ALBERT

Description: Introduction Within the first weeks of ICU admission, 10-30% of medical/surgical patients develop venous thromboembolism (VTE), and up to 60% of trauma patients. VTE has significant morbidity including longer mechanical ventilation, ICU and hospital stay. The Caprini VTE risk assessment model (Ann Surg, 2010; 251[2]:344-50) is based on clinical factors such as age and comorbidities. Previous data from our lab has demonstrated the role of soluble P-selectin (sPsel), D-dimer, CRP, von Willebrand factor activity (VWF), and ADAMTS13 in the diagnosis of DVT. The aim of this study is to determine levels of biomarkers and clinical characteristics in relation to Caprini risk level in patients both with and without DVT upon admission to the ICU. Methods We performed a prospective cohort study at the University of Michigan with patients from the surgical, neurological, trauma and medical ICUs. Inclusion criteria: age 〉 18 and at risk for DVT (i.e. not on therapeutic anticoagulation) when admitted to the ICU. At baseline, clinical information including demographics and comorbidities, Wells’ and APACHE score was collected, blood was drawn for biomarkers and upper and lower extremity duplex ultrasound (DVU) was performed. Blood samples and DVU were repeated every 7 days. Subjects were recruited within 24 hrs of ICU admission and stratified into low/moderate (Group 1), high (Group 2), and highest (Group 3) risk groups according to the Caprini model; Group 4 were DVT positive on screening ultrasound. Patients were followed until DVT positivity, floor transfer, or 35 days. All patients received VTE prophylaxis and had imaging performed as clinically indicated. ELISAs were performed for sPsel, D-dimer, CRP, and VWF activity, and slightly modified FRETS-VWF73 assay for ADAMTS-13 activity. ANOVA and t-test were used to compare continuous, and Chi-square categorical, variables between the groups. Spearman correlation coefficients were performed to assess the relationship between Caprini score and other variables. Results From 12/08-10/12, 145 subjects were recruited. The age range was 20-88 years. DVT positive patients on screening ultrasound were significantly older, more likely to be male, and have a personal history of VTE. There was no difference in BMI or family history of VTE. DVT positive subjects (Group 4) had a significantly higher baseline mean D-dimer, sPsel, day 1 Wells’ and APACHE score compared to the lowest risk group (Group 1), and a significantly lower ADAMTS13 level (see Table). Baseline D-dimer, Wells’ and APACHE scores rose with increasing Caprini risk group and were also significantly higher in Group 4 vs. Groups 2 and 3. There was a trend towards increasing baseline sPsel and declining ADAMTS13 level with increasing Caprini score. Increasing baseline CRP and VWF was also significantly correlated with increasing Caprini score. Eight patients (6.2%) developed a new DVT within the 35 day observation period (average day 10); 6/8 (75%) were in the highest risk group (Group 3). There was a trend towards higher baseline D-dimer, CRP, Wells’ and APACHE score in subjects who developed DVT vs. stayed negative. In patients who developed DVT, these markers did not significantly increase over time with serial sampling. There was a significantly higher BMI (mean, SE) 35.9 ± 3.3 vs. 29.4 ± 0.6 kg/m2, p

Description: Abstract 3398 INTRODUCTION: VTE is common in patients with cancer and causes significant morbidity and mortality. Clinical risk models and biomarkers including C-reactive protein (CRP), soluble P-selectin (sPsel), and D-dimer have been used to predict VTE in diverse groups of cancer patients at varying risk for VTE. The applicability of these findings to specific high risk subtypes of cancer has not been established. Therefore, we sought to identify the value of clinical factors, plasma biomarkers, and risk models in predicting VTE in patients with pancreatic cancer, a malignancy with a