Angela E. Hawley

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.

D-dimer, P-selectin, and microparticles: Novel markers to predict deep venous thrombosis: A pilot study

Current plasma markers for diagnosis of deep venous thrombosis (DVT) allow for exclusion of the diagnosis, but lack adequate specificity to establish the diagnosis. Thus, a prospective study was performed to determine the sensitivity and specificity of plasma assays for D-dimer, soluble P-selectin (P-selectin), and total microparticles in patients with documented DVT by duplex ultrasound. Three groups of individuals were examined: 30 normals; 22 positive for DVT on duplex ultrasound (Group 2); and 21 symptomatic, but negative on duplex ultrasound for DVT (Group 3). Group 1 individuals had D-dimer values of 1.53 +/- 0.12 mg/l and P-selectin values of 0.34 +/- 0.05 ng/mg total protein. Group 2 vs. Group 3 individuals had D-dimer values of 7.57 +/- 2.03 vs. 3.19 +/- 0.79 mg/l, p = 0.02; P-selectin values of 0.98 +/- 0.11 vs. 0.55 +/- 0.08 ng/mg total protein, p < 0.01; and micro-particle values of 129 +/- 17% vs. 99 +/- 12% of control, p = ns. Using a logistic regression model with dichotomous variables, we determined a sensitivity of 73%, specificity of 81%, and accuracy of 77% when combining D-dimer, soluble P-selectin, and total microparticles to differentiate Group 2 from Group 3 patients. Logistic regression using continuous variables yielded similar results (p = 0.05). This study demonstrates that plasma markers for DVT can be developed and achieve moderate sensitivity and specificity in diagnosing DVT. However for clinical applicability, the sensitivity/specificity will need to be improved. These studies also suggest the importance of soluble P-selectin in assessing DVT in humans.

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.

Interleukin-6: A potential target for post-thrombotic syndrome

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

Evaluation of Soluble P-Selectin as a Marker for the Diagnosis of Deep Venous Thrombosis

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.

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

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

Proteomics of microparticles after deep venous thrombosis

BACKGROUND Microparticles (MP) are submicron size membrane vesicles released from activated cells that are associated with thrombosis and inflammation. MP present diverse biological expressions that may be linked to a unique subset of proteins derived from their origin cells. METHODS To identify these proteins, plasma samples were taken from 9 patients with deep venous thrombosis (DVT) documented by duplex ultrasound, 9 with leg pain but negative for DVT by duplex, and 6 healthy controls without a history of thrombosis, for fold variation. MP were extracted from platelet-poor plasma, digested separately with trypsin and tagged using iTRAQ reagents. The digests were subjected to 2-D LC separation followed by MALDI tandem mass spectrometry. Peak lists were generated and searched against all human sequences. For protein identification, a minimum of two peptides at 95% confidence was required. Later, iTRAQ ratios were generated comparing relative protein levels of DVT patients to baseline. The proteomic analysis was performed twice for each blood sample. Proteins were considered elevated or depressed if the iTRAQ ratio (R) deviated by 20% change from normal and a p-value less than 0.05. RESULTS Two proteins (Galectin-3 Binding Protein, [Gal3BP], R=1.76 and Alpha-2 macroglobulin [A2M] R=1.57) were differentially expressed on DVT patients. Nine proteins were depleted including fibrinogen beta and gamma chain precursors (R=0.65). CONCLUSIONS These proteins influence thrombosis through inflammation, cell shedding, inhibition of fibrinolysis and hemostatic plug formation. Further studies are needed to confirm the mechanistic role of these proteins in the pathogenesis of venous thrombosis in humans.

Thrombogenesis with continuous blood flow in the inferior vena cava: A novel mouse model

Several rodent models have been used to study deep venous thrombosis (DVT). However, a model that generates consistent venous thrombi in the presence of continuous blood flow, to evaluate therapeutic agents for DVT, is not available. Mice used in the present study were wild-type C57BL/6 (WT), plasminogen activator inhibitor-1 (PAI-1) knock out (KO) and Delta Cytoplasmic Tail (DCT). An electrolytic inferior vena cava (IVC) model (EIM) was used. A 25G stainless-steel needle, attached to a silver coated copper wire electrode (anode), was inserted into the exposed caudal IVC. Another electrode (cathode) was placed subcutaneously. A current of 250 muAmps over 15 minutes was applied. Ultrasound imaging was used to demonstrate the presence of IVC blood flow. Analyses included measurement of plasma soluble P-selectin (sP-Sel), thrombus weight (TW), vein wall morphometrics, P-selectin and Von Willebrand factor (vWF) staining, transmission electron microscopy (TEM), scanning electron microscopy (SEM); and the effect of enoxaparin on TW was evaluated. A current of 250 muAmps over 15 minutes consistently promoted thrombus formation in the IVC. Plasma sP-Sel was decreased in PAI-1 KO and increased in DCT vs. WT (WT/PAI-1: p=0.003, WT/DCT: p=0.0002). Endothelial activation was demonstrated by SEM, TEM, P-selectin and vWF immunohistochemistry and confirmed by inflammatory cell counts. Ultrasound imaging demonstrated thrombus formation in the presence of blood flow. Enoxaparin significantly reduced the thrombus size by 61% in this model. This EIM closely mimics clinical venous disease and can be used to study endothelial cell activation, leukocyte migration, thrombogenesis and therapeutic applications in the presence of blood flow.

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

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