ALBERT

Description: Abstract 3406 Background: Accurate and early diagnosis of DVT is crucial to prevent untreated DVT from progressing to fatal PE. Clinical scoring systems used in combination with D-dimer for DVT diagnosis can exclude but not confirm DVT, and vascular ultrasound is still needed. Diagnostic algorithms are challenging in cancer, where the burden of VTE is higher, clinical scores less accurate and baseline D-dimer frequently elevated. Soluble P-selectin (sPsel) is expressed upon activation of platelets and endothelial cells by a thrombotic stimulus. sPsel is known to be important in predicting VTE in cancer patients. We previously showed that elevated sPsel in combination with Wells' score can confirm lower extremity (LE) DVT with a positive predictive value (PPV) of 100% in a general patient cohort (Clin Appl Thromb Hemost, 2011; 17:425–31). To date, no validated biomarker can establish VTE in cancer patients. The primary objective of this analysis was to identify a biomarker that alone or in combination confirms the presence of acute DVT in cancer patients. Secondary objective was to apply the previously derived model to patients with active cancer. Methods: Patients referred to the diagnostic vascular ultrasound lab with signs or symptoms concerning for acute LE DVT were enrolled in a prospective cohort study. Following informed consent, patients underwent LE doppler, clinical assessment, and blood draw for biomarkers sPsel, D-dimer, and CRP. Exclusion criteria were age 〈 18, isolated calf vein DVT, pregnant, nursing, or on therapeutic anticoagulation. Only subjects with active cancer, defined as actively receiving anti-tumor therapy, or with physical or radiological evidence of malignancy within 6 months of enrollment, were included in this subset analysis of previously published data. Continuous and categorical values were compared with t-test and chi-squared test, respectively. PPV and NPV were estimated from cut points. Results: Between 2006 to 2012, 442 patients with LE DVT were eligible for analysis; 99 had active cancer. 60 cases (63% male) had a confirmed proximal LE DVT; 39 controls (56% male) had leg pain but negative for VTE. Cases had increased prior history (p=0.001) and family history of VTE (p=0.039). No difference in mean age (62.4 vs. 61.8 years), BMI, or active chemotherapy was seen. Cancer types (%) included: breast (4), lung (10), hematologic (22), melanoma (3), GI (16), GU (16), Gyn (11), brain (4), other (6). sPsel, D-dimer, CRP, and Wells' score were all elevated in cases versus controls at time of DVT diagnosis. Application of our previously derived model of sPsel + Wells' with cut point of 2 yielded a similar PPV but lower NPV in active cancer versus non-cancer patients. Had imaging not been available, we could have ruled in or out 27.5% (27/98) patients for LE-DVT with this rule. D-dimer + Wells' had a superior NPV, albeit in a low number of patients. Raising the Wells' cut point to 3 (to accommodate a point assigned in the score for active cancer) improved the PPV for both the sPsel and D-dimer models, at the expense of the NPV in the D-dimer model. 38% (37/98) cancer patients could be ruled in or out for LE-DVT without imaging in this sPsel model. Conclusions: D-dimer is the most commonly used marker to rule out acute DVT. However, due to its low specificity and elevated baseline value in patients with cancer, it cannot be used to confirm clot presence. In patients with active cancer presenting with potential symptoms of LE DVT, sPsel, D-dimer, CRP and Wells' criteria were all elevated in cases versus controls. A combination of sPsel ≥ 90 ng/mL + Wells' ≥ 3 was superior to the D-dimer + Wells score to rule in clot at the time of presentation. D-dimer remained superior to rule out clot in this population. In the future, rapid VTE diagnosis through the use of novel biomarkers such as sPsel may help improve morbidity and mortality. Disclosures: No relevant conflicts of interest to declare.