high predilection for VTE. METHODS: Patients seen at the University of Michigan Comprehensive Cancer Center (UMCCC) and previously consented and enrolled in a prospective cohort study were eligible. Inclusion criteria are diagnosis of pancreatic adenocarcinoma, evaluation at UMCCC, no VTE within a month prior to cancer diagnosis, and documentation in the Electronic Medical Record (EMR) at least every 6 months until death. Primary objective was to identify factors predictive of VTE. Secondary objectives were to develop a VTE predictive model, assess the utility of published VTE risk models, and evaluate factors associated with overall survival (OS). Demographics, clinical data, and VTE (deep vein thrombosis [DVT], portal vein thrombosis [PVT], or pulmonary embolism [PE]) rate were obtained from the EMR. ELISAs were performed for CRP, D-dimer, Mac-2 binding protein, soluble E-selectin (sEsel), and sPsel using banked plasma specimens drawn at diagnosis. A retrospective cohort study was performed including univariate and multivariate regression analysis. The utility of predictive models by Khorana, et al (Blood, 2008. 111:4902–4907), which includes cancer site, body mass index (BMI), hemoglobin (Hb), platelet (plt) count, and white blood cell count, and the expanded model by the Vienna Cancer and Thrombosis Study (CATS) (Blood, 2010. 116:5377–5382), which additionally includes sPsel and D-dimer, were assessed. RESULTS: Between 2005 and 2011, 89 patients were eligible for analysis. Median follow-up was 268 (18–2433) days. Twenty (22%) cases had a VTE; 10 (50%) DVT, 2 (10%) PE, 4 (20%) PVT, and 4 (20%) multiple VTEs. Mean (SD) age was 63.4 (8.9) in cases and 65.3 (11.2) in controls. Women accounted for 55% of cases and 48% of controls. Higher BMI (median 28.8 [21.2–44.7] in cases vs. 25.4 [16.4–43.3] in controls, p=0.03) and lower plt count (median 241 [145–323] in cases vs. 289 [97–648] in controls, p=0.001) were associated with VTE on univariate analysis. On multivariate regression analysis, lower plt count (β −0.01, SE 0.004) and lower Hb (β −0.43, SE 0.20) were predictive of VTE after adjusting for BMI, tumor location, and treatment with surgery, chemotherapy or radiation (AUC 0.78). None of the biomarkers were significantly associated with VTE on univariate analysis, although there was a trend with D-dimer (p=0.09). The Khorana score was determined in 85 patients; 48 were intermediate (2 points) and 37 high risk (≥3 points) with VTE rates of 20.8% and 24.3%, respectively (p=0.70). The AUC of this model was 0.63. The risk score from CATS was calculated for 84 patients; 54 were intermediate (2 or 3 points), 17 high (4 points), and 13 highest risk (≥5 points). VTE incidence was not different among these groups and the AUC was 0.65. Factors associated with poor OS on univariate analysis were: age (per 10-year increment) (HR [95% confidence interval], p-value) (1.35 [1.07–1.71], 0.013), chronic kidney disease (5.67 [2.62–12.25],

Description: Introduction: Accurate and rapid diagnosis is essential in acute venous thromboembolism (VTE) to help prevent significant morbidity and mortality. Soluble P-selectin (sP-sel) is a cell surface ligand that aids in cell adhesion. It is released from activated platelets and damaged endothelial cells resulting in release of procoagulant molecules from leukocytes and aiding in thrombus generation. Our lab has previously shown elevated sP-sel is highly predictive of VTE in combination with clinical probability (Wells score). Current standards for ruling out VTE include combining Wells Score and plasma D-dimer; however, D-dimer increases with age and is less useful in the elderly. Age adjusted D-dimer, defined as 10*age in patients ≥50 years old, has been described as being more sensitive, which could rule out more clots in the elderly, sparing them from other diagnostic tests. Methods: We performed a prospective cohort study of patients ≥18 years old who presented with symptoms of