have not been associated with clinical DVT. SETTING: From December 2008 to August 2010, patients were screened through the University of Michigan Diagnostic Vascular Unit and were divided into three distinct groups: 1) the DVT positive group, consisting of patients who were symptomatic for DVT, which was confirmed by compression duplex ultrasound (n=47); 2) the DVT negative group, consisting of patients that present with swelling and leg pain but had a negative compression duplex ultrasound, (n=28); and 3) a control group of healthy non-pregnant volunteers without signs or symptoms of active or previous DVT (n=19). Patients were excluded if they were less than 18 years of age, unwillingness to consent, pregnant, on an anticoagulant therapy, or diagnosed with isolated calf vein thrombosis. METHODS: Blood was collected for circulating DNA, CRP, D-dimer, VWF activity, myeloperoxidase (MPO), ADAMTS13 and VWF. The Wells score for a patient's risk of DVT was assessed. The Receiver Operating Characteristic (ROC) curve was generated to determine the strength of the relationship between circulating DNA levels and the presence of DVT. A Spearman correlation was performed to determine the relationship between the DNA levels and the biomarkers and the Wells score. Additionally the ratio of ADAMTS13/VWF was assessed. RESULTS: Our results showed that circulating DNA (a surrogate marker for NETs) was significantly elevated in DVT patients, compared to both DVT negative patients (57.7±6.3 vs. 17.9±3.5ng/mL, P<.01) and controls (57.7±6.3 vs. 23.9±2.1ng/mL, P<.01). There was a strong positive correlation with CRP (P<.01), D-dimer (P<.01), VWF (P<.01), Wells score (P<.01) and myeloperoxidase (MPO) (P<.01), along with a strong negative correlation with ADAMTS13 (P<.01) and the ADAMTS13/VWF ratio. The logistic regression model showed a strong association between plasma DNA and the presence of DVT (ROC curve was determined to be 0.814). CONCLUSIONS: Plasma DNA is elevated in patients with deep vein thrombosis and correlates with biomarkers of DVT. A strong correlation between circulating DNA and MPO suggests that neutrophils may be a source of plasma DNA in patients with DVT.

Rosuvastatin reduced deep vein thrombosis in ApoE gene deleted mice with hyperlipidemia through non-lipid lowering effects.

INTRODUCTION: Statins, particularly rosuvastatin, have recently become relevant in the setting of venous thrombosis. The objective of this study was to study the non-lipid lowering effects of rosuvastatin in venous thrombosis in mice with hyperlipidemia. MATERIALS AND METHODS: An inferior vena cava ligation model of venous thrombosis in mice was utilized. Saline or 5mg/kg of rosuvastatin was administered by gavage 48hs previous to thrombosis. Blood, the inferior vena cava, thrombus, and liver were harvested 3, 6hours, and 2days post-thrombosis. Thrombus weight, inflammatory markers, and plasminogen activator inhibitor-1 expression and plasma levels were measured. Also, neutrophil migration to the IVC was assessed. RESULTS: Rosuvastatin significantly decreased thrombus weight, plasminogen activator inhibitor-1 expression and plasma levels, expression of molecules related to the interleukin-6 pathway, and neutrophil migration into the vein wall. CONCLUSIONS: This work supports the beneficial effects of rosuvastatin on venous thrombosis in mice with hyperlipidemia, due to its non-lipid lowering effects.

Novel anticoagulants: a discussion of clinical use in the treatment and prevention of venous thromboembolism.

Traditional therapeutic oral anticoagulation strategies often require invasive dosing or monitoring. Vitamin K antagonists (VKAs) have a large number of interactions, delayed onset requires frequent dose monitoring, and they have a small margin between therapeutic dose and bleeding complications. Novel oral anticoagulants, such as dabigatran, rivaroxaban and apixaban, are being developed to prevent those VKAs drawbacks. Besides oral bioavailability, those compounds are designed to require minimal to no monitoring and have a favourable safety profile. This review reports efficacy and safety data of these compounds throughout clinical development, as well as new approaches for oral pharmacological management of venous thromboembolism.

Decrease in fibrin content of venous thrombi in selectin-deficient mice.

