Shirley K. Wrobleski

Extracellular DNA traps promote thrombosis

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

P-selectin and leukocyte microparticles are associated with venous thrombogenesis.

OBJECTIVES P-selectin inhibition has been found to limit venous thrombosis. We hypothesize that elevated levels of P-selectin will amplify thrombosis, mediated by procoagulant microparticles (MPs). METHODS Male mice (Mus musculus, n659), 20 to 25 grams, underwent IVC ligation to induce thrombosis. Groups consisted of wild type (WT) C57BL/6 controls, mice with high circulating levels of soluble P-selectin (CT), P-selectin gene-interrupted knockout mice (PKO), and E- and P-selectin gene-interrupted mice (EPKO). Additional groups were used to evaluate the ability of a P-sel antagonist (rPSGL-Ig) and an antibody directed against PSGL-1 to downregulate the effects of P-sel in CT mice and WT mice administered soluble P-sel at time of thrombosis. Animals were sacrificed on days 2 and 6 after IVC ligation. Thrombus mass (TM), vein wall morphometrics, and serum leukocyte/platelet microparticles (MPs) were evaluated by means of double-stained fluorescence-activated cell scanning analysis, and soluble P- and E-sel protein determination by ELISA. RESULTS At days 2 and 6 in phase I of the experiment, significant differences (P <.01) in TM were noted between groups, with CT animals having the largest thrombi (50% and 57% increase in TM compared to WT at days 2 and 6) while EPKO mice had the smallest thrombi. Statistically, greater levels of neutrophils and total inflammatory cells were noted in the vein walls of CT animals at day 2 compared with WT and PKO animals. A significant difference was noted between CT and EPKO for neutrophils, monocytes, and total inflammatory cells, also at day 2. At day 6, the only statistically significant difference was found for monocytes, with a higher number in the CT animals than in WT animals. The evaluation of MPs revealed that the CT mice had a mixed leukocyte (MAC-1) and platelet (CD41) MP population that was also present in WT and PKO mice on day 2 and day 6. EPKO mice revealed a primarily platelet-derived MP population. Of interest, the CT mice with the highest TM showed a high amount of mean channel fluorescence for MAC-1 (phycoerythrin) antibody, indicative of leukocyte MPs. CT mice revealed statistically higher levels of soluble P-selectin at days 2 and 6. In phase 2, an antibody directed against PSGL-1 was more effective than rPSGL-Ig in decreasing TM and limiting leukocyte-derived MP fluorescence. CONCLUSIONS This study demonstrates that high circulating levels of P-selectin are associated with increased thrombosis, whereas a lack of P-selectin and E-selectin is associated with a lessening of thrombosis. Additionally, leukocyte MPs are associated with venous thrombus formation. These data suggest the importance of selectins to venous thrombogenesis and show that P-selectin and leukocyte-derived MPs should be good targets to limit venous thrombus formation.

Venous Thrombosis–Associated Inflammation and Attenuation With Neutralizing Antibodies to Cytokines and Adhesion Molecules

Thrombosis and inflammation are closely related. However, the response of the vein wall to venous thrombosis has been poorly documented. This study examines the hypothesis that venous thrombosis is associated with an inflammatory response in the vein wall. In a rat model of inferior vena caval thrombosis, vein wall was temporally examined for inflammation by assessment of histopathology, leukocyte morphometrics, and cytokine levels. Animals were killed 1 hour and 1, 3, and 6 days after thrombus induction. Our findings demonstrated an early (day 1) neutrophil infiltration into the vein wall followed by a later (days 3 and 6) monocyte/macrophage and lymphocyte response. Cytokines were elevated only under conditions of venous thrombosis. Levels of epithelial neutrophil activating protein-78 (ENA-78), tumor necrosis factor-alpha (TNF), interleukin-6, and JE/monocyte chemoattractant protein-1 (JE/MCP-1) increased over the 6-day period, while macrophage inflammatory protein-1 alpha (MIP-1 alpha) peaked at day 3 after thrombus induction. Additionally, rats were passively immunized with neutralizing antibodies to TNF, ENA-78, MIP-1 alpha, JE/MCP-1, intercellular adhesion molecule-1 (ICAM-1), and CD18 compared with control antibodies. The most effective antibody early after thrombus induction for attenuating vein wall neutrophil extravasation was anti-TNF (P < .01). The monocyte/macrophage extravasation was inhibited most by anti-ICAM-1 followed by anti-TNF (P < .01). These findings demonstrate that venous thrombosis is associated with significant vein wall inflammation that is partially inhibited by neutralizing antibodies to cytokines and adhesion molecules.