deep venous thrombosis (DVT) in upper or lower extremities from December 2008 to July 2013. Exclusion criteria included isolated calf DVT, superficial thrombosis, indeterminate duplex scans, pregnancy or nursing mothers, therapeutic anticoagulation, and symptoms of simultaneous upper and lower extremity (LE) clot. After informed consent was obtained, biomarkers were drawn and duplex ultrasound was used to confirm or deny presence of acute clot. Our objective was to examine the accuracy of biomarker combinations and clinical probability score to rule in or rule out acute venous thrombus. Results: We recruited 461 patients to the study. Patients with positive lower extremity DVT were significantly more likely to be male, have a prior history of DVT, have active cancer or history of cancer, and be inpatient. Table 1: Biomarkers and Clinical Probability Score of Patients Presenting with Symptoms of LE DVT Table 1:. Biomarkers and Clinical Probability Score of Patients Presenting with Symptoms of LE DVT There were no significant differences in biomarkers between upper and lower extremity VTE aside from non-significance of sP-sel in upper extremity clots. We calculated the specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) of several combinations of biomarkers and clinical probability assessment. Table 2: Specificity and PPV Table 2:. Specificity and PPV Table 3: Sensitivity and NPV Table 3:. Sensitivity and NPV Using the age adjusted D-dimer did not improve the overall sensitivity when compared to the traditional cut off. However, sub-analysis demonstrated using D-dimer age 50, but its utility increased with age, making it a promising biomarker to safely rule out thrombosis in the elderly. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1345 Introduction Thrombus stability is provided by very large polymers adhering to platelets and anchoring the thrombus to the vessel wall. The best described polymers are fibrin and von Willebrand Factor (VWF). Activated neutrophils and other leukocytes can form an extracellular fibrous network which is composed of DNA, histones, and granular proteins. These neutrophil extracellular traps (NETs) are present in various inflammatory diseases. In deep vein thrombosis (DVT) inflammation closely cooperates with thrombosis. Here we examine whether NETs provide a new means to support the adhesion and recruitment of platelets and whether NETs are present in DVT. Methods and Results: To study the interaction of platelets with NETs, we isolated human neutrophils, induced NET formation and perfused over the NETs human platelets in plasma or whole blood anticoagulated with the thrombin inhibitor PPACK. Microscopic analysis revealed that under flow platelets adhere avidly to NETs. Perfusion of whole blood at physiological shear resulted in formation of thrombi on NETs in a time dependent manner. Addition of DNase1 degraded NETs and removed all platelets and thrombi demonstrating their adhesion to NETs. Thrombus formation on NETs was absent if blood was supplemented with EDTA indicating the requirement for divalent cations. Perfusion of NETs with heparinized blood dismantled NETs and prevented thrombus formation. Incubation of NETs with heparin alone released histones from NETs, indicating that heparin destroys the chromatin backbone of NETs. Furthermore, immunocytochemistry revealed that NETs were able to bind platelet adhesion molecules VWF and fibronectin from human plasma. Immunohistochemical analysis of a baboon deep vein thrombus showed abundant extracellular chromatin which co-localized with fibronectin and VWF. Conclusions: We show that extracellular traps are able to promote thrombosis in vitro and are abundant in vivo in DVT. We propose that extracellular chromatin provides a new type of scaffold that promotes platelet adhesion, activation, and aggregation and may be important for thrombus initiation or stability. Disclosures No relevant conflicts of interest to declare.