The purpose of this study was to quantify the fibrin content of thrombi produced in a mouse model of venous thrombosis and correlate this to thrombus mass. The role of P-selectin, E-selectin, and IL-10 on thrombus fibrin content was analyzed using knockout (KO) mice. Five groups of mice were evaluated: control (N = 10), P-selectin KO (N = 7), E-selectin KO (N = 5), combined E-/P-selectin KO (N = 12), and IL-10 KO (N = 10). Venous thrombosis was induced by ligation of the infrarenal IVC. Mice were sacrificed on postoperative days (POD) 2 and 6. Thrombus mass was calculated. Sections of IVC were stained with an antibody that cross reacts with mouse fibrin. The distribution of RGB color pixels was generated from digitized micrographs of the thrombus of each animal. The mean pixel value for each group was compiled and analyzed using 2-way ANOVA. Mean pixel value per group was correlated with the mean thrombus mass per group. Color analysis demonstrated significant decreases in the analyzed fibrin content on POD-2 between the control vs E-/P-selectin KO (P < 0.05) and control vs IL-10 KO (P < 0.05) groups. In addition, significantly less fibrin staining was noted on POD-6 between the control vs E-selectin KO (P = 0.03), control vs P-selectin KO (P = 0.01), and control vs E-/P-selectin KO (P < 0.01). There was a strong overall correlation between the mean pixel value for each group and the thrombus mass (R = 0.964; P < 0.01). This study demonstrates a difference in fibrin content of thrombi produced in animals deficient in E-selectin, P-selectin, and IL-10, supporting their importance in thrombus amplification, fibrin formation, and the mass of thrombus formed.

Triplex ultrasound: elasticity imaging to age deep venous thrombosis.

Deep venous thrombosis (DVT), and its sequela, pulmonary embolism (PE), is the leading cause of preventable in-hospital mortality in the USA and other developed countries. After it is detected, acute clots must be differentiated from chronic DVT for appropriate treatment. However, there are no reliable thrombus staging methods presently available in clinical practice. In this study, we tested the hypothesis that blood clots can be detected and staged using a triplex ultrasound (US) test. Triplex US is based on a "gold standard" duplex US technique augmented by US-based reconstructive elasticity imaging. Fibrin-composed blood clots harden with development and organization. By imaging clot elasticity, it may be possible to both detect and differentiate clots and, therefore, provide an urgently needed noninvasive means of DVT staging.

Venous thrombosis incidence in burn patients: preliminary results of a prospective study.

There are few prospective data on the incidence of deep venous thrombosis (DVT) in burn patients. In an on-going prospective study, hospitalized burn patients 18 years or older with an expected hospital length of stay more than 72 hours were imaged with baseline venous duplex ultrasound of all extremities within the first 48 hours after admission and weekly until discharge. Patient demographics and clinical risk factors for DVT were assessed. At the time of submission, 40 patients met screening criteria, and 30 were enrolled. Ultrasound diagnosed seven patients with 11 acute DVT for an incidence of 23%. One pulmonary embolism was documented. DVT patients had a mean age of 49 +/- 23 years with an average TBSA burn of 15 +/- 4% compared with those without thrombosis with a mean age of 44 +/- 17 years (P = NS) and TBSA burn of 18 +/- 25% (P = NS). There were no statistically significant differences for DVT patients in terms of age, number of central line days, hospital length of stay, or TBSA burned. Given the preliminary findings of this small study, we believe that all hospitalized burn patients are at risk for DVT. On-going investigation will be helpful in defining level of risk and improved prevention strategies for thromboembolic complications in burn patients.

Low molecular weight protamine as nontoxic heparin/low molecular weight heparin antidote (III): preliminary in vivo evaluation of efficacy and toxicity using a canine model.