Critical Review of Mouse Models of Venous Thrombosis

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

Neovascularization during venous thrombosis organization: A preliminary study

PURPOSE Thrombus organization after venous thromboembolism leading to recanalization occurs at a variable rate. The angiogenic chemokine interleukin-8 (IL-8) has been found in thrombus months after thrombus initiation. We hypothesize that thrombus organization involves neovascularization and leukocyte influx and that IL-8 administered at thrombus induction will promote thrombus organization. METHODS A group of rats underwent inferior vena caval occlusive thrombosis. At thrombus induction and every 24 hours, the rats were administered IL-8 (1 microgram) or serum albumin. The rats were killed at either day 4, day 8, or day 12, and, at death, colloidal carbon was perfused via the heart. The inferior vena cava was isolated, measured, weighed, and formalin fixed. The sections were stained with anti-polymorphonuclear leukocyte antibody, the endothelial marker factor VIII-related antigen, and with hematoxylin and eosin. Thrombus neovascularization (colloidal carbon) with morphometric analysis was normalized to the total thrombus area. In addition, the rats underwent perfusion with fluorescein isothiocyanate dextran (molecular weight, 150,000) at death to correlate with colloidal carbon perfusion, and thrombus fluorescence was determined. RESULTS Thrombus cellularity initially involved neutrophils, followed by monocytes. Significantly more neutrophils, monocytes, and cells that were defined as spindle shaped (fibroblasts and endothelial cells) were noted in the animals treated with IL-8. Neovascularization was significantly increased at day 4 in the animals treated with IL-8 versus the animals treated with serum albumin and was corroborated with a significant increase in thrombus fluorescein isothiocyanate dextran fluorescence at day 4 in the rats treated with IL-8. Colloidal carbon perfusion was noted within vascular channels without extravasation and colocalized with factor VIII-related antigen. CONCLUSION This study shows that thrombus organization involves neovascularization and that IL-8 augments thrombus organization.

Anti-P-selectin antibody decreases inflammation and thrombus formation in venous thrombosis

PURPOSE Venous thrombosis and inflammation are interrelated. P-selectin contributes to activation of leukocyte-mediated inflammation. Therefore, we hypothesized that the neutralization of P-selectin would decrease vein wall inflammation and thrombosis. METHODS Twelve baboons underwent infrarenal inferior vena caval balloon occlusion to induce thrombosis. Two groups of four baboons received neutralizing intravenous anti-P-selectin antibody (PSab) GA6 or CY1748 before occlusion and at days 2 and 4. Four baboons received saline control injections. One baboon per group was killed at days 2, 6, and 13, and at 2 months. Analysis included phlebography, ultrasound, gadolinium (Gd)-enhanced magnetic resonance venography (reflecting vein wall inflammation), and histologic, morphometric, and protein evaluation of the vein wall. Thrombus presence or absence was assessed. RESULTS By day 2 in PSab baboons, vein wall Gd enhancement was decreased in the mid-inferior vena cava and the right iliac vein (p < 0.05; GA6 vs control baboons), normalizing by 2 months. The mid-inferior vena cava revealed fewer neutrophils and total leukocytes in PSab baboons; however, for GA6 in the right iliac vein these decreases were not present despite the absence of Gd enhancement; they were decreased with CY1748. PSab baboons demonstrated significantly less thrombus than control baboons (p < 0.01, GA6 and CY1748 vs control baboons). CONCLUSIONS Anti-P-selectin antibody decreases vein wall inflammation and thrombus formation. Inhibition of P-selectin may be useful in venous thrombosis prophylaxis.

Venous thrombosis prophylaxis by inflammatory inhibition without anticoagulation therapy

OBJECTIVE This study was performed to determine the effectiveness of recombinant P-selectin glycoprotein ligand Ig (rPSGL-Ig) pretreatment to decrease thrombosis and inflammation in experimental venous thrombosis. rPSGL-Ig, a unique mucin-like glycoprotein, has a high affinity for P-selectin. METHODS Twelve juvenile baboons underwent inferior vena cava (IVC) thrombosis with temporary 6-hour IVC balloon occlusion. Before balloon placement, the animals received rPSGL-Ig (4 mg/kg; n = 8) or saline solution for control (n = 4). The animals underwent evaluation with duplex ultrasound scan imaging, magnetic resonance venography (MRV), phlebography, coagulation profile, and tissue analysis at death for cytokines and vein wall leukocyte morphometrics. With the MRV results, thrombus development, thrombus resolution, and inflammation (gadolinium; square millimeters of enhancement) were assessed. RESULTS Each animal provided two time points for evaluation (days 2 and 6 after balloon occlusion). A significant decrease in IVC thrombosis between balloons was found in the rPSGL-Ig animals (1 of 16) versus the control animals (5 of 8; P <.01). The MRV results showed significantly less enhancement in the rPSGL-Ig animals at days 2 and 6 (P <.05). Spontaneous thrombus resolution (including balloon sites) was significantly greater from day 2 to day 6 in the rPSGL-Ig animals versus the control animals (23% vs 2%; P <.001), without pulmonary embolism. Lower interleukin-8, platelet factor IV, and monocyte chemotactic protein-1 levels were found in rPSGL-Ig vein walls without significant differences in vein wall leukocyte morphometrics. There were significantly lower D-dimer levels in the rPSGL-Ig-treated animals (P <.05), but there were no differences in measurements of coagulation. Adequate circulating rPSGL-Ig levels were documented. CONCLUSION Pretreatment with rPSGL-Ig results in: (1) a significant inhibition of thrombosis and vein wall inflammation; (2) a decrease in vein wall cytokine expression; and (3) a promotion of thrombus resolution. Inflammatory inhibition by rPSGL-Ig without anticoagulation therapy provides effective venous thrombosis prophylaxis in experimental venous thrombosis.

Statins, inflammation and deep vein thrombosis: a systematic review

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

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

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

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.