Description: The diagnosis of deep venous thrombosis (DVT) is mostly dependent upon duplex ultrasound (DUS) examination of the affected extremity. Data from our laboratory implicates P-Selectin and microparticles (MPs) in the pathogenesis of venous thrombosis. A prospective study was performed to determine the sensitivity and specificity of plasma assays for D-Dimer, soluble P-selectin (P-selectin), and total MPs in patients with documented DVT by DUS. Three groups of individuals were examined: Group 1, 30 normal volunteers; Group 2, 22 patients positive for DVT on DUS (DVT); and Group 3, 21 patients with symptoms of leg pain but negative DUS for DVT (SMP). D-Dimer was measured by the Advanced D-Dimer assay (Dade-Behring, Deerfield, IL), P-Selectin was measured by ELISA (R&D, Minneapolis, MN.) and MPs were assayed by flow cytometry. Total MPs are cell fragments detected by anti-CD41 and anti-CD11b that are 〈 1 micron, as seen on flow cytometry. Group 1 individuals have D-Dimer levels of 1.53±0.12 mg/dl, P-Selectin of 0.34±0.05 ng/mg total protein (TP), and total MP of 370,103±41,910 particles/200 microliters of platelet-poor plasma. No differences in age (mean=48 vs. 51 yrs, respectively), weight, BMI, use of OCP/HRT, smoking, family history of DVT, or trauma history were noted between DVT and SMP patients. Patients with DVT were more likely to have traveled recently or have a malignancy present. 100% of DVT patients were at highest risk (score≥5) for thrombosis by thrombosis risk assessment (Caprini, JA, J. Thromb. Thrombolysis, 9:253, 2000) while only 62% of SMP patients were at highest risk. Group 2 individuals (DVT) have D-Dimer values of 8.20±2.03 mg/dl, P-Selectin of 0.98±0.11 ng/mg TP, and MPs 129±17% of control. Group 3 individuals (SMP) have D-Dimer values of 3.12±0.79 mg/dl, P-Selectin of 0.55±0.08 ng/mg TP, and MPs 99±12% of control. Differences were statistically significant between groups 2 and 3 for D-Dimer (p=0.01) and P-selectin (p

Description: Background: Previous studies have shown that inhibition of E-selectin can decrease thrombus formation and associated inflammation. E-selectin (CD-62E) is a cell adhesion molecule that is expressed on activated endothelial cells and plays an important role in leukocyte recruitment to the site of vascular injury. GMI-1271 is designed to mimic the bioactive conformation of the sialyl-Lex carbohydrate binding domain of E-selectin and is a specific E-selectin inhibitor. There remains an unmet medical need for a translatable therapeutic that can treat VT in combination with lower, safer levels of low molecular weight heparin (LMWH) anticoagulation. We hypothesize that E-selectin inhibition combined with LMWH will permit lower doses of therapeutic LMWH for the treatment of VT without increasing adverse bleeding events. Methods: Male C57BL/6J mice, 10 weeks old (23-28 grams, n5), underwent our electrolytic IVC model (EIM) to produce a non-occlusive thrombosis, via electrical free radical stimulation (250 µAmp) for 15 minutes. Experimental groups included non-treated controls (CTR-No Tx), animals given LMWH (3-6 mg/kg, SQ, once daily (qd), the E-selectin inhibitor GMI-1271 20/kg intraperitoneal (IP) twice daily (BID), and a combination of the agents. The dose range of LMWH that produced anti-Xa levels in the therapeutic range (0.5-1.0IU/mL) and significantly decreased thrombus weight in this mouse VT model was 5 and 6 mg/kg. Treated mouse groups received the first dose of experimental therapy immediately following thrombus induction and through day 2. Animals were euthanized 2 days post-thrombosis for tissue harvest and blood collection for the following evaluations: thrombus weight (grams), anti-Xa testing, and tail vein bleeding time (seconds). Results: GMI-1271 Works in Combination with LMWH to Decreases Venous Thrombosis : LMWH dosed at 6mg/kg and 5mg/kg alone, significantly decreased venous thrombus weight at 2 days, versus non-treated controls (73.0±7.5, 62.8±1.9 vs. 186.8±63.9 x10-4 grams, P