Description: Leukocytes and leukocyte-derived microparticles contain low levels of tissue factor (TF) and incorporate into forming thrombi. Although this circulating pool of TF has been proposed to play a key role in thrombosis, its functional significance relative to that of vascular wall TF is poorly defined. We tested the hypothesis that leukocyte-derived TF contributes to thrombus formation in vivo. Compared to wild-type mice, mice with severe TF deficiency (ie, TF–/–, hTF-Tg+, or “low-TF”) demonstrated markedly impaired thrombus formation after carotid artery injury or inferior vena cava ligation. A bone marrow transplantation strategy was used to modulate levels of leukocyte-derived TF. Transplantation of low-TF marrow into wild-type mice did not suppress arterial or venous thrombus formation. Similarly, transplantation of wild-type marrow into low-TF mice did not accelerate thrombosis. In vitro analyses revealed that TF activity in the blood was very low and was markedly exceeded by that present in the vessel wall. Therefore, our results suggest that thrombus formation in the arterial and venous macrovasculature is driven primarily by TF derived from the blood vessel wall as opposed to leukocytes.

Description: Background : Selectins, among other adhesion-mediated functions, facilitate and augment thrombosis; standard anticoagulants address thrombosis but also increase bleeding risk. Previous work in animal models showed inhibiting E-selectin decreases venous thrombosis (VTE) and vein wall inflammation without adverse bleeding events, making E-selectin inhibition a favorable therapeutic candidate for VTE. GMI-1271 is a potent, specific E-selectin antagonist. Here we report final analysis of safety, PK, biomarker and bleeding risk profile for GMI-1271 in 2 phase 1 studies of healthy subjects. Methods: The first study was a blinded single ascending dose (SAD) evaluation of 2, 5, 20, or 40mg/kg of GMI-1271 (n=4/cohort), vs placebo control saline (n=4) or active control Lovenox 1mg/kg (n=4). The second was an open-label multiple ascending dose (MAD) study of 10 (n=3) or 20mg/kg (n=3) of GMI-1271 QD d 1-5 vs Lovenox 1mg/kg (n=2) d 1-5. Assessments included safety (adverse events [AEs], clinical labs, bleeding time, PT/PTT, vitals, exam); PK (plasma and urine); and biomarkers. Biomarkers included ELISAs (CRP, D-dimer, IL-10, MPO, Prothrombin fragment 1.2, soluble E-selectin (sEsel), soluble P-selectin (sPsel), sICAM, Thrombin-antithrombin complex (TAT), Tissue Factor (TF), TNFα, VWF activity, and sCD40L); PicoGreen Assay for circulating DNA; flow cytometry (Platelet Monocyte Aggregates (PMA), Mac-1, LFA-1, and CD44) and Thromboelastography (TEG). See Table 1 for functional description. SAD remains blinded to GMI-1271 or placebo (GMI-1271/p). In SAD, we measured biomarker values at baseline, 8 and 24 h after dosing. Analysis was performed of biomarkers at each dose level and then pooled by GMI-1271/p vs Lovenox. In MAD, we measured biomarker values at baseline and day 4. Comparisons were made using unpaired t-test. Results: In total, 32 subjects enrolled and received GMI-1271/placebo (GMI-1271/p; 20), GMI-1271 (6) or Lovenox (L; 6). Safety: All subjects completed dosing uneventfully. Safety findings for GMI-1271/p were unremarkable. No moderate or serious AE were seen. AE overall were as expected in healthy volunteers. In SAD, only 1/20 GMI-1271/p subjects experienced an AE possibly related to study drug (mild transient headache); 0/6 in MAD. In the L group we saw expected mild transaminitis and injection site bruising. In all studies, GMI-1271 had no effect on bleeding time, PT, or PTT. PK: Plasma levels, Cmax, and AUC increased in a linear manner. Cl, Vz, and t ½ were not dose dependent; no accumulation was seen with multiple dosing (Fig 1 and 2). TEG: In SAD, there was a tendency to increase R, K, and decrease A and MA with no change in % lysis in L vs GMI-1271/p. In MAD, we saw a similar trend as SAD except for an increase in % lysis in L. In the pooled SAD analysis, there was a statistically significant difference between GMI-1271/p and L (higher values) for R (p