Heparin employed in cardiovascular surgeries often leads to a high incidence of bleeding complications. Protamine employed in heparin reversal, however, can cause severe adverse reactions. In an attempt to address this clinical problem, we developed low molecular weight protamine (LMWP) as a potentially effective and less toxic heparin antagonist. A homogeneous 1880-d peptide fragment, termed LMWP-TDSP5 and containing the amino acid sequence of VSRRRRRRGGRRRR, was derived directly from protamine by enzymatic digestion of protamine with thermolysin. In vitro studies demonstrated that TDSP5 was capable of neutralizing various anticoagulant functions of both heparin and commercial low molecular weight heparin preparations. In addition, TDSP5 exhibited significantly reduced crossreactivity toward mouse sera containing antiprotamine antibodies. TDSP5 showed a decrease in its potential in activating the complement system. All of these findings suggested the possibility of markedly reduced protamine toxicity for TDSP5. In this article, we conducted preliminary in vivo studies to further demonstrate the feasibility and utility of using LMWP as a nontoxic clinical protamine substitute. Dogs were chosen as test animals because they were known to magnify the typical human response to protamine. By using a full spectra of biological and clinical assays for heparin, including the anti-IIa and anti-Xa chromogenic assays and the activated partial, thromboplastin time and TCT clotting assays, TDSP5 showed that it could completely neutralize all these different anticoagulant functions of heparin in dogs. Although administration of protamine in dogs produced a significant reduction in mean arterial blood pressure (-14.9 mm Hg) and elevation in pulmonary artery systolic pressure (+5.0 mm Hg), the use of TDSP5 in dogs did not elicit any statistically significant change in any of the variables measured. Furthermore, the use of LMWP also significantly reduced the protamine-induced transient thrombocytopenic and granulocytopenic responses. The white blood cell counts and platelet counts decreased to 82.1% and 60.0% of baseline, respectively, in dogs given intravenous protamine compared to 97.8% and 88.6% of baseline in dogs receiving TDSP5. These preliminary findings indicated that LMWP could potentially provide an effective and safe means to control both heparin- and protamine-induced complications.

Computed tomographic venography is specific but not sensitive for diagnosis of acute lower-extremity deep venous thrombosis in patients with suspected pulmonary embolus.

PURPOSE: Duplex ultrasound scanning (US) is the accepted standard means of diagnosis for lower-extremity suprageniculate deep venous thrombosis (LE-DVT). Computed tomographic venography (CTV) has been proposed as an alternative modality for diagnosis of LE-DVT in patients with suspected pulmonary embolism (PE). This study compared CTV with US as a means of diagnosing acute LE-DVT. METHODS: A retrospective review of US and CTV scans from 136 patients with suspected PE who underwent both studies to exclude acute LE-DVT at a single institution was performed. Studies were reviewed and coded in a blinded manner. US was considered to be the reference test. Direct costs of each study were determined by using commercial software. RESULTS: The sensitivity and specificity rates of CTV were 71% and 93%, respectively. The positive predictive value, negative predictive value, and accuracy rates of CTV were 53%, 97%, and 90%, respectively. DVT localization was the same in eight of 10 cases in which the results of both US and CTV were positive. CTV costs and charges per study were greater than those of US by $46.88 and $602.00, respectively. CONCLUSION: CTV is specific, but has a lower sensitivity rate and positive predictive value for the diagnosis of acute LE-DVT compared with US. Additionally, CTV is more costly than US scanning. Because of the lower sensitivity rate and positive predictive value and the increased cost of CTV, US remains the screening study of choice in cases of suspected acute LE-DVT.

Gadolinium-enhanced MR angiography (Gd-MRA) of thoracic vasculature in an animal model using double-dose gadolinium and quiet breathing.

OBJECTIVE: To evaluate a gadolinium-enhanced MR angiography (Gd-MRA) imaging protocol for the assessment of thoracic vessels using double-dose gadolinium and quiet breathing. An animal model was used to simulate imaging in infants and young children. MATERIAL AND METHODS: Six baboons (Papio anubis), mean weight 5.7 kg, were sedated and intubated. After the injection of double-dose Gd-DTPA (0.2 mmol/kg) through a peripheral vein, a coronal spoiled 3D gradient-echo volume acquisition was obtained during quiet breathing. Two radiologists reviewed the images for visualization of aortic arch, brachiocephalic vessel origins, pulmonary arteries (central, upper lobe and descending branches), and pulmonary veins (upper and lower). RESULTS: Visualization was excellent for the aortic arch, brachiocephalic vessel origins, and pulmonary arteries, including the hilar branches. Visualization was excellent for the lower and right upper pulmonary veins and fair for the left upper pulmonary vein. There was excellent agreement between radiologists. CONCLUSION: Imaging of thoracic vessels with Gd-MRA using double gadolinium during quiet breathing was effective in our animal model. The advantages of this technique include a short imaging time and depiction of vascular segments--branches of pulmonary arteries and intraparenchymal segments of pulmonary veins--not optimally visualized with other non-invasive imaging techniques.