Description: Neutrophils are involved in venous thrombogenesis, through P-selectin and neutrophil endothelial traps (NETS). In order to determine the importance of neutrophils to thrombogenesis, neutrophil depletion was performed in our rat model of stasis-induced deep venous thrombosis (DVT). Animals were treated with control serum or rabbit anti-rat PMN serum administered perioperatively and sacrificed at two and seven days. At two days, neutropenic rats had 1.6-fold larger thrombi (P = 0.04) and 1.4-fold higher femoral venous pressures by manometry (P = 0.008) without a difference in thrombus neovascularization. By seven days, DVT sizes were similar, but vein wall injury persisted in neutropenic rats with a two-fold increase in vein wall stiffness by microtensiometry (P 〈 0.05), as well as a 1.2-fold increased thickness (P = 0.04). Vein wall and intrathrombus uPA by Western immunoblotting, as well as intrathrombus MMP-9 gelatinase activity was significantly less in neutropenic rats than controls (P 〈 0.001). Conversely, MMP-2 was significantly elevated in neutropenic inferior vena cava (IVC) at two days after DVT.1 P-selectin inhibition has been found to limit venous thrombosis in mice. Animals with high circulating levels of sP-selectin (^CT) were compared to selectin gene-deleted animals (PKO, EPKO) and wild-type (WT) mice. ^CT mice showed a significant 50% increase in thrombosis in our IVC ligation model while EPKO mice had the smallest thrombi. A significant difference was noted between ^CT and EPKO for neutrophils, monocytes, and total inflammatory cells at day two. Microparticle (MP) analysis revealed that in the ^CT, WT and PKO mice, a mixed leukocyte (MAC-1) and platelet (CD41) MP population was present. EPKO mice (with the smallest thrombi) revealed primarily a platelet-derived MP population, suggesting the importance of leukocyte-derived MPs in venous thrombogenesis. Of interest, the ^CT mice with the highest TM showed an elevated level of mean channel fluorescence for MAC-1 antibody, indicative of leukocyte derived MPs.2 Through processes that also involve the initial activation of leukocytes and platelets, neutrophils initiate and amplify thrombosis through the formation of NETS, which are extracellular fragments of DNA containing histones and antimicrobial proteins.3,4 In vitro and in vivo, NETs provide a scaffold and stimulus for thrombus formation.5 In order to investigate if plasma DNA (surrogate for NETS) is elevated in patients with DVT and to determine correlations with other biomarkers of DVT, we studied patients presenting to our diagnostic vascular laboratory. From December 2008 to August 2010, patients were divided into three distinct groups: (1) DVT positive, patients symptomatic for DVT confirmed by ultrasound (n = 47); (2) DVT negative, patients with leg pain but negative by ultrasound (n = 28); and (3) control healthy non-pregnant volunteers without signs or symptoms of active or previous DVT (n = 19). Blood was collected for biomarkers and the Wells score risk of DVT was assessed. Results showed that circulating DNA was significantly elevated in DVT patients, compared with both DVT-negative patients (57.7 vs. 17.9 ng/mL; P 〈 0.01) and controls (57.7 vs. 23.9 ng/mL; P 〈 0.01). There was a strong positive correlation with C-reactive protein (P 〈 0.01), D-dimer (P 〈 0.01), VWF (P 〈 0.01), Wells score (P 〈 0.01) and MPO (P 〈 0.01), along with a strong negative correlation with ADAMTS13 (P 〈 0.01) and the ADAMTS13/VWF ratio. The logistic regression model showed a strong association between plasma DNA and the presence of DVT (ROC curve 0.814) suggesting a role for DNA in venous thrombogenesis.6 References: 1. Varma MR, Varga AJ, Knipp BS, Sukheepod P, Upchurch GR, Kunkel SL, Wakefield TW, Henke PK. Neutropenia impairs venous thrombosis resolution in the rat. J Vasc Surg. 2003;38:1090-1098 2. Myers DD, Hawley AE, Farris DM, Wrobleski SK, Thanaporn P, Schaub RG, Wagner DD, Kumar A, Wakefield TW. P-selectin and leukocyte microparticles are associated with venous thrombogenesis. J Vasc Surg.2003;38:1075-1089 3. Fuchs TA, Brill A, Duerschmied D, Schatzberg D, Monestier M, Myers DD, Wrobleski SK, Wakefield TW, Hartwig JH, Wagner DD. Extracellular DNA traps promote thrombosis. Proceedings of the National Academy of Sciences. 2010;107:15880-15885 4. von Brühl M-L, Stark K, Steinhart A, Chandraratne S, Konrad I, Lorenz M, Khandoga A, Tirniceriu A, Coletti R, Köllnberger M. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. The Journal of Experimental Medicine. 2012;209:819-835 5. Massberg S, Grahl L, von Bruehl M-L, Manukyan D, Pfeiler S, Goosmann C, Brinkmann V, Lorenz M, Bidzhekov K, Khandagale AB. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. Nature Medicine. 2010;16:887-896 6. Diaz JA, Fuchs TA, Jackson TO, Kremer Hovinga JA, Lämmle B, Henke PK, Myers Jr DD, Wagner DD, Wakefield TW. Plasma DNA is elevated in patients with deep vein thrombosis. J Vasc Surg. 2013;1:341-348 Disclosures No relevant conflicts of interest to declare.