Description: Deep vein thrombosis (DVT) and its complication, pulmonary embolism, are frequent causes of disability and mortality. Although blood flow disturbance is considered an important triggering factor, the mechanism of DVT initiation remains elusive. Here we show that 48-hour flow restriction in the inferior vena cava (IVC) results in the development of thrombi structurally similar to human deep vein thrombi. von Willebrand factor (VWF)–deficient mice were protected from thrombosis induced by complete (stasis) or partial (stenosis) flow restriction in the IVC. Mice with half normal VWF levels were also protected in the stenosis model. Besides promoting platelet adhesion, VWF carries Factor VIII. Repeated infusions of recombinant Factor VIII did not rescue thrombosis in VWF−/− mice, indicating that impaired coagulation was not the primary reason for the absence of DVT in VWF−/− mice. Infusion of GPG-290, a mutant glycoprotein Ibα-immunoglobulin chimera that specifically inhibits interaction of the VWF A1 domain with platelets, prevented thrombosis in wild-type mice. Intravital microscopy showed that platelet and leukocyte recruitment in the early stages of DVT was dramatically higher in wild-type than in VWF−/− IVC. Our results demonstrate a pathogenetic role for VWF-platelet interaction in flow disturbance-induced venous thrombosis.

Description: Introduction: Despite efforts toward prevention, thousands of patients suffer from venous thromboembolic disease (VTE), consisting of deep vein thrombosis (DVT) and pulmonary embolism (PE), each year; this results in significant morbidity and mortality. Clinical prediction rules, such as the Wells score, in conjunction with biomarkers like the D-dimer, are suggested by national guidelines to aide in evaluating for DVT. Substantial interest exists in developing improved diagnostic testing strategies and algorithms to limit the need for more costly, time consuming, and/or invasive testing. Novel biomarkers, like the soluble P selectin (sP-Sel), a cell adhesion molecule that functions to mediate thrombus formation and amplification, have shown promise in this area. While cancer patients suffer from a high burden of VTE, the D-dimer and Wells score are less helpful in this group. This is due to non-specific D-dimer elevations in these patients, and cancer being part of the Wells risk stratification schema. We sought to identify a more specific combination of biomarkers and Wells score, for the diagnosis of DVT that would apply to cancer patients as well. Methods: We previously performed a prospective cohort study of adults presenting with symptoms of DVT afflicting the upper or lower extremities (Vandy et al. J Vasc Surg Venous Lymphat Disord., 2013). Patients were enrolled from December 2008 to July 2013. Those with isolated calf DVT, superficial venous thrombosis, pregnant or breastfeeding, on therapeutic anticoagulation, or with symptoms of simultaneous upper and lower DVT were excluded. After informed consent was obtained, clinical characteristics and biomarkers (D-dimer, C-reactive protein (CRP), Von Willebrand Factor activity (VWF), and sP-Sel) were collected, and duplex ultrasonography was performed to determine if DVT was present. In this subset analysis, cancer patients were compared to non-cancer patients with regards to thrombotic risk factors, biomarker values, and to assess the test characteristics of various combinations of biomarkers and Wells score. Results: A total of 373 patients were enrolled, 151 (40%) in the cancer group, compared to 222 (60%) in the non-cancer group. Cancer patients were more likely to have a DVT on confirmatory testing (58.9% vs. 43.2%). Cancer patients tended to be older, male, have a lower BMI, more recent acute illness, and more central lines, relative to the non-cancer group. Non-cancer patients were more likely to have other potential risk factors for thrombosis, i.e. recent surgery, oral contraceptive use, and a family history of thrombosis. Biomarker values for CRP and sP-Sel were similar, however VWF and D-dimer values were significantly higher in the cancer patients (see table). D-Dimer 〉 500 with Wells score ≥ 2 was less specific for the diagnosis of DVT among cancer patients compared to non-cancer patients (p=0.003). However, D-Dimer ≥ 500 with sP-Sel ≥ 90 showed not only high specificity, but that specificity was not different between the cancer and non-cancer populations (p=0.88). sP-Sel ≥ 90 and Wells ≥ 2 had similar performance characteristics in both groups as well (p=0.54 for specificity, p=0.14 for positive predictive value (PPV)). Conclusion: Co-morbid risk factors for thrombosis among cancer patients included an older age, male gender, and an increased use of central lines. This group was less likely to have other potential thrombotic risk factors like family history or recent surgery. Our results concur with the finding that the D-dimer, combined with a clinical prediction rule, is not as helpful for DVT in cancer patients. Moreover, this study further supports sP-Sel as a specific test for DVT, that can be combined with clinical information (Wells score) or other laboratory data (D-dimer) to reflect the presence of DVT and potentially rule in clot; the test seems equally useful for both cancer and non-cancer populations. This could potentially be used to guide initial patient management in scenarios where imaging or further testing is not immediately available, such as in an outpatient clinic or rural area. Further exploration of the optimal strategy for utilizing biomarkers and clinical prediction rules in the diagnosis of DVT in cancer is needed. Table Table. Disclosures Sood: Bayer: Research Funding.