A clinical comparison of pneumatic compression devices: the basis for selection.

PURPOSE: The five pneumatic compression devices (PCDs) that are marketed provide mechanical protection from deep venous thrombosis (DVT). They differ with respect to patterns of compression and the length of the sleeve. Evidence linking differences to clinical outcomes is lacking. Our purpose was twofold: to evaluate each of the marketed PCDs with respect to effectiveness, compliance, and patient and nursing satisfaction and to determine whether there is a clinical basis for the selection of one device over another. METHODS: Each of the marketed devices was used exclusively for a 4-week period. Patients participated in an evaluation including venous duplex ultrasound scan, DVT risk assessment, and device evaluation. Vascular laboratory records were used to document DVT. Compliance was measured by meters installed on all pumps. A ranking matrix was stratified by compression pattern: rapid graduated sequential compression, graduated compression, and intermittent compression, and each device was rated by patients and nurses. RESULTS: The PCDs were used in 1350 cases with a DVT rate of 3.5% ranging from 2% to 9.8% depending on the method of compression. Patients with DVT were older (58 vs 54 years), had better compliance (67% vs 50%), and had more compression days (11 vs 7.2). When thigh-length sleeves were used, a greater proportion of DVT occurred in the proximal segments (71%) as compared with the number of proximal DVT when the calf-length devices were used (52%; P =.21). Devices W, X, and Y had comparable rates of DVT, which were lower than those for V and Z. Compression device W, [correction] with calf and thigh sleeves, achieved the best overall ranking largely because of high scores for patient and nurse satisfaction. CONCLUSION: Our data appear at odds with commonly held beliefs. We were unable to show a difference in DVT incidence based on the length of the device or the method of compression. Randomized studies are needed to confirm our findings and evaluate hypotheses derived from this study.

Renal artery aneurysms: a 35-year clinical experience with 252 aneurysms in 168 patients.

OBJECTIVE: To define the relevance of treating renal artery aneurysms (RAAs) surgically. SUMMARY BACKGROUND DATA: Most prior definitions of the clinical, pathologic, and management features of RAAs have evolved from anecdotal reports. Controversy surrounding this clinical entity continues. METHODS: A retrospective review was undertaken of 168 patients (107 women, 61 men) with 252 RAAs encountered over 35 years at the University of Michigan Hospital. Aneurysms were solitary in 115 patients and multiple in 53 patients. Bilateral RAAs occurred in 32 patients. Associated diseases included hypertension (73%), renal artery fibrodysplasia (34%), systemic atherosclerosis (25%), and extrarenal aneurysms (6.5%). Most RAAs were saccular (79%) and noncalcified (63%). The main renal artery bifurcation was the most common site of aneurysms (60%). RAAs were often asymptomatic (55%), with a diagnosis made most often during arteriographic study for suspected renovascular hypertension (42%). RESULTS: Surgery was performed in 121 patients (average RAA size 1.5 cm), including 14 patients undergoing unilateral repair with contralateral RAA observation. The remaining 47 patients (average RAA size 1.3 cm) were not treated surgically. Operations included aneurysmectomy and angioplastic renal artery closure or segmental renal artery reimplantation, aneurysmectomy and renal artery bypass, and planned nephrectomy for unreconstructable renal arteries or advanced parenchymal disease. Eight patients underwent unplanned nephrectomy, being considered a technical failure of surgical therapy. Dialysis-dependent renal failure occurred in one patient. There were no perioperative deaths. Late follow-up (average 91 months) was available in 145 patients (86%). All but two arterial reconstructions remained clinically patent. Secondary renal artery procedures included percutaneous angioplasty, branch embolization, graft thrombectomy, and repeat bypass for late aneurysmal change of a vein conduit. Among 40 patients with clearly documented preoperative and postoperative blood pressure measurements, 60% had a significant decline in blood pressure after surgery while taking fewer antihypertensive medications. Late RAA rupture did not occur in the nonoperative patients, but no lessening of this group's hypertension was noted. CONCLUSION: Surgical therapy of RAAs in properly selected patients provides excellent long-term clinical outcomes and is often associated with decreased blood pressure.