Description: Background: GMI-1271 is a potent, specific E-selectin antagonist which targets interactions between cancer cells and the bone marrow niche thereby increasing tumor sensitivity to chemotherapy; avoids HSC mobilization; and inhibits thrombosis without increasing bleeding risk. Delayed inflammatory cell recruitment into the vein wall post thrombus induction indicates a possible decrease in leukocyte activation, which may be crucial in treating both acute and cancer associated thrombosis. Here we report first-in-human evaluation of GMI-1271 for safety (including bleeding), PK, and biomarker data for effects on adhesion and mobilization. Methods: Two Phase I single dose escalation DBRCT in healthy subjects evaluated 2, 5, 10, and 20 mg/kg IV doses of GMI-1271. Study 1 is complete and unblinded (GMI-1271 vs placebo). Study 2 is ongoing, remains blinded (GMI-1271 vs placebo) with open-label positive control (Lovenox), and is reported in aggregate. In both studies, assessments included safety (adverse events [AEs], clinical labs, bleeding time, PT/PTT, vitals, and exam); PK (drug levels in plasma and urine); and biomarkers (plasma soluble E-selectin [sEsel], sPsel, sICAM-1 levels; and blood CD34+ counts). Only sEsel and CD34+ are reported for Study 1; sEsel, sPsel, sICAM-1 for Study 2. Comparisons to baseline were made by ANOVA and paired t-test models; PK analyses were for total clearance (CL), volume of distribution (Vz), elimination half-life (t½), fraction excreted (Fe), and renal clearance (CLr). Results: Forty-six subjects enrolled; 30 received GMI-1271 (10 each at 2 and 5 mg/kg; 6 at 10 mg/kg; and 4 at 20 mg/kg) and 16 received control (13 placebo, 3 Lovenox). Safety: All subjects completed the studies; labs, vitals, and exam were unremarkable. Bleeding times and PT/PTT were unaffected by E-selectin inhibition. No serious AEs were seen. For Study 1, AEs were seen in 12/18 (66.7%) of those on GMI-1271 and 5/10 (50%) on placebo. AEs were mostly mild events at the infusion site (i.e. tenderness or erythema), and occurred in both groups. One moderate AE occurred (vasovagal reaction) in the 2 mg/kg GMI-1271 cohort. Three AEs were possibly related to drug: 2 in the 2 mg/kg cohort (infusion site bruise, lightheaded), and 1 in the 5 mg/kg cohort (metallic taste). For Study 2, AEs were seen in 12/15 (80%) of those on GMI-1271/placebo and 3/3 (100%) on Lovenox. All AEs were mild. In GMI-1271/placebo group, all AEs were considered unrelated to study drug; 3 were infusion site bruise (1 per dose level). In the Lovenox group, AEs were bruise at injection site (related) and blood in urine (possibly related). PK: Plasma levels, Cmax, and AUC increased in a consistent dose-related manner in both studies (figure). CL, Vz, and t½ were not dose dependent; the latter averaged ~2.3 hours. ~66% of the dose was excreted unchanged in the urine independent of dose level, and CLr averaged 86 mL/min, less than estimated CrCl, suggesting tubular reabsorption is one component of CLr. Biomarkers: In Study 1, the absolute CD34+ and %CD34+ cell counts were normal across all cohorts. There was no dose-response for changes in peripheral CD34+ counts after administration of GMI-1271 (doses 2-10 mg/kg). Plasma concentrations of sEsel were relatively constant in the placebo and GMI-1271 2 mg/kg cohorts, with significant reduction (vs baseline, p=0.012) at 48hrs post-2mg/kg dose only. In the 5 and 10 mg/kg cohorts sEsel decreased significantly post-dose (p