Description: Malignancy is a major risk factor for venous thromboembolic events (VTE), but not all patients with malignancy develop this complication. Who best to aggressively prophylax is thus not well defined. In the current study, 960 consecutive patients, 523 men and 437 women, admitted to the University of Michigan with malignancy between 1992–2000 were identified using ICD-9CM codes. Factors including cancer stage, type, therapy, and patient vital status were obtained from a database maintained prospectively by the University of Michigan Cancer Registry as well as a review of the medical record. Acute VTE, confirmed on radiological or ultrasound studies, occurred in 408 patients and were compared to 552 patients who did not experience any VTE using logistic regression analysis. Factors associated with VTE include solid tumors (Odds Ratio 5.0; 95% confidence interval 1.65–14.9, P =.004), infection (4.9; 1.2–19.8, P =.025), and advanced age (1.05; 1.03–1.08, P

Description: Abstract 3599 Poster Board III-536 Introduction P-selectin is a pro-inflammatory molecule that increases neutrophil recruitment to the vein wall at the site of thrombosis. Aptamers are short single-stranded oligonucleotides with the ability to bind small molecules and diverse proteins such as P-selectin. P-selectin could be targeted by a specific inhibitor or novel aptamer to limit inflammation following deep vein thrombosis (DVT). In this study, the effect of a novel anti-P-selectin aptamer on reducing DVT associated inflammation was evaluated. Methods Male C57BL/6J mice (20-25g) were placed into groups: anti-P-selectin aptamer ARC5690 (APSA, 2mg/kg, 5mg/kg, and 10mg/kg intraperitoneal [IP]), anti-P-selectin control aptamer “scrambled sequence” (PSCA 1mg/kg and 10 mg/kg IP), anti-P-selectin antibody (APSB 0.2mg/kg IP), ligation only (LO), or true control (TC). Inferior vena cava (IVC) ligation, below the renal vein, was performed on all groups except TC. Groups PSCA and APSA received daily injections starting two days pre-IVC ligation and continued up to 3 days post-IVC ligation. Groups TC and LO did not receive test compounds. At 3 days post-IVC ligation, mice were euthanized. The IVC was harvested and weighed or submitted for vein wall morphometric inflammatory cell histological analysis. Blood was collected via cardiac puncture for plasma soluble P-selectin (sP-sel) protein analysis. Data was analyzed using a student t-test and Kruskal-Wallace test with a Dunn's multiple comparison test. Results The total leukocyte counts in APSA groups 5 mg/kg and 10 mg/kg were significantly reduced compared to the PSCA groups and APSA group 2 mg/kg (p

Description: Key Points We determined the location of gal3bp and gal3 and their role in promoting VT and leukocyte/endothelial cell interactions for the first time. Gal3bp and gal3 have the potential to be used as targets for future VT therapies.