Description: Abstract 3273 Introduction: E- and P-selectin have structural similarities and both facilitate white blood cell tethering on vascular endothelium. The beneficial effect of combined E- and P-selectin inhibition in decreasing venous thrombosis (VT) by gene deletion in a mouse model of VT has been demonstrated. GMI-1070 is a pan-selectin inhibitor designed to mimic the bioactive conformation of the sialyl-Lex carbohydrate binding domain of E-selectin and the sulfate interactions of P- and L-selectins. GMI-1070 has primary activity against E-selectin, with second and tertiary activity against P- and L-selectin. It has passed Phase I clinical trials showing no serious adverse events, and has a serum half-life in humans of 7 to 8 hours. In this study, the effect of pan-selectin inhibition by GMI-1070 on reducing VT was evaluated. Methods: Male C57BL/6J mice (20-25grams) underwent our electrolytic IVC model (EIM) to produce a non-occlusive thrombosis via electrical free radical stimulation (250 μAmp) for 15 minutes. Experimental groups included the following: GMI-1070 delivered continuously by mini osmotic pump (300 mg/ml), and mice administered saline via the same protocol served as controls (SAL CTR). Continuous delivery of GMI-1070, or saline, began one day pre-thrombus induction. Mice were euthanized 2 and 6 days post-thrombosis for tissue harvest and blood collection for the following evaluations: thrombus weight (grams); plasma soluble E- and P-selectin (ng/mg total protein); vein wall E- and P-selectin protein by ELISA (pg/mg total protein); and vein wall inflammatory cell counts per high powered field. Results: Continuous GMI-1070 administration significantly decreased venous thrombus weight (WT) two days post thrombosis (78±8 vs. 216±97 ×10−4 grams, P

Description: Abstract 3422 Introduction: Selectins function in venous thrombosis presumably by binding and activating immune cells to initiate the coagulation cascade. E-selectin (CD62E) is known to bind and activate both monocytes and neutrophils. GMI-1271 is a small molecule antagonist that specifically inhibits E-selectin and is rationally designed to mimic the bioactive conformation of the sialyl-Lex carbohydrate ligand. Here we determine whether specific inhibition of E-selectin is sufficient to inhibit acute venous thrombosis and associated inflammatory events in both prophylactic and treatment protocols without causing the broader effects of increased bleeding time. Methods: Male C57BL/6J mice underwent our electrolytic inferior vena cava (IVC) model to produce a non-occlusive thrombosis via electrical stimulation (250 μAmp). Animals were divided into prophylactic or treatment groups. Both groups included the following: non-thrombosed animals (TC, no surgery or drug), 2 Day sham (needle inside the IVC and no current or drug), 2 Day CTR (current and no drug), 2 Day GMI-1271 (10mg/kg IP BID), and LMWH (Lovenox®, 6mg/kg SQ QD). Animals were divided into prophylactic or treatment groups. Mice in the prophylactic group were dosed one day pre-thrombus induction through day 1. Animals in the treatment groups received the first dose of the drug following thrombus induction on day 1. Mice were euthanized 2 days post-thrombosis for tissue harvest and blood collection for the following evaluations: thrombus weight; vein wall inflammatory cell counts per high power field; vein wall-thrombus histology; and intra-thrombus polymorphonuclear cell (PMN) counts. A separate group of mice received IV administration of compounds for tail bleeding time evaluation (seconds). Results: GMI-1271 Significantly Decreases Venous Thrombus Weight (Figure 1). Treatment with GMI-1271 decreased venous thrombus formation in a dose-dependent manner with significant inhibition at 10mg/kg (P=0.0271). Treatment with LMWH significantly decreased thrombus formation 2 day post induction at 6mg/kg (P=0.0203). All mice pre-treated prophylactically with GMI-1271 or LMWH followed the same pattern of decreasing thrombus weight 2 days post injury (P